Animal models have supported clinical studies in humans, with epidemiological data pointing out the potential side effects of EDCs on both the male and female reproductive systems, as well as increased disorders and functional problems. Over a considerable period, especially during the EDCs rush, there has been an observable correlation between EDCs exposure and reproductive health in both males and females. However, the most important aspect of human health in this context is the latent period, since exposure during the developmental stage may not have immediate consequences, and it may be difficult to detect the consequences until the body matures later in life. Additionally, fetal tissue examination is not an option for studying the effects of EDCs. ⁹

There has been a debate among the population regarding EDCs. Studies have shown that the early onset of puberty and decreased average age in ethnic groups in many countries can be observed as an effect of environmental EDCs. Heating plastics can produce hazardous chemicals that can cause hair loss in women and bear growth in men. In the 1960s, synthetic progestins and estrogens, such as oral contraceptives, were highlighted; however, these synthetic hormones are now extensively used beyond their original purpose. They are now used for commercial purposes, such as increasing cattle for meat and dairy production, as well as in humans for birth control. Antiestrogens, aromatase inhibitors, and anti-androgens are also used in cancer therapy. ^{8, 9} We are surrounded by intentional and unintentional EDCs in our daily lives, ranging from paracetamols to parabens in plastic toys. Both synthetic and natural EDCs are equally prevalent, such as phytoestrogens, which are notably used for pain relief in postmenopausal women.

As Diethylstilbestrol (DES) causes havoc in wildlife, it also has a dramatic effect on humans. From the 1940s or specifically (as mentioned in some literature), 1948 until 1971, it was prescribed to approximately 7 million women to prevent miscarriage in the first trimester. The first case of vaginal cancer in a newborn daughter was reported in 1971. Further investigations led to the discovery of its harmful side effects, which immediately ceased. PCBs were introduced in 1929, and their production ceased in 1972, but they came into the limelight in Japan in 1968 when the population was poisoned by consuming rice oil contaminated with PCBs, particularly affecting thyroid hormone synthesis. Similarly, PBDEs also exhibit thyroid-disrupting activity. Early exposure to these chemicals directly correlates with breast cancer in females and prostate cancer in males. Nonetheless, many more recurring diseases, such as obesity, can alter physiology. TBT tributyltin (TBT) have not been fully elucidated, but it is used in the drug rosiglitazone, which has potential effects on humans, as the one consuming this drug showed increased body weight and fat cell numbers. ¹⁰

When discussing Male reproductive health issues, such as hypospadias, cryptorchidism, diabetes, prostate cancer, and testicular germ cell cancers, it is important to note the dramatic increase in these diseases since the onset of environmental EDC exposure. Furthermore, occupational studies have provided evidence of poor semen quality due to pesticide exposure in specific areas. The concentration of pesticides in urine is inversely related to degraded sperm quality and motility, as shown by the ORs. Testosterone and androgen are hormones responsible for puberty and reproductive system maturity in males. Sertoli and Leydig cells play a major role in sperm formation and androgen release. Therefore, EDCs that attack these cells directly interfere with their function and may lead to testicular dysgenesis. Studies conducted in the US have shown that EDC monobutylphthalate has catastrophic effects on male reproductive health, as it leads to decreased sperm quantity and quality. Another non-environmentally friendly chemical, dioxin, has yielded similar results. During the prepubertal stage (1-9 years of age), poor sperm quality was observed, while slight changes were observed in the age group–10-17. However, no difference in sperm quality was observed in males aged 18-27, and no health issues were observed. In terms of Female reproductive health, previous texts have discussed examples such as DES. In summary, pregnant females exposed to DES had daughters born with typical cervicovaginal cancer and other reproductive tract and organ issues. The fertility rate decreased, and there was an increased rate of ectopic pregnancies, early menopause, PCOS, and POF, among other common female reproductive health concerns. Animal and human studies over the past years have provided a combined view that supports conclusive views of induced reproductive abnormalities and chemicals mimicking critical developmental periods, affecting gonad organogenesis. ¹¹ Although there is no evidence to prove the hypothesis that EDCs affect general follicular development, there are cases in which they increase the rate of aneuploidy. Exposure of females to such chemicals leads to delayed menarche, altered cyclicity of menstruation, and decreased fecundity. Studies with DES provide prominent clues about the effects of such chemicals on female reproductive health. Although experimental studies might not have provided a clearer explanation of the mechanism, they have provided a brief overview of the effects of chemical exposure on human health. Two studies have described the effects of DES on female reproductive health, and one study concluded that there was no significant relationship between the exposure of EDC DES to uterine fibroids or leiomyomas (non-cancerous growth of the uterus) in the majority of born female children, while some showed a sensitive relationship. ¹² It was concluded that the differences observed might be due to the sensitivity of the techniques used for the evaluation; however, there were some definitive signs of increased rates of uterine fibroids. Unopposed estrogen signalling may also be one of the causes of increased tumor rates and obesity in females.

Numerous compounds, such as dioxins, methylchlor, DDT, and its derivatives, which widely affect living tissues, particularly in developed countries, have shown consequences in laboratory animal models and estrogen-sensitive cells, causing autoimmune diseases in women and affecting the brain-pituitary-ovarian axis. On the other hand, males exposure to estrogen agonist compounds often leads to prostate hyperplasia. These chemicals can affect psychomotor skills and development and impair visual recognition. EDCs cause bioaccumulation and disturb fauna in the environment. They create immunological, metabolic, and neurological effects, as well as directly attack genes, and have an epigenetic impact on adult or oocyte development. However, studies have shown that environmental pollutants have decreased potent estrogenic agonist activity. ¹² Figure 2 describes the epigenetic impacts affecting adults.

Experimental studies conducted in animal models serve as a foundation for further human model studies of the same compound. Evidence has been proposed to demonstrate the potential for similar effects of the same compound in both animals and humans. Endocrine-disrupting chemicals (EDCs) can cause permanent damage and alter developmental patterns. Additionally, the timing of EDC exposure plays a vital role. If exposure occurs in adulthood, the effects may not be as severe and may require high doses of EDCs. However, maternal exposure before pregnancy can lead to critical problems in the early stages of embryonic fetal life or reveal relevant visible functional problems until maturity or middle age. ¹³

Studies conducted in the United States on animals have shown permanent changes in tissues after exposure to hormone doses. In the 1970s, the biocide tributyltin (TBT) and in the 1980s, an accidental spill of the pesticide Dicofol at Lake Apopka greatly affected aquatic wildlife. Fish and birds in and near the Great Lakes of North America showed abnormal thyroid functions, decreased fertility, demasculinization, and feminization in male fish, and defeminization and masculinization in female fish, birds, and gastropods. Alterations in immune function were also observed, in addition to physical and hormonal changes. EDCs affect reproduction and some signs of early mortality. Intentional EDCs such as Diethylstilbestrol (DES) and unintentional EDCs such as DDT have been studied in various models. DES, a synthetic nonsteroidal estrogen agonist, was first synthesized in 1938 and tested in rodents and female mice. Rodents showed abnormal growth or development of the prostate, whereas female mice showed cornification in the vaginal epithelium. In the 1940s, o, p’-DDT was introduced into the environment on a large scale and banned in the USA in 1972. Studies have shown that eggshell thinning and cracking in bald eagle eggs affect embryonic survival. DDT mimics the action of endogenous estrogen and disrupts the estrogen pathway. It has been banned in most countries owing to its half-life of 57.5 years in temperate soils. Animal studies are necessary when EDCs, such as the phthalate Syndrome Model, have not been directly studied in humans. The rats were fed an androgen-blocking chemical compound and observed. The model showed a non-descendant testis. ¹³

Perturbation by EDCs is common in the new generations. Neonatal susceptibility was higher than that of adults. Direct exposure here defines the immediate contact of EDCs in adults, parents, and the first generation, that is, offspring. The utero-developmental period is very sensitive, and exposure during that time may create subtle changes that cannot be differentiated until later in life. ¹⁵ However, recent findings from the non-monotonic drug response (NMDR) are presented in Figure 3a and 3b.

Genome replication mechanisms have a high degree of fidelity. Through generation and evolution, this crucial, complicated, and critical mechanism has maintained its stability through accessory molecules. Molecules and compounds are needed for programming of each cell to transform into functionable and differentiated cells, that is, from stem cells to be the part of tissue or organ system, also termed as Epigenetic markers. ¹⁶

Researchers have suggested that the body is more vulnerable to low radiation doses. As the saying goes “Doses makes potion” is seems to come thorough. The misunderstanding that higher doses contribute more and harm more is not always true, as proven by these NMDR curves based on a model for risk assessment and toxicity check. Toxicity was based on the scale of high dose to no observed adverse effect level (NOAEL), whereas low doses were based on standard toxicology testing recommendations. In the late 90’s, there was a study showed reproductive anomalies in rodents when higher doses of phthalates were administered, but another study proved that pregnant women showed antiandrogenic activity even at general population doses. These differences may be due to differences in cell proliferation or organ development. When the offspring are born, they are yet to have a fully developed body, that is, they have time to reach their maturity period, which is the crucial time as it provides room for the environmental chemicals to attack, and the body is clearly more susceptible and vulnerable at that time. The effects may include the length of gestation period, head circumference of the newborn, or weight of the child at the time of birth. The latent period can be of any duration, and it might or might not show effects such as cognitive and developmental deficiencies or anomalies. The individual’s milieu, duration, and method of exposure to EDCs affect the biological activity and chemical metabolism of molecules/compounds. For example, Organophosphorus Pesticides (OPs) are detoxified by activated PON 1(Paraoxonase) activity, but not until the age of 9. ¹⁷

They are categorized as severe pollutants; therefore, food safety and quality standards have been formulated accordingly. The reported certified information was retrieved from organizations such as the National Institute of Standards and Technology (NIST). To date, the most monitored organochlorine compound is DDT and its metabolic transformants, such as DDE and DDD. These OCs are subjected to bioaccumulation and biomagnification via the food chain. There have been limits on its use to prevent environmental hazards and human health imposed by consumption advisories. Quantitative and qualitative analyses were carried out in biological tissues, such as breast milk and tissues. The sample preparation workflow contains a generalized plan of action. After this, instrumental analysis was carried out. Soxhlet extraction is the standard approach for solvent extraction; however, as it has its own certain disadvantages such as time consumption and the requirement of a large amount of solvent, other strategies have also been used for mass quantification, such as pressurized liquid extraction, and lipid removal methods should not be used as OCs. Polar solvents are used because they are non-polar compounds. Hydrophobic contaminants, such as OCs, are best analyzed using GC-MS, MS/MS, and HRMS. Although earlier GC-ECD was considered the best instrument for the analysis of such OCs, although it had been sensitive, its detector detects all other halogenated compounds and therefore lacks specificity. ²⁰ Moreover, advancements are still ongoing in their specific analyses, as the current techniques have major disadvantages such as cost and time consumption.

These properties are more or less similar to those of OCs, as they are also subjected to accumulation in biological tissues, that is, bioaccumulation, as well as their persistence and toxicity. Their presence has been detected at very low levels in lipid-rich biological tissues, such as fish and wildlife. Compounds such as PCBs and PCDD/DFs are usually found in biological tissues at concentrations of parts per trillion or quadrillion. The most challenging part of their analysis is when they are present with other co-contaminants, which are present in higher concentrations. ²¹

When discussing PCBs, the preferred approach for their quantification is freezing the samples in the original wet state, and depending on the matrix, the sample can be subjected to various extraction processes. Preferred samples or matrices are lipid-rich tissues and milk, although removing co-extracted lipids is one of the obligatory steps before analyses so that there is no detectable interference in further processing. To eliminate any possible insensitivity and selectivity for the final quantification process, different techniques are used in combination or individually. GC-EI-MS is the currently used analytical method rather than GC-MS, which was used before the 2000s as it is not specific but sensitive. The adopted separation technique and type of data requirement define the choice of detector. ^{21, 22}

PCDD/DFs analysis process steps are more or less similar to PCBs. Compared with PBC, they are used at lower concentrations, and OCs and halogenated compounds are co-extracted with them, which h is why HRGC/HRMS are preferable methods of determination. Recently, more advanced techniques have been developed that provide more precise data in terms of sensitivity and selectivity. ²³ In addition, with the help of such techniques and advanced instruments, new rules and regulations are formulated for toxicology potency levels reflected in different areas, such as food regulatory rules.

It includes compounds such as PBBs and PBDEs, which are similar to OCs and PCBs. General extraction methods include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, and pressurized liquid extraction. Solid-phase extraction (SPE) is commonly used to measure Blood Serum and urinary markers. GC-MS and GC-MS/MS remained the standard instruments for its analyses, although different compounds have their own need for instrumentation for precise results, such as tetra-bromo bisphenol A (TBBPA) analyses, which are performed by LC-MS/MS. ²³

Skin-care products, cosmetics, and other domestic and commercial products contain considerable amounts of PFAS. Even the compounds used for their manufacture and the formed products and by-products were included in this range. The technological shift in the 2000s from GC-MS to LC-MS increased its detection range considerably by decreasing the sample volume requirements. The availability of HRMS for its estimation has led to increased investigation of PFAS using nontargeted approaches. However, various novel classes of PFAS, precursor compounds, and transformation products are coming into the limelight as the approachability and availability of instruments is increasing. ²⁴

Nonylphenol (NP), 4-NP, or para-isomer of 4-NP is the most threatening and concerning APE in terms of endocrine disruption, as they are present in plasticware, reagents, solvents, etc. Another Endocrine disruptor, APE, is octyl phenol (OP), which is present at lower concentrations than NP, and is also present in environmental matrices, so it is less threatening and of lesser concern. Monitoring NPs such as NPECs and NPEOs compounds requires permissions because their investigation is bounded by jurisdiction. Alkylphenols like NP and OP wastewater extractions occurs usually extracted from wastewater using SPE or LLM. GC-MS is used for derivatization, although it is a skippable step, and LC-MS/MS with ESI is preferred for analysis, which eliminates the need for derivatization. Alkylphenol ethoxylates (APEOs) are NPEOs extracted by the SPE method, whereas alkylphenol ethoxy carboxylates which are NPECs extracted via liquid-liquid extraction, are the predominant APEs in the environment. ^{25, 26} However, the environmental analysis of these compounds is difficult in terms of selecting appropriate standards, which h is why analytical standards are required commercially.

Phthalic acid esters (PAEs), including DMP, DEP, and DBP, have been the most frequently monitored PAEs for the last 15 years. They are primarily found in things made up of plastics and resins. Recently discovered compounds are comparatively more concerning molecules, including monoester metabolites, which are phthalate metabolites metabolized in humans within hours, and other compounds such as DiDP and DiNP. Therefore, urine is more traceable and is generally studied for its presence, as blood serum has the necessary enzymes to degrade it. These molecules require more precise and selective analytical tools and instruments for discovered and suspected metabolites/compounds. There are now limits imposed by food advisory committees on beverages and foods in terms of Tolerable Daily Intake (TDI) which is essential for human health and prioritizes the environment. The main approach for this assay is chromatography with mass spectroscopy. ²⁷

TDI has already been established as one of the most studied endocrine disruptors. Polycarbonate plastic bottles, containers, resins, coatings, etc., are materials responsible for BPA contamination in environmental matrices. LC-MS/MS and GC-MS were both used for the analysis. ²⁸

The uterotrophic assay is a widely accepted short-term screening technique to ascertain the estrogenic and anti-estrogenic characteristics of substances in vivo. This is based on the mechanism by which estrogen affects uterine tissue development. Immature and young adult rodents with ovariectomies (OVX) were used in two uterotrophic test models. It is possible to assess the sensitivity and reaction of uterine tissue to exogenous drugs because the hypothalamus-pituitary-ovarian axis is not operational in either model. Female mice or rats that were pre-pubertal (22 days old) were employed in the underdeveloped utero-trophic experiment. Young, mature rodents are OVX in the adult model, and sufficient time is provided for uterine tissue to regress. After three days of daily oral gavage or intravenous injection of the test material, the weight of the uterine tissue was assessed, and a histomorphometric analysis was carried out. ³⁰

Owing to its specificity and sensitivity, vitellogenin expression in male fish is frequently used as a biomarker for sensitivity to outdoor estrogens. Fish vitellogenin is a precursor protein for the yolk that is activated by estrogen and is produced in an estrogen-dependent manner. Vitellogenin is regarded as a distinctively feminine protein. Because of the relatively low levels in male fish, the non-physiological stimulation of vitellogenin in males is interpreted as an endocrine disruption caused by estrogen. There are international standards for the vitellogenin assay, an endocrine disruptor assay that is sensitive and well standardized. Several in vitro models are available for investigating the estrogenic and anti-estrogenic effects of EDCs. ³¹

The Hershberger Assay, a short-term in vivo screening assay, was used to identify drugs with androgenic and antiandrogenic activities. The accessory tissues of the male reproductive system in castrated rats are susceptible to weight changes caused by androgenic or antiandrogenic substances. Before they reached puberty, male rats in the pre-pubertal stage were castrated; after the operation, they were allowed 10 days to recover. Animals were administered the test compounds over a 10-day period, along with a known androgen (typically testosterone) as a positive control. The ventral prostate, seminal vesicles, paired Cowper’s glands, levator ani bulbocavernosus muscle, and glans penis are among the androgen-sensitive tissues collected, weighed, and ready for histomorphometry if further examination is necessary. The specifics of the method are described in the EPA guidelines. ^{32, 33}

Recently, molecular screening techniques for obesogens have been developed. Based on the 3 T3-L1 cell line, this highly standardized adipogenesis model was created. Nuclear receptors called Peroxisome Proliferator Activated Receptors (PPARs) control adipocyte development and lipid metabolism. PPARs can interact with unsaturated fatty acids as lipid sensors. Adipogenesis is typically induced by PPAR agonists. These nuclear transcriptional regulators can bind DNA response elements and regulate gene expression. ³⁴

Some EDCs show strong estrogen hormonal replacement activity, whereas others show it to a lesser extent. Besides the estrogen hormone, EDC molecules also mimic other hormones, many of which have not yet been discovered. Anthropogenic activities have led to increased concentrations of these EDCs in water bodies; therefore, determination of these EDCs is required in drinking water, surface water, etc. ⁴⁷

Surface water mainly receives EDCs flux from wastewater treatment plant (WWTP) effluents. It is important to monitor surface water, as it is one of the main sources of drinking water. It was observed that E1 concentrations are higher than those of E2 and E3, while EE2 is present in trace amounts but is persistent; therefore, its removal tactics require special attention. ⁴⁸

Groundwater is also one of the main sources of freshwater supply, contributing approximately 20% of it in the industrial and agricultural sectors. Urban areas are vulnerable to contamination; therefore, the protection of groundwater is of utmost priority for many nations. Although it is difficult to track contaminants in groundwater from the research performed to date, it has been concluded that BPA and NP are slightly more concentrated than E1 and E2. E3 and EE2. Although they have been banned from many nations for a very long time, there has been evidence of their contamination in groundwater at considerate levels or higher than the threshold limits defined by concerned environmental and food-based committees. ^{49, 50}

Drinking water is a possible unknown risk that directly affects the population. Proper rules and regulation imposition might prevent or help reduce EDCs threats. Drinking water treatment plants use the traditional method of chlorination for purification, which has led to transformational chlorinated products having a more severe EDC. ⁵¹ Therefore, reclaiming wastewater requires a different approach that focuses on complete removal rather than incomplete removal of contaminants.

Microorganisms play an important role in EDCs degradation. This is an easy and accessible approach, and for years, it has been the center of research for the removal of such compounds. The past decade has been proof of the bacterial degradation of EDCs. EDCs are divided into six main categories, for which degradation occurs via bacteria. The main pollutant reservoirs, that is, water bodies, have been studied for these purposes to a considerable extent from literature review. Discharge of WWTP effluents is mainly the collective base of such estrogen-interrupting EDCs. These biologically active micro-pollutants are constantly attacked by bacteria, which develop different catabolic pathways to degrade these EDCs for their energy source, leading to EDCs transformation into new products. The six categories include NP and BPA (both xenoestrogens) and are industrial chemicals, estrone (E1), 17β-estradiol (E2), Estriol (E3), and 17α-ethinylestradiol (EE2). where E1, E2, and E3 are female hormones in humans and animals, respectively, and EE2 is a synthetic steroid that is mainly used as an oral contraceptive. BPA and NP have long been known to threaten the environment. NP, a xenobiotic compound, also mimics E2 and disrupts EDCs for natural estrogen metabolism. ⁵²

E1, and E2 degradation

E2 exhibited four degradation patterns. Degradation pattern I had bacteria degrading EDCs as a carbon source, and there was complete degradation with no left traces of toxicogenic by-products on the basis of GC-MS analysis. Degradation pattern II includes bacterial species that convert E2 to E1, and further E1 degradation occurs. Deterioration trend III. Unlike degradation pattern 2, isolates cannot break down E1; instead, they can convert E2 to E1. This implies that the rate-limiting step in the conversion of E2 to non-estrogenic metabolites or the final product may be the degradation of E1. Degradation trends IV. Isolates can convert E2 into different degradation products, some of which may also include phenolic groups and do not undergo further degradation. In addition to degrading E1, Nitrosomonas europaea, a well-known ammonia-oxidizing bacterium (AOB), has also been shown to degrade E2. However, it is not known whether the breakdown products possess endocrine-disrupting properties. Sphingomonas sp. converted E2 into 4-OH-E2 (a novel inter-metabolite), 4-OH-E1, ketoE1, keto-E2, and an unidentified product. ⁵³ Additional work is required to verify these results.

When studying bacterial E1 degradation, E2 degradation is always present as well. In other words, the majority of E1-degrading bacteria can also break down E2. A few studies have found bacteria that can break down both E1 and E2, such as Sphingomonas sp., Sphingobacterium sp., and Pseudomonas putida, however they have not precisely outlined the degradation pattern of E1 or E2. Although E1 is thought to be more resistant to biodegradation than E2, E2 is not the primary target estrogen in most studies. Contrary to the frequently cited transformation of E2 into E1 under aerobic environments, only two isolates have been shown to convert E1 to E2. Methylobacterium sp. and Rhodococcus sp. are two genera. ⁵⁴ This conversion may have a negative impact on the biological therapy process by increasing estrogenic potency. However, it also has the potential to reduce the biodegradability of estrogens by converting E1 into the more readily biodegradable E2, which is a positive outcome of the same process. The relative quantity of each type of estrogen-converting bacteria would ultimately determine the outcome, given the two opposing paths for E1 and E2 conversion. ⁵⁵ To confirm whether aerobic E1-reducing bacteria are sufficiently prevalent in a biological treatment process performed under various conditions to play a significant role in the net removal efficiency of estrogens, more research is required. ^{53, 54}

E3 degradation

It was noted that Nitrosomonas europaea converted E3 into an unidentified product that was not degraded further. The E3-degrading activities of Rhodococcus equi and Rhodococcus zopfii were found to be strong and rapid, and their kinetic rate constants for E3 degradation were the highest among the bacteria listed in this review. In the case of Agromyces sp., E3 underwent a brief metabolic conversion to 16-hydroxyestrone. Interestingly, E3 did not decay when the initial concentration was less than 80 mg/L. Given that aquatic environments have substantially lower E3 quantities, this may have practical implications. Therefore, it is not possible to directly extend the laboratory results to environmental conditions. ⁵⁵ The ability to transform or metabolize E3 was found in a combination of isolates Achromobacterxylosoxidans and Ralstoniapickettii, Sphingobacterium sp., and Pseudomonas sp. However, these studies were not concerned with the depth, specific process, or compounds of E3 degradation. Only the degree of E3 degradation was considered for Novosphingobium tardaugens, Shewanella baltica, and Rhodococcus sp . The following cases provide more details regarding bacterial E3 degradation. According to some studies, E3 is a metabolite of E1 and E2, and women excrete it at a rate that is significantly higher than that of E1 and E2. However, in contrast to E2, E3 is less frequently chosen as the primary target estrogen for research on bacterial breakdown. Few studies have examined bacterial E3 degradation and those that have almost always focused on bacterial E1 and/or E2 degradation. As a result, the above isolates that can degrade E1 and/or E2 were conjointly studied, and it was discovered that they can also alter E3. ⁵⁶

EE2 degradation

The Synthetic estrogen EE2 is far less likely to degrade in water than natural estrogens. There are three main methods of bacterial degradation based on the involvement of co-substrates and bacteria in the decomposition processes of EE2. The following is a description of the first three bacterial degradation mechanisms.

Mechanisms of bacterial breakdown 1. Isolates can co-metabolize EE2 with other organic substances. This study is the first to discuss how bacteria co-metabolize EE2 when metabolizing structural counterparts E1, E2, and E3. Six isolates from four different genera ( Acinetobacter sp., Phyllobacterium myrsinacearum, Pseudomonas sp., and Ralstonia pickettii—were recovered from compost in this study and were shown to completely co-metabolize EE2 when E1, E2, and E3 were present, indicating that these aromatic rings were broken. Transformation also occurred at doses ranging from mg/L to nanograms per liter. These results suggest that xenobiotic EE2 will only be co-metabolized with natural substances if there is an acceptable quantity of natural estrogens. ⁵⁷ Future studies must urgently close the knowledge gap related to the co-metabolism of EE2 in the presence of natural estrogens.

Mechanisms of bacterial breakdown 2. Isolates can oxidize specific chemicals from low to high valences. Subsequently, the oxidation products were transformed into EE2. In other words, bacteria mediate the degradation of EE2. The manganese-oxidizing bacteria Leptothrix discophora and Pseudomonas sp. were found to degrade EE2 at trace concentrations into products with no estrogenic activity. However, they require the essential chemical, Mn2C. Similarly, the AOB Nitrosomonas europaea has been reported to transform EE2 rich in ammonia (N-NH4). However, two divergent conclusions were obtained regarding the mechanism of EE2 degradation by this bacterium. ⁵⁸

Mechanisms of bacterial breakdown 3. Isolates have the ability to metabolize EE2 when it is the only source of carbon or when a co-substrate is present. Sphingobacterium sp. utilized EE2 as its sole source of carbon and energy. According to the authors, EE2 is first oxidized to E1 and then to the other two ring-cleaved acids. However, it is recommended that the EE2-degrading activity of this isolate be increased. ⁵⁹ In contrast, Rhodococcus sp. barely metabolized EE2 in the absence of other carbon sources. Rhodococcus sp. significantly enhanced the breakdown rate of EE2 in the presence of co-substrates (adipic acid or glucose). ^{58, 59}

The metabolites identified by these researchers were phenol and an unidentified substance with a high molecular weight. This shows that EE2 is not mineralized and that ecological concerns may be caused by transformation products. To fully comprehend EE2’s fate and assess the hazards associated with its transformation products, it is imperative that the biotransformation pathway of EE2 be further studied. ⁶⁰