I.

Introduction

1.1- Brief overview of active pharmaceutical ingredients (APIs) and their widespread use

1.2- Importance of aquatic ecosystems and the potential impact of APIs on aquatic life

2. Sources of API contamination in aquatic ecosystems

2.1- Human pharmaceutical use and disposal

2.2- Agricultural runoff and veterinary use

2.3- Industrial effluent and wastewater treatment plants

3. Occurrence of APIs in fish

3.1- Examples of APIs detected in fish tissues (e.g. antidepressants, antibiotics, hormones)

3.2- Factors influencing API accumulation in fish (e.g. species, habitat, diet)

3.3- Potential effects of API exposure on fish physiology and behavior

4. Occurrence of APIs in amphibians

4.1- Examples of APIs detected in amphibian tissues (e.g. painkillers, anti-inflammatory drugs)

4.2- Factors influencing API accumulation in amphibians (e.g. habitat, diet, life cycle)

4.3- Potential effects of API exposure on amphibian development and survival

5. Environmental and ecological implications*

5.1 - Potential for API-induced changes in aquatic food webs and ecosystem dynamics

5.2 - Implications for human consumption of contaminated fish and amphibians

. Conclusion

. references
CHAPTER ONE

Introduction.

Active pharmaceutical ingredients (APIs) are the chemical-based compounds that have produced mainly
in the countries the USA, Europe, China, and India. APIs have pharmacological activity mainly used with
combination of other ingredients to diagnose, cure, mitigate, and treat the disease. However, in the
recent past years, many medicinal-based corporations have started importing these substances from
countries producing active ingredients to their home countries. Modern day medicines have been used
by people to prevent, treat, diagnose, and cure disease. Every single medication is composed of two
main components, i.e. the API, which is the major component, is chemically and biologically active that
has to do the work in your body and other component known as excipients like lactose or mineral oil in
the pill, which is chemically inactive that provides, e.g. volume, a sweet flavor, or a color. These
excipients helps in the delivery of APIs in the body system. Numerous chemical compounds and raw
materials are utilized in multi-step reaction to make an API. However, their main purpose is to treat the
disease directly by acting upon (via their pharmacological activity) along with combination of inactive
form. Nearly more than 1 lakh tones of pharmaceutical products are consumed all over the globe (e.g.
Europe alone covers up to 24% of the consumption of medicinal products). Therefore, the generation of
these APIs has stimulated the release of chemicals in the environment and thereby lead to the spreading
of pollution. Concurrently, this enormous generation of pollution has called the worldwide attention
that requires an immediate alteration in the policies and regulations.

Thereby, considering the negative impacts of these chemical channels that are responsible for
generating API, the pharmaceutical companies are rendering approval to the microbial-based
fermentation using bacteria or yeast bial-based biopharmaceuticals created the returns of around $100
billion in 2017, and the business is escalating at a substantial pace (6% CAGR) [5,6]. It is predicted that in
the year 2020 the market demand for fermentation-based protein drugs is expected to reach $60 billion
from $44 billion. Similarly, the demand of peptide hormones and vaccine, which was $10–19 billion,
respectively, is expected to reach $18–28 billion. Today pharmaceutical companies such as Bayer,
AbbVie, Biocon, GlaxoSmithKline, Eli Lilly, Sanofi, and Merck are depending upon microbial schemes and
arrangements for the production of biopharmaceutical products. In this review, the focused has been
laid upon the current and modern developments that taken place in chemical route of processing of
biomass relative to traditional methods with emphasis on the production of APIs.

things hazardous to human, water bodies (including drinking water), and other bio-based lives owing to
their uncontrolled contact to the environment . Furthermore, the presence of these compounds has
been detected in the trace levels from nanograms to micrograms in the last span of 10 years from
ground water, drinking water, and waste water . The recent finding was reported on the pervasiveness
of enormous amount of around 18 APIs in the Lake Victoria-Uganda (in the amount of 5600 ng
L−1) .Thereby, these APIs have been established as global contaminants. Although the formation of
these chemical compounds is not from the single source of reaction, rather, they are developed from
many chemical components that are usually initiated from a single intermediate . Furthermore, several
intermediates are formed during the process in order to convert any raw material into an API. These
several reactions usually pass through the long channels of purification during their developmental
engineering, which involves the usage of huge reactors . Concurrently, these APIs are then checked for
their purity before they are being sold to the drug manufacturers.

In order to generate the chemicals, one of the major biomasses is the carbohydrates, which constitute
about the largest (95%) amongst the organic compounds of the planet. These are basically exploited for
producing further products either using fermentation or chemical alteration. Biomass in the form of
starch, cellulose, hemicellulose, pectin, and lignin usually exists in the form of feedstock . In the earlier
era of traditional processing starch and other carbohydrate-rich feedstock were usually being exploited
as a raw material by the vivid chemical industries. Undoubtedly, various products in the form of
chemical and polymers can be generated by using any alteration in the procedure (fermentation) in
order to achieve the desired derivative nation toward production of fuel, which is more than the
conversion to xylose and glucose . Since the earth’s biomass is largely composed of lignocellulose and
thus the entire crop left over in terms of wheat and rice straw, sugarcane bagasse and fibers of corn
husk are classified underneath of it. These crops are made of lignin, hemicellulose, and cellulose, which
are collectively called as lignocellulosic material. These lignocellulosic materials are processed by various
techniques, viz. chemical, thermal, or biological; they are converted into sugars, chemicals, or ethanol.

But in order to breakdown these lignocellulosic materials, some pretreatment in the form of hydrolysis,
delignification like the usage of sulfuric acid, alkaline treatment, high-temperature steaming, and
pressure-based homogenization are generally required for distorting the structured and organized
plants-based structure . Therefore, after the application of pretreatments of these lignocellulosic
materials, they can be readily converted to desired intermediates. In order to reduce the steps of
production or to reduce the cost by keeping the safety of the environment, we need to cross the various
technological and economic barriers for employing the potential applications of lignocellulosic
materials .

A number of multiple published reports focus on the production of ethanol rather than other product .
Thereby, there are limited number of life cycle assessments (LCAs) that can generate multiple products.
However, from the perspective of environments as well as economic, most of the bio-based fuels and
bioresources that are produced under the one canopy of the factories are still not a promising option.
Similarly, forestry-based biorefinery system is one of the specific examples . Concurrently, there are a
number of key factors that have been recognized considering the environment-favored performance for
the generation of bioethanol and biodiesel. In order to carry out the united production of chemicals, a
widened and well-adapted spectrum is needed. Nevertheless, there are still certain gaps of reasonable
prices that are required for the creation of large-scale biorefineries. Furthermore, the chemical
properties of biomass are not always favorable to biomass-based raw material; therefore, new
technologies are demanding and challenging amongst the existing companies.

Also, propionic acid and its related esters are produced in the amount of approximately 192,000 tons
throughout the year and also have several applications in the chemical industry (e.g. for the production
of thermoplastics, solvents in paints and resins as well) and in the usage of. animal feeds Further, from
the viewpoint of biorefinery its production can be elevated for the fermentation of glycerol and sugar
Also, economically, the conversion of glucose to propionic acid is anticipated to be 15% less than the
conversion of glycerol. Likewise, the other related upcoming feasibility is to employ the fermentation of
glycerol for the production of 3-hydroxypropionic acid, which is an essential building-block chemical
Secondly, it can be converted to 3-hydroxypropionic acid to acrylic acid, which comparatively has better
environment-based functioning. Concurrently, researchers from the National Renewable Energy
Laboratory have reported the development of an innovative pretreatment by employing the use of an
organic solvent in combination with water for the clarification of lignin and sugars derived from chemical
nature .

Moreover, the contemporary revolution in the advanced technologies (e.g. genetic and metabolic
engineering, enzymatic engineering) has opened up new avenues for creating vivid types of industry-
based products such as APIs from raw materials derived from plant . Owing to the preferential use of
renewable raw materials of biomass by the APIs’ chemical industries, the concept of biorefinery has
emerged and can be executed to produce different bioproducts with the replacement of chemical-based
manufacturing . Renewable resources are good sources as substrate for the production of green
chemicals, APIs, and key starting materials (KSMs), which come under a vast subject. Therefore, the
presented review entirely covers the major developments and possibilities of lignocellulose as feed
stock that occurred in the recent years in the capacities of renewable biomass as a prominent
foundation of chemicals and their related converted products.

Presently, owing to the considerable increase in the pollution threat to the environment, more and
more API-producing companies have been urged to follow the greener path in order to reduce the
generation of waste (in terms of chemicals, solvents as by-products) . Whereas API-producing
companies are always looking for the faster and economical methods, however, in reality, if we tend to
bring down the generation of waste, then the number of steps for producing API has to be reduced.
Because few and less tangible steps is the primary requisite with production of solvents or chemicals
generated for producing a single pure molecule of API. Likewise, in addition to cut down the steps,
manufactures are further needed to select nonhazardous types of solvents that possess the ability of
producing proficient and effectual results. Thereby, in order to achieve the results on greener guidelines,
manufacturing companies should employ contract development officers (CMOs) and further pass their
product through the services of contract development and manufacturing organization (DCMOs) so that
the formulation of the process of API can be planned out at an early stage with the help of additional
screening to avoid unavoidable changes or alteration in the later stage. This can be accomplished by
following the protocols of scale-up processing in the pilot plants where thorough supervision needs to
be administered for assessing time-to-time assessment and quality control. However, the establishment
of such a kilo lab comes with potential challenges of good manufacturing practices (GMPs) that require
planned budget and diligent supervision. And, by following these measures, we can reduce the
exploitation of raw materials and generation of by-products.

Concurrently, in the last year’s vivid methods of organic synthesis have been employed for generating
pharmaceutical products, which has strengthened the medical sector by reducing the causalities,
illnesses, and death. However, in order to achieve this accomplishment, if we are deteriorating the
environment simultaneously then all the efforts of pharmaceutical chemists will go in vain. Therefore,
the pathway of green chemistry is utmost desirable for minimizing the dreadful impact on the
environment. It is widely known that 80% of the wastage in the form of by-product by the
pharmaceutical industry is related to the solvent reported by GlaxoSmithKline . Thus, the production of
the significant amount of the contaminated solvents will generate the air and other pollutants.

These ways can only be achieved through employing the use of sustainable tools. These tools call for the
nib-to-nib strategy-based research that involves the following steps in making the processes threat free.
The ways of biomass-generated feedstocks, their bioconversion routes, use of selected harmless
chemicals, and channelized ways of technical processing are the key parameters that can lessen the
environmental issues to the minimal. Similarly, the inline concept of integrated biorefinery and the
continuous use of biosubstrates with the consumption of nonrenewable sources can safeguard the
environment with a profound relevance . In order to satisfy these parameters, the methodology of LCA
needs to be persuaded that consisted of complete assessment of the products and processes from start
to the end and quantify each environment-based quantification. Therefore, the only way to drop down
the expanding problem is the generation of green manufacturing practices from pharmaceutical
industries with thorough attentiveness on the selection, use, recovery, and disposal of the chemicals.

CHAPTER TWO

Sources of Api contamination in aquatic ecosystem

2.1 Human pharmacitical use and disposal

Human consumption of pharmaceuticals often leads to environmental release of residues via urine and
faeces, creating environmental and public health risks. Policy responses must consider the normative
question how responsibilities for managing such risks, and costs and burdens associated with that
management, should be distributed between actors. Recently, the Polluter Pays Principle (PPP) has been
advanced as rationale for such distribution. While recognizing some advantages of PPP, we highlight
important ethical and practical limitations with applying it in this context: PPP gives ambiguous and
arbitrary guidance due to difficulties in identifying the salient polluter. Moreover, when PPP does
identify responsible actors, these may be unable to avoid or mitigate their contribution to the pollution,
only able to avoid/mitigate it at excessive cost to themselves or others, or excusably ignorant of
contributing. These limitations motivate a hybrid framework where PPP, which emphasizes holding
those causing large-scale problems accountable, is balanced by the Ability to Pay Principle (APP), which
emphasizes efficiently managing such problems. In this framework, improving wastewater treatment
and distributing associated financial costs across water consumers or taxpayers stand out as promising
responses to pharmaceutical pollution from human use. However, sound policy depends on empirical
considerations requiring further study.

While pharmaceuticals are essential to human and animal well-being, their release into the environment
is a source of growing concern. Emissions of pharmaceutical residues may occur during drug
manufacturing, via urine and faeces following use in humans or domestic animals, in some cases via use
on plants, and through inappropriate disposal of unused drugs (Boxall et al., 2012). The great majority of
active pharmaceutical ingredients (APIs) are designed to interact already at low concentrations with
target molecules in humans or pathogens, molecules that are often well conserved across a range of
different non-target organisms (Gunnarsson et al., 2008). Accordingly, there is a growing body of
evidence for effects on wildlife associated with exposure to various APIs, including reproductive
disturbances, behavioural changes and disruption of organ development (Oaks et al., 2004; Kidd et al.,
2007; Brodin et al., 2013; Näslund et al., 2017). Environmental emissions of antibiotics are particularly
worrisome from a human health perspective because they can create a selection pressure favouring
resistant bacterial strains (Bengtsson-Palme et al., 2018; Larsson and Flach, 2022), potentially
exacerbating antimicrobial resistance, a major global threat to public health and economic development
(WHO, 2015; World Bank, 2017). These concerns have recently led key international actors to emphasize
the need for policy measures tackling pharmaceutical pollution (European Commission, 2019, 2022;
OECD, 2019; European Parliament, 2020, 2021; WHO/FAO/OIE, 2020)

2.2 Agricultural runoff and ventinary use

Globally, municipal sewage and agricultural runoff have been recognized as a growing threat to
freshwater systems due to the discharge of inorganic and organic contaminants (Nsenga Kumwimba and
Meng, 2019; Zhang et al., 2014c). This discharge leads to eutrophication of surface waters and excessive
growth of invasive plant species, which are the well-known issues of water environment quality
worldwide (Sharpley et al., 2008). It is therefore evident that suitable pollution control measures must
be taken to considerably reduce the effects of these contaminants. Although multifarious measures can
be taken to remediate eutrophic waters, however, highly efficient and low-cost biotechnologies with
resource recovery back into agriculture fields can be an alternative (Vymazal, 2007).
Source reduction strategies (from agricultural fields) have been used to control eutrophication of
surface waters. Nevertheless, even though upstream management practices reduce entry of pollutants
into lakes (Wu et al., 2017), it is unable to deal with pollution that is occurring already in-situ at field-
scale (within water bodies). However, the ability of aquatic macrophytes to absorb and reduce
pollutants from eutrophic water systems offers an excellent opportunity to the recovery and reuse of
nutrients. Also, biomass harvesting and potential use offers a profitable means of recycling lost nutrients
(Quilliam et al., 2015).

Phytoremediation is believed to be an emerging low-cost and environmentally-friendly technique that

mitigates contaminants from eutrophic waters and sediments by using green plant species (Rezania et
al., 2015). Plant species used for the application of phytoremediation must possess unique qualities: fast
growth with high biomass production, and high accumulation and uptake ability in biomass, and
appropriate traits to tolerate elevated pollutant concentrations (Kumwimba et al., 2017b). The inherent
adaptability even in extreme environments and nutritional qualities of invasive plant species such as
water hyacinth, Hydrilla verticillata and Lemna gibba make them feasible for use in livestock feed, green
matter and compost, comestics, therapeutics, biosorbent, biofuels, and human food, as well as potential
candidates to breakdown pollutants (Jain and Kalamdhad, 2018; Mazumder et al., 2020).

Since the time of discovery, many invasive alien species were introduced to every part of the world from
their native range (Ding et al., 2008). Some authors believed that some of the invasive alien species
were accidently transported for erosion control measure. The rate of biological invasion has significantly
climbed up over the past decades, largely due to global change, international trade, rising globalization
of the world economy, etc. (Wu and Ding, 2019).

Myriophyllum (Haloragaceae R. Br.), also known as “watermilfoil,” is a highly prolific and cosmopolitan
genus consisting of aquatic or semi-aquatic species. This weed is found in many water bodies including
lakes, rivers, ponds, irrigation channels, drainage ditches, and other waterways. The widespread invasive
species of the genus Myriophyllum, particularly M. spicatum has attracted more and more attention
throughout the world (illustrated in Fig. 1a and c, Fig. 3) (Moody and Les, 2010; Orchard, 1985).
Environmental disturbances due to the introductions of invasive watermilfoils are now well documented
and recognized as an increasing menace to international sustainability (Kumar Rai and Singh, 2020). The
threats posed by this weed in waterbodies are believed to be one of the main drivers of biodiversity loss
and thereby changing the ecosystem functioning and socio-economic environments via various
mechanisms; and interfering with terrestrial life (Madsen, 1998; Madsen et al., 1991; Smith and Barko,
1990). They are known to develop over a broad range of growth conditions through different
mechanisms of vegetative stem fragmentation which account for their success as an invader in a new
region (Smith and Barko, 1990). Based on their widely negative impacts on the aquatic environment all
over the world, species like M. spicatum, water hyacinth, M. aquaticum, Arundo donax, etc. have been
classified as “class one” Weed by the United States (Couch and Nelson, 1985).

sediment-retained pollutant, combined with extensive senescence of invasive watermilfoil from

chemical management has the detrimental impact of promptly releasing a substantial amount of
nutrients and other pollutants as plant tissues decay, becoming a source of pollutants to the water
column (Carson et al., 2018; Evans and Wilkie, 2010; Quilliam et al., 2015). These conditions could
undoubtedly lead to algal blooms further downstream systems. Collectively, these concerns have led
some environmental managers and scientists to propose that alternatives to control methods must be
more widely taken into account, especially in affected aquatic ecosystems where invasive watermilfoil
coverage is already widespread and suppression is purely impossible (Quilliam et al., 2015).

Ventinary uses

Veterinary Active Pharmaceutical Ingredients (APIs) are relative to finished preparations and are the
main components of finished preparations. Most APIs are a class of substances obtained by chemical
synthesis, biotechnology synthesis or extraction of natural drugs by raw material manufacturers.
Veterinary APIs are the upstream chemicals in the manufacture of finished drugs, and the quality of
finished veterinary drugs directly depends on the quality of the API.

2.3 Industrial effluent and wastewater treatments plant

It can be observed that, the generation of industrial wastewater is overwhelming due to rapid
development of various industries which produce its own particular combination of pollutants are
extremely contaminate the surrounding environment. Hence, environmental rules have directed all the
industrial units to implant effluent treatment plant to curb the direct release of untreated wastewater in
water bodies.
Here, are some of the major industries which need to implant Effluent Treatment Plant (ETP) to purify
the waste water before it gets discharged are:

1. Food and processing industries

2. Pharmaceutical industry

3. Textile and Paper industries

4. Automobile industries

5. Textile and dye industry

6. Dairy and Beverage industries

1. How does industrial effluent pollute the environment?

Industry is a vital source of water pollution. Industrial effluent diminishes the aesthetic quality of water
courses such as ponds, lakes, rivers and etc. It causes significant effect to human health. Moreover,
contaminated wastewater (untreated effluent) includes both organic and inorganic matter destroys
marine life and reduces its reproductive ability. Diversity of impurities in industrial effluents has been
tied to many serious health issues and damage to natural environments.

2. What is industrial effluent treatment?

Industrial effluent is any wastewater that is produced by various industrial activities. The quality of
industrial effluent/wastewater varies from one industry to another is responsible for the degradation of
receiving water sources. In other words, treating all kinds of effluents originating from various industries
using industrial ETP/Effluent Treatment Plant is extremely desirable.

Industrial effluent treatment is the effective process of treating waste water that is highly contaminated
by industrial wastes, making it fit for reuse or dispose/discharge back to the natural environment.

3. Different levels of treatment involved in Industrial Effluent Treatment Plant or ETP:

Wastewater treatment technology includes physical, chemical, and biological methods. Depending on
the type and extent of contamination, various treatment levels involved during the treatment of
industrial effluent .
CHAPTER THREE

Occurrence of APIs in fish

The increasing levels and frequencies at which active pharmaceutical ingredients (APIs) are being
detected in the environment are of significant concern, especially considering the potential adverse
effects they may have on nontarget species such as fish. With many pharmaceuticals lacking
environmental risk assessments, there is a need to better define and understand the potential risks that
APIs and their biotransformation products pose to fish, while still minimizing the use of experimental
animals. There are both extrinsic (environment- and drug-related) and intrinsic (fish-related) factors that
make fish potentially vulnerable to the effects of human drugs, but which are not necessarily captured in
nonfish tests. This critical review explores these factors, particularly focusing on the distinctive
physiological processes in fish that underlie drug absorption, distribution, metabolism, excretion and
toxicity (ADMET). Focal points include the impact of fish life stage and species on drug absorption (A) via
multiple routes; the potential implications of fish’s unique blood pH and plasma composition on the
distribution (D) of drug molecules throughout the body; how fish’s endothermic nature and the varied
expression and activity of drug-metabolizing enzymes in their tissues may affect drug metabolism (M);
and how their distinctive physiologies may impact the relative contribution of different excretory organs
to the excretion (E) of APIs and metabolites. These discussions give insight into where existing data on
drug properties, pharmacokinetics and pharmacodynamics from mammalian and clinical studies may or
may not help to inform on environmental risks of APIs in fish.

There are currently over 20,000 FDA-approved prescription drug products on the market, many of which
are released into the environment daily as a result of their extensive worldwide usage as therapeutic
agents. Aquatic systems are often the most significant receptors of these medicated discharges.
Pharmaceuticals enter water bodies by means of multiple routes, principally through direct introduction
via treated and untreated sewage (following patient use and excretion), pharmaceutical manufacturing
waste streams and improper disposal of unused or expired medicines. Active pharmaceutical ingredients
(APIs) have been detected widely including in groundwater, surface waters and wastewater treatment
plant (WWTP) effluents, ( Gramumnitz, Jungmabnd 2021) with their levels and frequencises of detection
generally showing a positive correlation with the extent of their usage. Equally, significant levels of APIs
may also develop as a result of persistent drug properties and/or inefficient sewage treatment.Recent
global surveillance of 1052 sampling locations across 104 countries revealed that analgesics (29%),
antidiabetics (20%) and antibiotics (15%) are the most common pharmaceutical pollutants of rivers in
low to middle income countries, while antidiabetics (25%), anticonvulsants (15%) and analgesics (11%)
predominate in the rivers of high-income countries. The aforementioned therapeutic classes, as well as
several cardiovascular agents, antidepressants and hormones are currently detectable in the waters of
all five United Nations regions, typically at levels in the low ng/L to low μg/L range.8 In some cases, APIs
have been detected at physiologically active concentrations in wild and feral fish populations and have
been causally linked to adverse reproductive, immune and behavioral effects in these organisms (see
next section). Moreover, the global consumption of human pharmaceuticals is increasing owing to
growing and aging populations and a general rise in chronic health conditions.These statistics are a
significant source of environmental concern for fish, which is further compounded by the likely
interactions of complex and highly dynamic API mixtures–which may have additive, synergistic
orantagonistic effects–with the potential to adversely affect fish physiology and behavior.

With many pharmaceuticals lacking environmental risk assessments (ERAs), there is a need to better
define and understand the potential risks that APIs (and their biotransformation products and mixtures)
may pose to fish, while also keeping the use of experimental animals to a minimum. There are both
extrinsic (environment- and drug-related) and intrinsic (fish-related) factors that make fish potentially
vulnerable to the effects of human drugs, but which are not necessarily captured in nonfish tests. In this
review, we first briefly describe biological effects that have been observed in fish following exposure to
environmentally relevant concentrations of human APIs. We then detail what is currently known about
human API fate in the aquatic environment and their bioavailability to fish. Subsequently, we explore
the factors that make fish, and different fish species with their distinctive physiologies and ecologies,
either more or less susceptible to the exposure and effects of pharmaceuticals. We do so by framing this
in relation to the absorption, distribution, metabolism, excretion and toxicity (ADMET) of APIs. It should
be noted that, while the potential hazards of many APIs have previously been estimated in fish based on
mammalian-derived ADME parameters, we focus on fish-specific ADMET, highlighting factors that
differentiate fish from humans (and mammalian models). In these analyses, we also illustrate where
existing data on drug properties, pharmacokinetics (PK) and pharmacodynamics (PD) from mammalian
and clinical studies may be used to inform on environmental risks of APIs in fish, and where not, thus
necessitating testing in fish (or alternatives).

Biological Effects of APIs in Fish for Environmentally Relevant Exposure Concentrations

Most human APIs occur at relatively low exposure concentrations in the environment with a small
likelihood of causing adverse effects, but their potent nature and ability to accumulate (in some cases)
could lead to chronic effects via sublethal modifications to physiological processes with subsequent
consequences on the behavior and fitness of wild fish.Ecological life history traits may render some
species more susceptible to chemical exposure than others, as shown by the higher susceptibility of
short-lived fish to the effects of endocrine active substances, compared to longer-lived species.Different
fish life stages may also have different susceptibilities to API exposure due to, for example, life stage-
specific expression of drug target proteins. (Brown A R 2023)

Levels of human pharmaceuticals detected in aquatic environments have, for most cases, not been
directly linked to immediate or long-term (chronic) adverse effects in fish. Exceptions to this include for
exposure to the persistent synthetic estrogen, 17α-ethinyloestradiol, causing feminization of male fish,
evidence for the deterioration in the general health of both rainbow trout (Oncorhynchus mykiss) and
brown trout (Salmo trutta) exposed to a low level of the nonsteroidal anti-inflammatory drug (NSAID)
diclofenac,and an indication for masculinization of female fish for exceptional cases of environmental
exposure to the antifungal azole, clotrimazole.( Peters J, Tyler C.R 2015)

It is also increasingly being recognized that many neuroactive pharmaceuticals, even at low,
environmentally relevant concentrations, can accumulate in fish brain tissues, causing alterations in
neurotransmitter levels19 and/or fish behavior. Exposure to oxazepam, for example, has been
causatively linked to behavioral alterations in some fish species.Various antidepressants may affect fish
behavior as well. Examples include disruptions in anxiety- and aggression-related behaviors in zebrafish
(Danio rerio) and Siamese fighting fish (Betta splendens) exposed to fluoxetine and suppressed foraging
behavior in zebrafish exposed to escitalopram. ( Nielsen S.V 2019)
Although evidence exists that some human pharmaceuticals can affect the physiological functioning
and/or behavior of individual fish, very little is known about their impact at population level. Indirect
impacts of APIs on fish via their effects on other trophic groups, and vice versa, have been indicated,but
this is not the focus of this review.Although evidence exists that some human pharmaceuticals can affect
the physiological functioning and/or behavior of individual fish, very little is known about their impact at
population level. Indirect impacts of APIs on fish via their effects on other trophic groups, and vice versa,
have been indicated, but this is not the focus of this review.

Susceptibility of Fish to Human APIs in the Environment – Extrinsic Factors

Pharmaceutical Fate in Aquatic Environments

The environmental fate and behavior of pharmaceuticals in aquatic systems will vary considerably, both
spatially and temporally, as a result of wide-ranging environmental variables, including water quantity,
temperature and physico-chemistry.Pharmaceuticals may remain unchanged, undergo biotic and/or
abiotic transformation, sorb/bind to suspended matter, dissolve in water and/or accumulate in
biological tissues,( Fent k, Weston A.A. 2006) all of which may affect their bioavailability and potency in
exposed fish. Some drugs can travel long distances from their source(s),particularly when they have long
degradation half-lives and low organic carbon/water coefficients (log KOC < 4.0 at pH 4–9), enhancing
their environmental persistence and mobility. The continuous release of pharmaceuticals into
waterways can moreover cause the exposure profiles of degradable drugs to mimic those of truly
persistent pollutants–a phenomenon known as pseudopersistence.

While many APIs survive biodegradation and enter receiving waters as parent compounds, others are
generated as metabolites and reactive intermediates. Some of these entities may be equally or even
more potent than their parent compounds, as is the case for salicylic acid and norfluoxetine, the active
metabolites of aspirin and fluoxetine, respectively Different chemical species of the same compound,
such as diclofenac and its nitroso derivative, may also elicit synergistic toxic effects.Furthermore,
conjugated biotransformation products of some APIs may be deconjugated by microbial enzymes in
WWTPs, rendering them biologically active again.( Payne M, Lapan P.R 2008)

APIs and their metabolites can participate in various chemical and biochemical reactions in the
environment that may affect their bioavailability and/or biological potency. At least half of all
pharmaceuticals in current use may undergo chiral inversion, for example, causing one enantiomer to
predominate in terms of environmental occurrence and toxicity.Additionally, pharmaceuticals in surface
waters may be subject to phototransformation, resulting in products with either lower or higher toxicity
potential, the latter of which has often been noted for NSAIDs such as diclofenac.While warmer water
temperatures can accelerate API biodegradation, it may, conversely, amplify the bioactivation and
toxicity of some pollutants by altering homeostatic processes. The interaction of pharmaceuticals with
dissolved organic matter has also been shown to affect bioavailability, either enhancing55 or reducing56
drug accumulation and toxicity in exposed aquatic organisms.

Physico-Chemical Properties of APIs Affecting Their Bioavailability in Fish

Currently approved pharmaceuticals occupy a very broad “chemical space” in terms of their molecular
and associated physicochemical properties ) which, as key aspects of drug ADME, will result in different
bioaccumulation and toxicity potentials. The potential for an API, as for all chemicals, to bioaccumulate
in fish is generally characterized by its bioconcentration factor (BCF), where Cfish and Cwater are the
chemical concentrations in the organism and water at steady state, respectively:

ERA requires BCFs for APIs with bioaccumulation potential (i.e., an n-octanol/water partition coefficient,
log KOW, ≥3), a criterion which is breached by about 54% of currently approved pharmaceuticals ,many
of which are yet to be tested. Consequently, although the data currently available suggest that most of
the tested pharmaceuticals pose a low bioaccumulation risk to aquatic organisms, empirical BCF data
are lacking, notably for anticancer drugs and API metabolites more generally. The tests required to
determine BCFs are time-consuming, costly and require large numbers of fish to be sacrificed (100–200
individuals or more for a single full aqueous exposure bioconcentration test).Moreover, experimental
BCF data on the same drug are often variable between and within studies, particularly for highly
lipophilic compounds.Researchers have consequently started looking into machine learning methods to
predict BCF values based on key physicochemical drug properties. Molecular weight (MW) and
lipophilicity (represented by log KOW for neutral compounds), for instance, are both inversely related to
water solubility, which affects the amount of drug freely available for absorption. With increasing log
KOW values (up to a value of 5), there is also increased partitioning into lipophilic biological membranes
and tissues, thereby facilitating uptake and accumulation. For ionizable compounds, correcting the KOW
by the fraction of neutral molecules or using the pH-dependent distribution coefficient (log D) as an
input parameter have been suggested as possible ways to improve BCF predictions. Considering the
assumptions implied in this approach as well as the inherent differences between natural fish lipids and
octanol, however, the membrane/water (log KMW) or liposome/water partition coefficient (log KLipW)
may be more reliable surrogates in this regard.Nevertheless, log D takes into account ionization state,
which is dependent on the acid dissociation constant (pKa) – another important determinant of drug
fate and bioavailability, especially considering that the majority of pharmaceuticals contain an ionizable
group While both neutral and ionized species are believed to contribute to the passive uptake of APIs
through the establishment of a concentration gradient, ions are less likely to cross lipid membranes and
hence the effect of environmental pH on ionization is critically important. Likewise, the pH of body fluids
will determine the degree of electrolyte dissociation within an organism, which will ultimately affect
how these compounds, particularly those with pKa values of approximately are dealt with by the body
and interact with drug targets. In addition to MW, log D and pKa, topographical polar surface area and
the number of nitrogen atoms have also been noted as important molecular descriptors to keep in mind
when making BCF predictions.

In support of the aforementioned, Chang and colleagues pplied a partial-least-squares regression model
to predict pharmaceutical uptake rate across an in vitro fish gill system and found that log D, MW and
pKa were some of the most significant drivers. This provides substantial evidence that models based on
a combination of physicochemical drug properties can be useful in understanding pharmaceutical
uptake and accumulation in biota and, in conjunction with other in vitro and in silico tools, could
potentially replace or at least significantly reduce the number of whole animals used in bioaccumulation
studies.

Susceptibility of Fish to Human APIs in the Environment – Intrinsic Factors

In addition to environmental factors and drug-related properties, certain intrinsic physiological factors
underlying drug absorption, distribution, metabolism, excretion and toxicity (ADMET) may render some
fish more or less susceptible to the exposure and effects of pharmaceuticals than other fish or
mammalian species. Here, we identify these factors to help highlight where fish testing is likely to be
required in ERA to ensure the optimal protection of fish populations. Particular attention is given to
teleost fish, which represent the majority (>26,000) of extant fish species (>30,000) and more than half
of all extant vertebrate species.

When assessing the risks of pharmaceuticals to nontarget organisms, one should first and foremost
discern the potential for target interaction and resultant pharmacological effects in the organism of
interest. We hence start the ADMET intrinsic analyses for API effects in fish with T (toxicity) and then
consider whether the ADME properties of relevant drugs are likely to increase or decrease the risk of
adverse effects.
3.1 Examples of APIs detected in fish tissues (e.g antidepressants, antibiotics, hormones)

Antidepressants

Tissue-specific bioconcentration of selected antidepressants was studied in rainbow trout

(Oncorhynchus mykiss) exposed to undiluted effluent from a Swedish municipal sewage treatment plant
for 13 days. Citalopram, sertraline and venlafaxine were found in the brains and livers of most fish, but
not in blood plasma or muscle. Venlafaxine was the only drug found in plasma (3/20 fish). Fluoxetine
was not detected in any fish tissue, in accordance with a low concentration in the effluent and a
comparably high limit of quantification in tissues. Concentrations of citalopram, sertraline and
venlafaxine in fish brain were up to 1/12, 1/8 and 1/26, respectively, of the lowest concentrations found
in the brains of mammals treated with therapeutic doses. Thus, given co-exposure to several
antidepressants and an assumed similar potency in fish, the margin of safety for target-related effects in
fish residing in effluent-dominated streams is relatively low. Furthermore, the non-detectable levels of
these drugs in blood plasma suggest that analyses of concentrations in target tissues (brain) would be
more informative in field studies and other studies with environmentally realistic exposure
concentrations.

Studies are however still scattered and more work is needed to define which water concentrations are
required to cause biological effects in aquatic organisms. SSRIs can also decrease the Na/K-ATPase
activity in the brain which has been proposed to be used as a biomarker for SSRIs (Lajeunesse et al.,
2011).

There are many streams contaminated by municipal effluents where aquatic organisms are exposed to
various xenobiotics, including antidepressants. Some antidepressants have been shown to accumulate in
channel catfish (Ictalurus punctatus), black cappie (Pomoxis nigromaculatus), bluegill (Lepomis
macrochirus), largemouth bass (Micropterus salmoides), common carp (Cyprinus carpio), white sucker
(Catostomus commersoni), brown trout (Salmo trutta) or bull sharks (Carcharhinus leucas) (Brooks et al.,
2005, Du et al., 2012, Gelsleichter and Szabo, 2013, Ramirez et al., 2007, Ramirez et al., 2009, Schultz et
al., 2010) that live in effluent-dominated streams or estuaries, or in long-term tests where brook trout
(Salvelinus fontinalis) were exposed to STP effluent (Lajeunesse et al., 2011). In some contrast to the
results of the above-mentioned experiments, no bioconcentration or effects on fish behavior were
observed during short-term aquaria tests with CIT (Holmberg et al., 2011). Similarly, no bioconcentration
of SSRIs was found in fish from ponds receiving reclaimed wastewater (Wang and Gardinali, 2012) and in
fathead minnow caged 10 m downstream the STP effluent (Metcalfe et al., 2010). The aim of this study
was to describe the tissue-specific bioconcentration of SSRIs and venlafaxine in fish (rainbow trout)
exposed to treated sewage effluent from a modern STP, thus containing concentrations of
antidepressants and other unknown chemicals that would be realistic for an effluent-dominated stream.
A comparison between tissue levels in fish and corresponding levels in humans taking the corresponding
drug would allow a preliminary assessment of risk.

- Antibiotics

In order to properly treat your fish, you must first determine which of the above infections your fish has,
and to do this, you’ll need to watch for certain symptoms. For instance, symptoms of a bacterial
infection include body slime, mouth fungus, open lesions and swelling, fin & tail rot, and blood spots
(without skin damage). Fungal infections are usually exhibited by cotton mouth, body slime, eye
clouding, and fin and tail rot, while symptoms of a parasitic infection include hole-in-the-head, small
regular spots or visible flukes hanging off your fish, swollen abdomen, and noticeable weight
loss/wasting.

Viral infections cannot be treated with medication, and are best combated with API® STRESS COAT and
API® AQUARIUM SALT after quarantining your fish from other healthy fish in your aquarium. All other
infections, however, should be treated as soon as possible.

For bacterial infections, we recommend API® MELAFIX, especially for repairing damaged fins and
treating ulcers and wounds. API® E.M. ERYTHROMYCIN, and FIN & BODY CURE also target symptoms of
bacterial infection, and any of these may be used to effectively fight your fish’s illness until you settle on
one that works best for your fish’s disease. As with antibiotics used to treat human disease, there is
usually one medication that works best for treating the particular strain of illness, so it may be necessary
to try more than one until you notice your fish’s health begin to improve.

For fungal infections, use API® PIMAFIX. This powerful treatment counteracts the harmful effects and
symptoms of fungal disease, but it is safe for delicate fish and saltwater species. It can be used not only
to treat disease, but to build your fish’s immune system and develop its resistance to future sickness.

Symptoms of parasitic infections are a bit more specific than symptoms of other types of disease. Two
very common parasitic illnesses are Ich and Velvet. Ich is also characterized as white spot disease, and
typically appears on the body, fins, and gills in the form of white nodules that look like grains of salt.
However, in some cases, Ich disease may not always be visible. You may still detect it, though, if your
fish is rubbing or scratching against the gravel or aquarium ornaments. Ich should be treated as soon as
possible with API® LIQUID SUPER ICK CURE, which specifically targets Ich by disrupting the lifestyle of the
disease.

Velvet is also a visible symptom of a parasitic infection that is usually spread by contaminated tanks, fish,
and tools (such as scrapers or nets). Velvet can cause serious harm or death to your fish if left untreated,
so it is important to recognize the signs of this parasitic infection. These may include darting around the
aquarium, scratching against aquarium items, fin clamping, and lethargy. You will also notice what
appears to be a velvety texture covering your fish, which is actually a coating of parasites that range in
color from gold to brown to green. The best way to target and treat Velvet is by using API® AQUARIUM
SALT in your fish’s water, and by keeping your tank in total darkness for several days since this parasite
gets much of its energy from photosynthesis during the “free swimming” phase of its life cycle. Another
helpful tactic is to raise the water temperature in your tank a few degrees to quicken the life cycle and
death of the parasite while actively treating, but you should make sure this action will be safe for your
particular species of fish before implementing.

API® offers a wide range of medications that treat a variety of diseases to get your fish healthy as soon
as possible. Keep your fish quarantined if it lives in a community tank, and ensure its environment is
conducive to healing. With the proper care, your fish will be feeling well again soon!

- hormones

The unobservable use of hormones in fish production is becoming an alarming issue worldwide. To
reveal the fact in Bangladesh, 144 fish samples (rui (Labeo rohita), catla (Catla catla), and monosex
tilapia (Oreochromis niloticus)) were collected from different fish farms and markets of Mymensingh
district. The market samples had two sources (Mymensingh and Rajshahi district). The steroid hormonal
(testosterone, estrogen, and progesterone) residue was analyzed by HPLC-UV detection. A standard
questionnaire survey was conducted where most farmers (80%) denied using the hormone in fish
production. Among the analyzed samples of all three fishes, hormonal residues were detected in
approximately 98% of samples, and around 92% contained residues above the ADI. Among the
contaminated samples, 70% of samples had a single residue and 30% had multiple residues. The
testosterone and progesterone hormonal residue was detected in all three fishes in both farm and
market samples and ranged (above ADI) from 2.1 to 16.96 μg/kg and 31.47–731.57 μg/kg (p < 0.05)
respectively. The estrogen hormone residue was only detected in market samples (Rajshahi district) of
rui and catla and no residue was detected in tilapia fish and the hormone level (above ADI) ranged from
8.23 to 40.13 μg/kg. This study revealed that the use of hormones varies on the attitude of farmers
based on the local culture pattern as estrogen hormone residue was only detected in market samples.
The consumption of contaminated fish at such concentrations may cause many health hazards in
humans, especially in children. Thus, this study reveals a new alarming fact to focus on, and an effective
monitoring system should be implemented as soon as possible for public health concerns.

The steroid hormonal use in fish production is becoming a concern worldwide. Steroid hormones
(estrogen, progesterone, testosterone) are used in fish farming to increase growth when one sex of a
species can grow bigger and faster than the other sex (Li et al., 2018). It is used to increase the growth,
weight, and size of the fish for more economic gain (Hoga et al., 2018). In fish species, steroid hormones
work by binding with the activated hormone-receptor complex in DNA that activates specific genes and
thus increases protein production (Khalil et al., 2011). However, the use of hormones in fish farming can
have harmful effects on humans, animals and the environment due to indiscriminate use and hormone-
dependent parameters.

Studies have shown that synthetic steroid hormones were widely used to increase growth in many fish
species, and among them carp fish and tilapia were the most common (Das et al., 2022; Islam et al.,
2015; Zhai et al., 2022). Regarding the hormone residue in fishes, Liu et al. (2017) found that the
steroidal hormone accumulated in crucian carp (Carassius auratus), carp (Cyprinus carpio) and silvery
minnow (Anabarilius alburnops) in aquaculture environments in Dianchi Lake, China. It was also
reported by Wang et al. (2012) and Cheng et al. (2012) that in yellow croaker (Larimichthys polyactis)
collected from local supermarkets in Changchun, China and in tilapia (Oreochromis mossambicus and
Oreochromis niloticus) purchased from supermarkets in Taipei City, Taiwan had steroidal hormone
residue in their tissue samples.

In Bangladesh, the demand for fish has increased in recent years due to population growth and the
constant search for a healthy diet (Tidwell and Allan, 2001). However, due to natural and man-made
hazards, natural fish populations have declined during the last several decades. It has increased the
effort in techniques development for more fish production in hatcheries and farms (Rottmann et al.,
1991). In Bangladesh, many fish species culture is now in progress on farms and rivers; such as rui
(Labeo rohita), catla (Catla catla), monosex tilapia (Oreochromis niloticus) and so on (Ahmed et al., 2013;
Hosen et al., 2019). But these fish species do not gain weight or undergo reproduction spontaneously in
the culture system (Rottmann et al.,1991). Thus, various techniques have been developed to improve
growth and spontaneous reproduction, such as hormone-induced breeding, growth and transgenesis
(Alimuddin et al., 2010).

Steroid hormone has many harmful effects on consumers. In children, it causes early puberty, advances
in bone age, modification of sexual characteristics and cancer development (Bergman et al., 2013). It
also causes feminization effects in males, infertility, reduced fertility, inhibition of the development of
sexual organs and sex reversal in females (Hoga et al., 2018). To prevent consumers from suffering from
possible health risks, the use of steroid hormones in fish culture has been banned or limited in many
countries and organizations (Li et al., 2018). The Joint FAO/WHO Expert Committee on Food Additives
(JECFA) established an acceptable daily intake of testosterone, progesterone, and estrogen at the rate of
2 μg/kg, 30 μg/kg, and 0.05 μg/kg body weight, respectively (JECFA, 2000).

Nevertheless, it is necessary to know the hormonal contamination level in fish flesh as these hormones
pose human health hazards if they exceed acceptable dietary intake (ADI). Many methods are used for
the analysis of steroid hormones in fish, but in this study, the HPLC-UV detection method was used for
analysis. The main objective of this study was to investigate whether or not fish have steroid hormones
in their flesh at a harmful concentration at the time of marketing and also know the farmers' knowledge,
attitudes and practices regarding hormone use.

3.2 Factors influencing API accumulation in fish