FACULTY OF HEALTH SCIENCES

UNIVERSITI TEKNOLOGI MARA, CAWANGAN SELANGOR,

KAMPUS PUNCAK ALAM

BACHELOR’S IN ENVIRONMENTAL SAFETY AND HEALTH (Hons.)

ENV 411

ENVIRONMENTAL MICROBIOLOGY

ASSIGNMENT 2: BIOREMEDIATION OF PHARMACEUTICAL EFFLUENT

WITH THE USE OF EFFECTIVE MICROFLORAL CONSORTIA

PREPARED FOR:

COURSE: ENVIRONMENTAL MICROBIOLOGY

LECTURER’S NAME: DR. MUHAMMAD AFIQ BIN ZAKI
SUBMISSION DATE: 22 JANUARY 2022

PREPARED BY:

STUDENT NAME ID NUMBER GROUP

AMIRAH HUDA NAJIHAH BINTI FAUZI 2021614796 HS243 3C
DAMIA ATHIRAH BINTI MAHADI 2022838734 HS243 3C
CHE MUHAMMAD NAIM BIN CHE ABDULLAH 2022604616 HS243 3D

1
1.0 INTRODUCTION

Pharmaceutical companies are widely dispersed and medication research is responsible for the
steady rise in the usage of powerful medicines worldwide. There are real worries about the
potentially harmful environmental effects of this pollution given that a range of medications may
now be found in surface, ground, and drinking waters. The wastewater may contain organic
materials that are biodegradable or not, inorganic materials, heavy metals, and possible
inhibitors that ultimately find their way into groundwater or a water catchment region as landfill
leachates. Industrial culture uses pharmaceutical chemicals for many beneficial purposes, yet
these businesses also release harmful toxins into the environment (Kumar et al., 2022).
Pharmaceutical goods are among the growing contaminants, whose effects on the environment
have not been fully assessed and are now mainly unregulated in regards to environmental
health (Silva et al., 2015).

Growing awareness of the connections between pollution, public health, and the environment is
the result of rapid industrialization and global expansion. The general health of employees,
residents who live close to chemical synthesis factories, farmers, and field workers is negatively
influenced by environmental contamination carried on by industrial effluents (Asamudo et al.,
2005). In addition to their toxic effect, pharmaceutical compounds also have genotoxic and
pathogenic properties, which demand for an increased level of concern over their impacts on
public health and the environment. These pollutants have an impact on the health of people and
other living things when they are released into the ground or into bodies of water (Nadal et al.,
2004). Rivers have suffered greatly due to the rising pollution load from industrial water streams,
which also poses serious health hazards to those who directly bathe in or consume river water
(Seth et al., 2013).

Pharmaceutical firms utilize a variety of technologies to recycle waste and control environmental
contamination. Physical, chemical, and thermal techniques have been used for wastewater
treatment since ancient times. However, many treatment options have limitations, such as high
maintenance costs, limited efficiency, and large equipment requirements. Pharmaceutical firms
now use a range of treatment techniques, including primary chemical and physical processes,
secondary biological processes, and tertiary advanced oxidation procedures (Deegan et al.,

2
2011) An innovative technique called as "bio remediation" has been created and research is
ongoing for improved results in order to maximize effectiveness in treatment of waste water and
reuse the water. Bioremediation is one of the newest and most popular methods for treating the
wastewater from the pharmaceutical industry.

1.1 Problem Statement

Biodegradation studies of pharmaceuticals are initially done to assess which pharmaceuticals

are persistent in the environment (James, N. et al., 2020). However, due to the increased
findings of pharmaceutical components in drinking water, the bioremediation of pharmaceuticals
has taken a new route in ensuring the safety of water for human consumption. Although
bioremediation could be a cost-effective technology for removing pharmaceutical waste,
biodegradation of pharmaceutical compounds can be challenging, given their diverse and
complex chemical structures and relatively low environmental concentrations. Additionally, the
hazardous nature of these compounds and the process's own high dependability towards the
surroundings are vital factors that need to be addressed precisely as an uncontrolled
environment may result in the transformation of pharmaceuticals into harmful end products. The
environment includes pH, temperature, oxygen, nutrient levels and the sudden alteration in
toxicity of influent.

2.0 CONTAMINANTS INVOLVED IN PHARMACEUTICAL EFFLUENT

Scientific discovery and development are the primary drivers of the pharmaceutical industry. It is
an essential component of the health-care system, primarily contributing to the structure by
producing pharmaceutical active substances for human and animal welfare. The
pharmacological active substances may be composed of natural products or synthetic drugs.
Natural products are typically derived from microbial, plant, and animal resources, whereas
synthetic drugs are created using chemical processes. Antibiotics, steroids, peptide hormones,
vitamins, enzymes, prostaglandins, and pheromones are some examples of naturally derived
pharmaceuticals (Yakubu, 2017).

3
The presence of active pharmaceutical components, solvents, intermediates, and raw materials
in pharmaceutical industry effluents is currently the main cause of concern. Pharmaceutical
effluents have a vital impact on the environment and public health, particularly in areas that are
close to industrial pharmaceutical regions (Yakubu, 2017). A recent study discovered extremely
high levels (mg/L) of several drugs in the effluents of a local wastewater treatment plant. These
medications included nonsteroidal anti-inflammatory drugs such as ibuprofen, naproxen, and
diclofenac, as well as some other solids, biodegradable and non-biodegradable organic
compounds. Furthermore, the same study found that a high level of broad-spectrum antibiotics
in a pharmaceutical product could promote the development of antibiotic-resistant
microorganisms (Serjig et al., 2021).

In addition, health-care facilities and hospitals, like other industries, produce large amounts of
effluent on a daily basis that contains harmful and toxic pharmaceutical residual compounds as
a result of uncontrolled drug use. Several antibiotic groups, including ciprofloxacin, ofloxacin,
sulfa pyridine, trimethoprim, and metronidazole, as well as their metabolites, have been found to
be more concentrated in hospital effluents. The aquatic system also receives a high
concentration of pharmaceutical residues, more than 14,000 g/L, from treatment plants, in
addition to other surface water or even drinking water in Indian cities (Nadeem et al., 2021).

Similarly, antibiotic soil sorption can have a significant impact on important factors such as
manoeuvrability, sensitivity to environmental conditions such as contaminants, and microbial
disintegration availability. Typically, wastewater from homes and pharmaceutical manufacturing
sites is routed to treatment plants (Ahmad et al., 2021). Some of the compounds in the water
may be biologically active, toxic, or persistent, but treatment plants are not always capable of
removing all of the substances before discharge into streams or rivers (Surabhi et al., 2020).

The use of specific microorganisms has grown in popularity in applied environmental

microbiology. Fungi- and bacteria-based treatments have been identified as promising
technologies for pharmaceutical remediation. Fungi are eukaryotic organisms that include
microorganisms such as moulds, yeasts, and mushrooms. Some fungi are parasitic or
saprophytic chemoheterotrophic organisms. The majority are filamentous organisms, with some

4
being unicellular with cell walls. Fungi were successfully used to treat water samples
contaminated with micropollutants, particularly pharmaceuticals. They have long been known for
their ability to convert a wide range of recalcitrant substances using both non-specific
intracellular and extracellular oxidative enzymes. Mycelial fungi's physiology and colonisation
strategy allow them to withstand sudden changes in pH or humidity more easily, as well as
degrade complex organic compounds more efficiently, despite being limited by a long growth
cycle and spore formation (Akerman-Sanchez & Rojas-Jimenez, 2021).

Fungi can degrade at very low or non-detectable concentration levels and tolerate a wide range
of pH, which increases their degradation capability. However, certain controlled conditions must
exist for mycoremediation to continue as a reliable and effective process. The presence of
nutrients, pH, immobilisation on different supports, and agitation/static growth conditions can all
have a significant impact on fungi oxidative metabolism. Trametes versicolor, Irpex lacteus,
Ganoderma lucidum, and Phanerochaete chrysosporium, for example, were tested in the
removal of ibuprofen, clofibric acid, and carbamazepine at the same time. The findings
suggested that Trametes versicolor's cytochrome system degraded clofibric acid and
carbamazepine intracellularly.

3.0 THE EFFECTS TO ENVIRONMENT, AQUATIC LIFE AND HUMAN HEALTH

3.1 Significant organic content into water sources

Insufficient oxygen content in lakes is more vulnerable to pollution compared to rivers as rivers
are capable of self-purification. These wastewater discharges have little to no effect when they
are discharged in tiny volumes, and they may even be advantageous as sources of organic
pollutants that benefit rivers' aquatic life by providing them with nutrients. By lowering the
oxygen content of the water and reducing the amount of light available for photosynthesis, these
pollutants can impair the ecosystem as a whole when they are discharged in large amounts.
Human activities have resulted in point-source discharges as well as non-point loadings of
limiting nutrients like nitrogen and phosphorus into aquatic ecosystems accelerating the pace
and extent of eutrophication. (Chislock, M. F et al., 2021). A process known as eutrophication
occurs when a water body receives an excessive amount of nitrogen and phosphorus
molecules, which encourages the fast growth of plants and algae. Pharmaceutical effluents

5
contain organic materials including acetone and alcohols that are utilized as solvents and
denaturants in denatured alcohol.

3.2 Enhanced antibiotics resistance to microbe

Antibiotic resistance is currently on the rise due to the discharge of pharmaceutical wastewater.
Despite the fact that pharmaceutical pollutants are to be blamed for the rise in bacterial
resistance, the use and prescription of antimicrobial medications at an increasing rate has also
increased the amount of pharmaceuticals in freshwater, which pose detrimental effects on
human health. Varying kinds of antibiotics have different effects on wastewater treatment
efficiency such as ampicillin-containing wastewater has the largest number of bacteria that are
resistant to antibiotics. (Abbassi, B. E et al., 2016). Due to the active pharmaceutical ingredients
being thrown into rivers which directly influence naturally present microbes, antibiotic resistance
has developed. This may also be of the consequence from the release of drug-resistant strains
in wastewater as well as possible gene transfers caused by continued interaction with
antimicrobial compounds.

3.3 Slow-degrading organic substances

Hydrophobic heads and hydrophilic tails distinguish the amphiphilic monomers known as
surfactants. Remaining surfactants are dispersed in many environmental compartments such as
water and soil as a result of adopting only one treatment method which they eventually wind up
in wastewater either because they aren't treated at all or are only partially treated. When
surfactant loading exceeds 15 mg LAS/g dss, adverse metabolic responses, such as reduced
microbial respiration and a disruption of phosphorus absorption will occur (Dereszewska, A et.,
2015). Due to their unique functional characteristics, surfactants are used in the pharmaceutical
sector for a variety of purposes, such as cleaning up after drug production processes, improving
drug solubility and stability, strengthening the texture of semi-solid, besides preparing,
supporting, processing, and enhancing physical and chemical pharmaceutical drugs.

6
3.4 Feminization of Male Fish

It has been demonstrated in several studies that estrogen and chemicals that mimic it may alter
the female-to-male ratios and have a feminizing effect on male fish. Both postmenopausal
hormone therapy and birth control pills are included in this type of estrogen. Due to the river's
pollution in various places, the Potomac River in the United States is well recognized for having
a number of intersex fish or fish that exhibit both male and female features. More than the year
of hatching or the current environmental conditions in the studied rivers, the duration of
exposure to effluent was associated with the incidence and severity of the intersex condition
(Gross-Sorokin, M. Y et al., 2006). As a result of higher estrogen levels in the river's
downstream water, there are more female and intersex fish downstream from the plants that
pollute the river. Furthermore, it has been found that fish living downstream of wastewater
treatment facilities suffer from brain tissue highly concentrated on popular antidepressants.

3.5 Interrupting the Sewerage Process's Regular Operations

Antibiotics are routinely used to treat infections. They include elements that could affect the
sewage treatment procedure and the microbial ecology of the surface water. Antibiotics used in
sewage treatment systems later severely limit the activities of sewage bacteria, hence
negatively affected the decomposition of organic waste. Antibiotics are also harmful to nitrifying
bacteria since the chemical compounds might pose negative impacts on them throughout the
sewage treatment process. The antibiotics inclusion reduced the system's capacity to oxidize
ammonium and increased its capacity to remove nitrogen which was linked to a drop in
autotrophic ammonium-oxidizing bacteria (Gonzalez-Martinez, A et al., 2018). Residual
antibiotics are regarded as compounds that degrade slowly in wastewater treatment systems
under normal working conditions. Additionally, their presence in wastewater treatment plants
may affect how well those facilities treat the effluent. Reusing treated wastewater that includes
antibiotic residues that are partially decomposed will cause problems for the water cycle and the
food chain.

7
4.0 BIOREMEDIATION: PRINCIPLE INVOLVED

Bioremediation is a low-cost, economically feasible method that aids in cleaning contaminated

sites by using natural biological (microbe) activity (Gupta et al., 2020). In certain instances,
microorganisms may already be present on the site or removed from elsewhere and introduced
to the treated material, using bioreactors as an example (Das et al., 2012). The main principle is
degrading and converting pollutants in the environment to less-toxic or non-toxic forms by using
microorganisms. There are two contributing factors which are biotic and abiotic conditions.
These factors determine the rate of degradation of the organic compounds, in this case, the
pharmaceuticals. Environment can include soil, groundwater, sludge, and solids whereas
microorganisms refer to microscopic organisms such as fungi, algae and bacteria. The
microorganisms break down the pharmaceuticals by using them as an energy source or co-
metabolizing them with an energy source through a process known as metabolic process. Most
metabolic processes end with the production of harmless gases like carbon dioxide and water
which is the converted form of the pollutants.

5.0 BIOREMEDIATION: STRATEGIES AND TECHNIQUES

Bioremediation is classified into a number of different approaches in the utilization of various

biological sources like bacteria, fungi and algae to break down harmful substances into less
toxic or non-toxic compounds. Here are the two methods with highest efficiency in the treatment
of pharmaceutical wastes in the environment:

8
5.1 Microbial remediation

A process of employing microorganisms to lower the bioavailability of pollutants, especially

organic contaminants so as to make them less toxic to the ecosystem. This technique uses
aerobic or anaerobic microorganisms to remove or neutralize environmental pollutants through
their natural metabolic process. Microbial bioremediation strategies can be either ex-situ or in-
situ. Ex-situ techniques require the transportation of pollutants from polluted site to another site
for treatment while in-situ techniques treat polluted substances at the site itself. Examples of in-
situ strategies are bioventing, biosparging, biostimulation and bioaugmentation. Meanwhile, ex-
situ techniques can be learnt through the application of compost and land treatment.

In the process of treating subsurface water, recovery wells are used to remove contaminated
groundwater, which is treated aboveground, utilizing a bioreactor that contains microorganisms
which are acclimated to the contaminant. This would be considered as an ex-situ treatment.
Following bioreactor treatment, the clean water is then supplied with oxygen and nutrients,
before it is reinjected into the site. The reinjected water provides oxygen and nutrients to
stimulate in-situ biodegradation.

In another circumstance, in-situ techniques are applied in remediation of soil pharmaceuticals.

Bioventing enhances the activity of indigenous bacteria and archaea thus stimulates the natural
in-situ biodegradation of hydrocarbon derivatives (for instance; halothane and propellant) by
inducing air or oxygen flow and, if necessary, nutrients, into the unsaturated zone. During
bioventing, oxygen may be supplied through direct air injection into residual contamination in
soil. By adopting similar concept, biosparging works its way into the saturated zone, supplying
the surface with oxygen and nutrients to boost the biological activity of local microorganisms.

9
5.2 Mycoremediation

It is a form of bioremediation in which fungi-based remediation methods are employed to

decontaminate the environment. Fungi are able to degrade pollutants at very low or non-
detectable concentration levels and withstand a wide range of pH which further enhances their
degradation capability, as previously mentioned above. Both aquatic and white-rot fungi can be
promising candidates for the treatment of pharmaceutical wastes. Among these, the aquatic
fungus Mucor hiemalis has been proven to be able to eliminate the acetaminophen from
contaminated water bodies.

Fungi are long recognized for their abilities to convert a broad range of recalcitrant or
uncooperative compounds using non-specific intracellular and extracellular oxidative enzymes,
which also include enzymes produced by certain types of mushroom. The physiology and
colonization technique of mycelial fungi allow them to withstand sudden changes in pH or
humidity, as well as to degenerate complex organic compounds more efficiently, albeit being
limited by a long growth cycle and spore formation.

In most cases, the use of isolated enzymes through the application of enzymatic bioremediation
is preferred instead of fungi biomass in order to reduce the treatment time, to refrain from
having to deal with the lag phase of fungal growth, to reduce sludge production, and to facilitate
process control. Trametes versicolor, a white-rot fungus is frequently found to be effective in the
removal of different pharmaceuticals (Marco-Urrea, E. et al., 2009).

However, an eventual contamination with bacteria can adversely affect the efficiency since it
can develop competition for the substrate, impair fungi mycelium, disrupt biomass growth, and
undermine fungi activity (Asif, M. B., et al., 2017). Therefore, it is favorable to operate under
acidic conditions, since the optimum pH for fungi growth is lower than that of most bacteria,
giving upper hand for the fungi population to survive. Sterility conditions do not appear to be a
mandatory requirement for fungal application as they are mostly able to partially or completely
remove pharmaceuticals either under sterile or non-sterile conditions (Cruz-Morató, C., 2013).

10
6.0 CONCLUSION

In conclusion, due to the high complexity nature, low degradability, high concentration
deposition, and recalcitrant nature of pharmaceuticals in effluents, advanced treatment of
effluents is essential. There are many kinds of advanced treatment methods in which each
method has its own texture. The urban wastewater treatment industry is facing challenges, such
as the fate of pharmaceuticals, which induce the development of wastewater treatment
alternatives. From this review, a deduction can be made regarding fungi and bacteria potential
to remove pharmaceuticals by microbial remediation and mycoremediation. Bacteria present
more fascinating advantages when compared to fungi as they grow at a faster rate and are able
to remove both pharmaceuticals and nutrients, which is fairly important since tertiary treatments
are often needed for this purpose in domestic wastewater treatment. Microbial remediation and
mycoremediation studies proved to be eco-friendly and low-cost promising alternatives for
pharmaceutical removal when compared to conventional methods applied for the same
purpose. New knowledge in genetic engineering should be introduced in order to select and
amplify the most effective bacteria or fungi strains for pharmaceutical removal. Several
microorganisms contain key metabolic genes that could be introduced into other organisms.
Genetically modified fungi and bacteria, armed with new or increased capacities for degrading
various compounds, will likely have a significant future in this field for its capacity to effectively
remove pharmaceuticals from wastewaters as well as to allow their continuous use, considering
legal limitations, and a pragmatic market and cost rationale. The future will bring out the
development of growingly sophisticated effluent treatment solutions for a safer elimination of
potent hazards and reach toward the high-quality-level zero-discharge strategy.

11
 7.0 REFERENCES

Abbassi, B. E., Saleem, M. A., Zytner, R. G., Gharabaghi, B., & Rudra, R. (2016). Antibiotics in
wastewater: Their degradation and effect on wastewater treatment efficiency. J Food Agric
Environ. Retrieved from https://www.researchgate.net/profile/Bassim-Abbassi
2/publication/308764362_Antibiotics_in_wastewater_Their_degradation_and_effect_on_was
tewater_treatment_efficiency/links/5e333276458515072d70f5a4/Antibiotics-in-wastewater-
Their-degradation-and-effect-on-wastewater-treatment-efficiency.pdf

Andreia, S. et al., 2019. The Use of Algae and Fungi for Removal of Pharmaceuticals by
Bioremediation Biosorption Processes: A Review. Water, 11(8), 1555.

Asamudo, N. U., Daba, A. S., & Ezeronye, O. U. (2005). Bioremediation of textile effluent using
Phanerochaete chrysosporium. African Journal of Biotechnology. Retrieved from
https://www.ajol.info/index.php/ajb/article/view/71767

Asif, M. B. et al., 2017. Degradation of Pharmaceuticals and Personal Care Products by White-rot
Fungi - A Critical Review. Curr. Pollut. Rep., 3, 88 - 103.

Chislock, M. F., Doster, E., Zitomer, R. A., & Wilson, A. E. (2013). Eutrophication: causes,
consequences, and controls in aquatic ecosystems. Nature Education Knowledge. Retrieved
from https://www.wilsonlab.com/wp-content/uploads/2021/05/2013_NE_Chislock_et_al.pdf

Cruz-Morato, C. et al., 2013. Degradation of Pharmaceuticals in Non-Sterile Urban Wastewater by

_Trametes versicolor_ in a Fluidized Bed Bioreactor. Water Res, 47, 5200 - 5210.

Deegan, A. M., Shaik, B., Nolan, K., Urell, K., Oelgemöller, M., Tobin, J., & Morrissey, A. (2011).
Treatment options for wastewater effluents from pharmaceutical companies. International
Journal of Environmental Science & Technology. Retrieved from
https://link.springer.com/article/10.1007/BF03326250

Dereszewska, A., Cytawa, S., Tomczak-Wandzel, R., & Medrzycka, K. (2015). The Effect of Anionic
Surfactant Concentration on Activated Sludge Condition and Phosphate Release in
Biological Treatment Plant. Polish Journal of Environmental Studies. Retrieved from

12
 https://www.researchgate.net/profile/AlinaDereszewska/publication/270217202_The_Effect_
of_Anionic_Surfactant_Concentration_on_Activated_Sludge_Condition_and_Phosphate_Rel
ease_in_Biological_Treatment_Plant/links/54dc77340cf2a7769d963b98/The-Effect-of-
Anionic-Surfactant-Concentration-on-Activated-Sludge-Condition-and-Phosphate-Release-
in-Biological-Treatment-Plant.pdf

Gonzalez-Martinez, A., Margareto, A., Rodriguez-Sanchez, A., Pesciaroli, C., Diaz-Cruz, S.,
Barcelo, D., & Vahala, R. (2018). Linking the effect of antibiotics on partial-nitritation
biofilters: performance, microbial communities and microbial activities. Frontiers in
microbiology. Retrieved from
https://www.frontiersin.org/articles/10.3389/fmicb.2018.00354/full

Gross-Sorokin, M. Y., Roast, S. D., & Brighty, G. C. (2006). Assessment of feminization of male fish
in English rivers by the Environment Agency of England and Wales. Environmental health
perspectives. Retrieved from https://ehp.niehs.nih.gov/doi/full/10.1289/ehp.8068

James, G. S., 2020. Remediation Technologies. Natural Water Remediation: Chemistry and
Technology, 263 - 303.

Kumar, V., Agrawal, S., Bhat, S. A., Américo-Pinheiro, J. H. P., Shahi, S. K., & Kumar, S. (2022).
Environmental Impact, Health Hazards, and Plant-Microbes Synergism in Remediation of
Emerging Contaminants. Cleaner Chemical Engineering. Retrieved from
https://www.sciencedirect.com/science/article/pii/S2772782322000286

Marco-Urrea, E. et al., 2009. Ability of white-rot fungj to remove selected pharmaceuticals and
identification of degradation products of ibuprofen by _Trametes versicolor_. Chemosphere,
74, 765 -772.

Nadal, M., Schuhmacher, M., & Domingo, J. L. (2004). Metal pollution of soils and vegetation in an
area with petrochemical industry. Science of the total environment. Retrieved from
https://www.sciencedirect.com/science/article/abs/pii/S0048969703005023

Pharma’s potential impact on water quality. (2020). ScienceDaily. Retrieved from

https://www.sciencedaily.com/releases/2020/03/200325110904.htm

13
Seth, R., Singh, P., Mohan, M., Singh, R., & Aswal, R. S. (2013). Monitoring of phenolic compounds
and surfactants in water of Ganga Canal, Haridwar (India). Applied Water Science.
Retrieved from https://link.springer.com/article/10.1007/s13201-013-0116-z

Silva, B., Costa, F., Neves, I. C., & Tavares, T. (2015). Psychiatric pharmaceuticals as emerging
contaminants in wastewater (pp. 1-96). Cham: Springer International Publishing. Retrieved
from https://link.springer.com/book/10.1007/978-3-319-20493-2

Smer, L. et al., 2022. A Comprehensive Study of Bioremediation for Pharmaceutical Wastewater

Treatment. Cleaner Chemical Engineering, 4, 100073.

Treatment of a Pharmaceutical Industrial Effluent by a Hybrid Process of Advanced Oxidation and

Adsorption. (2020). ACS Omega. Retrived from
https://pubs.acs.org/doi/10.1021/acsomega.0c04139#

Treatment of drug residues (emerging contaminants) in hospital effluent by the combination of

biological and physiochemical treatment process: a review | Emerald Insight. (2021).
Frontiers in Engineering and Built Environment, 1(1), 1–13. Retrieved from
https://doi.org/10.1108\/FEBE

Yakubu, O. H. (2017). Pharmaceutical Wastewater Effluent—Source of Contaminants of Emerging

Concern: Phytotoxicity of Metronidazole to Soybean (Glycine max). Toxics, 5(2). Retrieved
from https://doi.org/10.3390/toxics5020010

14