Heliyon 10 (2024) e29165

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Factors affecting the performance of a pharmaceutical wastewater

treatment plant: Characterization of effluent and
environmental risk
Md Anowar Hossen , G.S. Sattar , M.G. Mostafa *
Water Research Lab, Institute of Environmental Science, University of Rajshahi, Rajshahi 6205, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: Pharmaceutical industries produce a huge volume of emerging pollutants (EPs) that pose a threat
Biological oxygen demand (BOD) to the aqueous environment. Biological processes have shown their inefficacy in treating many
Biological treatment pharmaceutical products. The study assessed physicochemical parameters, EPs, heavy metals in
Chemical oxygen demand (COD)
pharmaceutical industrial wastewater, and the removal efficiency (RE) of an aerobic biological
Emerging pollutants (EPs)
Wastewater
treatment plant. The study also assessed the contamination levels and risk using several indices,
such as the Canadian Council of Ministers of the Environment Water Quality Index (CCME-WQI),
heavy metal pollution index (HPI), heavy metal evaluation index (HEI), and risk quotients index
(RQs). The study found that the treated water quality was poor, having antibiotics, nonsteroidal
anti-inflammatory drugs, and others, along with several transformation products (TPs) and heavy
metals, which were unsafe for consumption with high environmental risk. The analysis results
showed that the RE for TSS, BOD5, COD, TDS, and EC were found to be 91.80%, 86.81%, 72.29%,
72.20%, and 65.60%, respectively, where the values of BOD5, COD, NO−3 , and PO3− 4 in the effluent
were still higher than the permissible limits of the ECR (2023). However, the RE for heavy metals
was in the order of Cu (84.62%) > Fe (65.04%) > Mn (63.3%) > Zn (60.58%) > Cd (53.85%) >
Ni (54.12%) > Pb (42.42%) > Cr (38%), where Cr and Cd concentrations were still higher than
the permissible limit of DoE (2019). The Pearson correlation and PCA suggested that EC, TDS,
TSS, DO, BOD5, and COD were the most correlating and contributing variables. This study argued
that metal-ligand behaviors mainly affect the removal efficiency of the treatment plant by
lowering the removal rate of heavy metals and pharmaceutical products.

1. Introduction

Pharmaceutical emerging pollutants (EPs), especially antibiotics, steroids, and hormones in aquatic environments, have become a
global environmental issue as they are found to be toxic, persistent, and non-biodegradable, which pose significant risks to aquatic life
and human health [1]. The traditional wastewater treatment processes have proved to be less efficient at entirely removing EPs, such
as selected pharmaceuticals, personal care products (PCPs), perfluorinated compounds (PFCs), and pesticides, due to their variety,
extreme low concentrations, and unique characteristics [2]. Due to the limited effectiveness of traditional wastewater treatment plants
(WWTPs) and the direct discharging of untreated wastewater to make more profit, pharmaceutical EPs, especially antibiotics, are

* Corresponding author.
E-mail address: [email protected] (M.G. Mostafa).

https://doi.org/10.1016/j.heliyon.2024.e29165
Received 27 January 2024; Received in revised form 31 March 2024; Accepted 2 April 2024
Available online 4 April 2024
2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license
(http://creativecommons.org/licenses/by-nc/4.0/).
M.A. Hossen et al. Heliyon 10 (2024) e29165

directly discharging into water bodies continuously. The adverse effects caused by the increasing amount of antibiotics released into
the environment cannot be ignored, as antibiotic-resistant microbes may evolve [3]. Alimba et al. (2019) suggested that the pollutants
in pharmaceutical effluents are emerging carcinogens and mutagens that are capable of increasing genome instability, altering blood
cell indices, and causing pathological lesions in fish tissues [4]. Pharmaceutical effluent also contains toxic heavy metals and met­
alloids, including Cr, Ni, Co, Cu, Cd, Pd, and As [4,5]. These toxic heavy metals are found to be extremely toxic to aquatic organisms
even at very low concentrations, which can cause significant histopathological alterations in the tissues of aquatic organisms, such as
fish [6,7]. Though the conventional biological WWTP is the most cost-effective method, it is found that only polar contaminants are
removed from the final discharged effluent in the biological process [8]. Many researchers have shown excellent COD removal effi­
ciency by biological WWTPs. Khan and Mostafa (2011) showed that about 75% removal of COD from pharmaceutical wastewater can
be achieved by employing hydraulic residence times of 15 days in an aerobic biological reactor [9]. Zhou et al. (2006) showed that the
COD removal efficiency of biological WWTP was as high as 97.8%, however, the removal efficiencies of two antibiotics, namely
ampicillin and aureomycin were, less than 10% [10]. It is more important to remove toxic nonbiodegradable chemicals, such as
antibiotics, by WWTPs than just to achieve high COD removal efficiency.
The pharmaceutical industrial effluent containing antibiotics is pre-treated by heating at a pH above 12 with sodium hydroxide
(NaOH) to break down β-lactam rings and convert simpler compounds before subjected to biological treatment (Fig. 1). Therefore, a
necessity exists to investigate antibiotic degradation associated with organic matter removal in wastewater treatment processes. This
study aimed to assess the physicochemical parameters, heavy metals, and EPs in pharmaceutical industrial wastewater and the
removal efficiency of the aerobic biological WWTP. This study investigated the impacts of the discharged effluent. The correlation
among the variables was assessed using Pearson correlation and PCA analyses.

2. Materials and methods

2.1. Sampling site

The study selected a pharmaceutical industry in Bogura city (24.83⁰ N and 89.37⁰ E), the northern part of Bangladesh. Several
industries discharge untreated effluent into the Korotoa River, which passes through the industrialized city of Bogura, and poses a huge
threat to the environment [6,11]. The pharmaceutical industry was purposefully chosen, and necessary permission was obtained from
the authority. The industry has an aerobic biological plant followed by multi-layer and activated carbon filters to treat wastewater with
a capacity of 5000 L per day.

2.2. Sample collection and extraction analysis

Two influent samples (one containing antibiotics and the other non-antibiotic tablets) and a combined effluent sample were
collected into separate high-density PVC bottles and were properly labeled, filtered, and preserved by adding sodium azide (1 g/L) and
kept at 4 ◦ C until the extraction process was started. The samples were extracted by the liquid-liquid extraction (LLE) process using
dichloromethane [12,13].

2.3. Analysis with LC-MS

The extracted samples were analyzed using a Liquid chromatography coupled with mass spectrometry (LC–MS 2020, Shimadzu,
Japan) in the laboratory of the Bangladesh Council of Scientific and Industrial Research, Rajshahi (BCSIR), Bangladesh, according to

Fig. 1. Sample collection point and operational diagram of the WWTP in Bogura city of Bangladesh.

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M.A. Hossen et al. Heliyon 10 (2024) e29165

the standard method of analysis [14,15].

2.4. Physicochemical parameters determination

The temperature, pH, EC, and DO were measured in situ using a digital multimeter (YSI Pro 1030 and Lutron PDO-519). The other
physicochemical parameters, such as biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total hardness (TH), total
suspended solids (TSS), total dissolved solids (TDS), chlorides, sulfates, nitrates, phosphates, sodium, potassium, calcium, and mag­
nesium were analyzed using the standard methods of APHA (2005) [16].

2.5. Heavy metals analysis

The concentrations of metals and metalloids were determined by using a flame atomic absorption spectrometer (AAS) (SHIMADZU,
AA-6800) in the Central Science Laboratory of the University of Rajshahi, Bangladesh, according to the standard methods of analysis
[17,18].

2.6. FTIR analysis

Different functional groups of organic compounds present in the influent and effluent were identified by an FT-IR (PerkinElmer
Spectrum 100 FTIR) machine at the Central Science Laboratory of Rajshahi University according to the standard method [12].

2.7. Indexing methods

Several water quality indices for the discharged effluent were calculated to assess the environmental impacts and the contami­
nation levels, which are stated below.

2.7.1. CCME-WQI
The treated water quality was assessed by the Canadian Council of Ministers of the Environment Water Quality Index (CCME-WQI)
method [19]. The CCME-WQI consists of three main factions, F1 (scope), F2 (frequency), and F3 (amplitude). These were calculated
using Eqs. (1), (2) and (6). Finally, CCME-WQI values were calculated using Eq. (7).
F1 is the percentage of the variables that exceeded their objective or standard limit relative to the total number of variables.
No. of failed variables
F1(scope) = × 100 (1)
Total no. of variables
F2 is the percentage of the total number of failed tests relative to the total number of tests, which is the sum of each variable tested
multiplied by the number of times that variable tested.
No. of failed tests
F2 (frequency) = × 100 (2)
Total no. of tests
F3 (amplitude) is calculated with the help of the normalized sum of excursion (nse), which is calculated either by Eq. (3) or (4)
depending on the conditions. When the test value must not exceed the objective or standard limit, Eq. (3) is used.
Failed test value
Excursion = − 1 (3)
Objective

In cases where the test value must not fall below the objective, Eq. (4) is to be used.
Objective
Excursion = − 1 (4)
Failed test value
The normalized sum of excursion (nse) is calculated by Eq. (5).
∑
n
Excursion
nse = i=1
(5)
Total no. of tests

Finally, F3 is calculated using Eq. (6).

nse
F3 (amplitude) = (6)
0.01(nse) + 0.01
The CCME-WQI water quality index value is then calculated using Eq. (7).
(√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ )
F12 + F22 + F32
CCMEWQI = 100 − (7)
1.732

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M.A. Hossen et al. Heliyon 10 (2024) e29165

The CCME-WQI criteria are as follows: CCME = 95–100 = Excellent; CCME = 80–94 = Good; CCME = 65–79 = Fair; CCME =
45–64 = Marginal; CCME = 0–44 = Poor [20].

2.7.2. Heavy metal pollution index (HPI) analysis

The overall water quality of the treated water and its suitability for human consumption were assessed using the heavy metal
pollution index (HPI) according to Eqs. (8)–(10) [6,21]. This study used Bangladesh standard permissible value for water quality
parameters [39]. The HPI values were calculated using the following equations:
∑
i=n
Wi × Qi
HPI = i=1 i=n (8)
∑
Wi
i=1

Where Qi is the subindex of the ith parameter, Wi is the unit weight of the ith parameter, and n is the number of parameters considered.
The unit weight of the ith parameter (Wi) and the subindex (Qi) of the ith parameter are given by the equations:
K
Wi = (9)
Si

(Mi − Ii )
Qi = × 100 (10)
(Si − Ii )

Where Mi is the monitored value of the metal of the ith parameter in ppb, Si is the standard permissible in ppb and Ii is the ideal value of
the ith parameter in ppb. The HPI value of water less than 100 is considered safe for drinking, while water is considered unsafe for
drinking, when the HPI value of water is greater than 100 [6].

2.7.3. Heavy metal evaluation index (HEI)

The HEI analysis was done to assess the level of pollution of the discharged effluent with respect to heavy metals, calculated based
on Eq. (11) [22].
∑n
HC
HEI = (11)
i=1
Hmac

Where Hc and Hmac are the monitored values and maximum admissible concentration (MAC) of the ith parameter in ppb, respectively.
The applied parameters and constants for the calculation of HEI were done according to WHO guidelines [40]. The proposed HEI
criteria are as follows: pollution level is considered low, when HEI < 10, medium, when HEI = 10–20, and high, when HEI > 20 [22].

2.7.4. Hazard quotients (HQs)

The Hazard Quotients (HQs), also called Risk Quotients (RQs), is calculated using Eq. (12).
MEC
RQ = (12)
PNEC

Where the unit for the measured environmental concentration (MEC) and the predicted no effect concentration (PNEC) is ppb. When
PNEC is not available, which is a constant for each chemical, estimated PNECs can be derived from the values of effect concentration,
EC50 or lethal concentration, LC50 reported in the literature, divided by an appropriate uncertainty factor (assessment factor, AF),
which varies from 10 to 1000 [23,24]. The RQ for the mixture based on MEC/PNEC ratios given by Eq. (13) is well-accepted and
extensively used model [25].
∑
n ∑n
MECi
RQMEC/PNEC = RQi = (13)
i=1 i=1
PNECi

The proposed RQ criteria are as follows: RQ < 0.1 meaning minimum risk; 0.1 ≤ RQ < 1.0 meaning intermediate risk and RQ ≥ 1.0
meaning high environmental risk [26].

2.8. Statistical analysis

Pearson’s correlation matrix and principal component analysis (PCA) were used to assess the correlations among the variables. For
PCA analysis, factors with an eigenvalue greater than 1 were extracted. The first principal component (PC1) is equal to the weighted
(factor loading) linear combination of the initial variables that account for the greatest variability [27,28]. Pearson correlations and
PCA were calculated using IBM SPSS Statistics software version 2022.

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3. Results and discussion

The descriptive results of the physicochemical parameters, heavy metal concentrations, and EPs are presented in Tables 1–5. To
assess the pollution level, the water quality parameters of the discharged effluent were compared with the standards of the Bangladesh
Environmental Conservation Rules 2023 (ECR, 2023) and the Department of Environment, Bangladesh Water Quality Standard 2019
(DoE, 2019) [41,42]. Since the two influents of the WWTP are combined before they are subjected to the biological treatment (Fig. 1),
the values of each parameter for the two influents were added to evaluate the performance of the WWTP except color, temperature, pH,
and DO.

3.1. Physicochemical parameters

This study exposed that all the physicochemical parameters for discharged effluent were found below the permissible limit set by
the ECR (2023) with the exception of BOD5, COD, nitrate, and phosphate (Tables 1, 2 and 5).

3.1.1. Physical parameters (color, temp., EC, TSS, TDS)

The discharged effluent was colorless and had a temperature of 31 ◦ C, which was just above the guidelines provided by the DoE
(2019). However, the ECR (2023) did not set any temperature range, instead, it suggested that the temperature of the discharged
effluent should be a maximum of 5 ◦ C higher than the temperature of the reservoir. The value of EC of the treated effluent was 501 μS/
cm, which was within the permissible standard (1200 μS/cm) of the ECR (2023). The TSS value of the effluent was found to be 47 mg/
L, which was within the permissible limit of the ECR (2023), whereas this value exceeded the standard permissible limit of the DoE
(2019) (Table 1). However, the TDS value for the effluent was found within the standard limits of both the ECR (2023) and DoE (2019)
(Table 1).

3.1.2. Chemical parameters (pH, TH, DO, BOD5, COD)

The pH of the discharged effluent was found to be acidic in nature (6.35), which was within the allowable range of both the ECR
(2023) and DoE (2019) (Table 1).
The TH value of the discharged effluent was 355 mg/L, suggesting that the water was very hard. However, this value did not exceed
the permissible limit of 500 for the TH according to the guidelines set by the DoE (2019) (Table 1). The DO value was found within the
standard range of 4–6 set by the ECR (2023), while the value was lower than the standard value of DO set by the DoE (2019) (Table 1).
Both the BOD5 and COD values for the effluent were found to be high above their standard permissible limits, suggesting that very high
concentrations of organic and metal-ligand complex compounds were present in the discharged effluent, which was confirmed by the
FTIR and LC-MS analyses (Tables 1 and 4 and Fig. 2).

3.1.3. Chemical parameters (anionic)

The nitrate concentration in the effluent was found to exceed the standard permissible limits of both the ECR (2023) and DoE
(2019) (Table 2). The values of chloride and sulfate were 160.20 and 113.61 mg/L, respectively, for the effluent, which were within the
standard limits set by both the ECR (2023) and DoE (2019). However, the phosphate concentration was found to be nearly three times
higher than the allowable limits set by both standards (Table 2).

3.1.4. Chemical parameters (cationic)

The effluent was found to have very little Na and K, which were far below their standard limits (Table 2). The concentration of Ca
was also found to be within the maximum allowable limit of the DoE (2019). However, the concentration of Mg (97.63 mg/L) was
found to be very high above the permissible limit of the DoE (2019) (Table 2).

3.2. Emerging pollutants in influent and effluent

The LC-MS analysis showed that both the influent and effluent had a very large number of EPs, though the number of EPs in the

Table 1
Physicochemical parameters of the influent and effluent.
Type Color Temp. (◦ C) pH EC (μS/cm) Concentrations (mg/L)

TSS TDS TH DO BOD5 COD

Influent-1 Greenish yellow 32 8.90 605 340 610 66 3.20 93.40 287.28
Influent-2 Brownish yellow 33 6.62 853 233 1235 740 1.60 410.55 515.69
Total – – – 1458 573 1845 806 2.40 503.95 802.97
Effluent Colorless 31 6.35 501 47 513 355 4.80 66.75 222.5
ECR (2023) 150 Hazen – 6–9 1200 100 2100 – 4–6 30 200
DoE (2019) 15 Hazen 20–30 – – 10 1000 500 6 0.2 4
RE (%) – – – 65.60 91.80 72.20 55.90 – 86.81 72.29

Influent-1 = antibiotics; Influent-2 = non-antibiotic tablets; RE = removal efficiency.

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Table 2
Physicochemical parameters of the influent and effluent.
Type Concentrations in mg/L

Na K Ca Mg NO−3 Cl− PO3-

4 SO2-
4

Influent-1 0.93 0.82 7.2 13.82 81 144.14 11.05 118.91

Influent-2 0.46 0.33 16.8 201.02 257 318.23 49.3 156.76
Total 1.40 1.15 24 214.84 338 462.37 60.35 275.67
Effluent 0.39 0.51 6.4 97.63 26 160.20 17.85 113.61
ECR (2023) – – – – 10 600 5 –
DoE (2019) 200 12 75 35 10 600 6 400
RE (%) 72.2 55.6 73.3 54.6 92.3 65.4 70.4 58.8

Table 3
Key findings from the LC-MS and FTIR analyses of the effluent and influent.
Category Influent-1 Influent-2 Effluent

No. of compounds found 70 35 28

No. of TPs 38 18 17
Common TPs Two TPs common to effluent at m/z = 58 and Two TPs common to effluent at m/z = 219 and 223; Four TPs common to
223; RT = 3.45 and 2.37 respectively; both in RT = 4.98 in negative ESI and 2.6 in positive ESI influents
positive ESI mode mode respectively
Average m/z 237.6 298.9 238.6
β-lactam ring Present Absent Present
FTIR: 1768 cm− 1 FTIR: 1785 cm− 1
LC-MS: Amoxicillin, Flucoxacillin etc. LC-MS: Amoxicillin,
Flucoxacillin etc.
Metal-oxygen bond (M − Present Present Present
1 1 1 1
O), metal-nitrogen FTIR: 622 cm− for M-N; 470 cm− for M-O FTIR: 594 cm− for M-N; 484 cm− for M-O FTIR: 594 cm− 1 for M-N;
bond LC-MS: TPs LC-MS: TPs 468 cm− 1 for M-O
(M − N) LC-MS: TPs

Table 4
LC-MS analysis data of effluent.
Possible Compounds Empirical Formula MW (g/mol) Mode RT (min) (M ± zA)z± (m/z) Intensity

Amoxicillin C16H19N3O5S 365 – 6.667 364 644

Benzyl benzoate C14H12O2 212 + 11.85 214 69673
Cetirizine C21H25ClN2O3 389 – 0.567 388 53451
Drotavarine HCl C24H32ClNO4 434 + 8.133 435 64737
Fluconazole C13H12F2N6O 306 + 2.433 307 528
Flucoxacillin C19H17ClFN3O5S 454 + 2.583 472 68534
Ibuprofen C13H18O2 206 + 9.183 245 57202
Lactic acid C3H6O3 90 + 5.35 108 11238
Losartan potassium C22H22ClKN6O 461 – 1.167 460 22522
Metformin C4H11N5 129 + 9.983 132 17485
Propylhydroxybenzoate sodium C10H12NaO3 202 + 2.283 203 125095
TP – – – 13.567 274 3432
TP – – + 12.2 117 15601
TP – – + 14.067 140 85417
TP – – + 3.05 402 83431
TP – – + 3.45 58 142925
TP – – + 7.617 191 105618
TP – – – 3.1 393 44952
TP – – – 3.517 171 54219
TP – – – 4.983 219 36382
TP – – – 7.883 80 36749
TP – – + 2.367 223 14506
TP – – + 5.15 210 14654
TP – – – 5.25 109 4180
TP – – – 7.117 85 4653
TP – – – 12.533 210 5356
TP – – – 2.567 405 19335
TP – – + 11 67 113336

z = 1,2,3, etc.; A = H and M = molar mass.

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Table 5
Heavy metal concentrations in influent and effluent.
Type Concentrations in mg/L

Cr Mn Fe Ni Cu Zn Pb Cd

Influent-1 0.036 0.061 0.754 0.039 0.006 0.046 0.014 0.009

Influent-2 0.064 0.059 0.824 0.046 0.007 0.058 0.019 0.004
Total 0.100 0.120 1.578 0.085 0.013 0.104 0.033 0.013
Effluent 0.062 0.044 0.552 0.039 0.002 0.041 0.019 0.006
ECR (2023) 0.50 2 3 1 3 5 0.1 0.02
DoE (2019) 0.05 0.1 1 0.1 1 5 0.05 0.005
RE (%) 38 63.3 65.04 54.12 84.62 60.58 42.42 53.85

Fig. 2. FTIR spectra of effluent and influent water residue.

effluent was reduced to 28 from 105 in the influent (Table 3). The data presented in Table 3 showed that nearly half of the EPs are
transformation products (TPs) in influent and effluent, of which only 4 TPs were common. The analyzed data suggested that the
discharged effluent contained β-lactam antibiotics (Table 4) as well as other toxic compounds, along with a number of TPs, which could
be the metal complexes of pharmaceutical ligands. EPs were characterized by LC-MS and FTIR analyses, as illustrated below.

3.2.1. FTIR analysis

The FTIR spectra of influent and effluent samples were found to have identical absorption bands, suggesting that the compounds
with similar functional groups were present in the influent and effluent. The broad absorption bands in the range of 3150–3600 cm− 1
denoted by ‘a’ in Fig. 2, corresponded to the asymmetric and symmetric stretching vibrations of O–H and N–H bonds present in al­
cohols, carboxylic acids, amines, amides, and imides [29,30].
Both influent and effluent contained this characteristic peak, suggesting that the discharged effluent carried these compounds and
was mixed with the aquatic system. The low-intensity peaks for the C–H stretches were in the range of 2900–2950 cm− 1 (b in Fig. 2).
The weak absorption bands at 2269 and 2296 cm− 1 were due to the stretching vibrations of C– – C, C–
– – N, and C–
– –S bonds, suggesting
that the different types of chemical compounds were present in the influent and effluent (c in Fig. 2). The absorption peaks at 1768 and
1785 cm− 1 were for the stretching vibrations of C––O bond in β-lactam rings, suggesting that β-lactam antibiotics were present in the
influent-1 and effluent (d in Fig. 2). The absorption peaks observed in the range of 1630–1660 cm− 1 (e in Fig. 2) were due to the
stretching vibrations, which indicated the presence of C––O bonds other than β-lactams, which was clear evidence for the existence of
different types of organic compounds, such as aldehydes, ketones, carboxylic acids, acid halides, amides, esters, etc. [31]. The
appearance of these peaks in relatively lower fields (Fig. 2), suggested that the metal-ligand coordination bonds were present [30,32].
The heterocyclic stretching vibration of the C––N bond was also observed in the range of 1630–1660 cm− 1, suggesting that organic
compounds with a C– –N bond might also be present [33]. The medium intensity absorption bands in the range of 1410–1485 cm− 1
were due to the stretching vibrations of the C––C bond (f in Fig. 2). The strong absorption bands in the fingerprint region within the
range of 1050–1100 cm− 1 (g in Fig. 2) were for the stretching or bending vibrations of the C–C, C–O, and C–N bonds [29,31]. Several

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absorption bands in the range of 1000–1400 cm− 1 suggested that C–F bond was also present [31]. The medium intensity absorption
bands observed between 600 and 650 cm− 1 (h in Fig. 2), due to the stretching vibrations of C–Cl, C–Br, and C–I vibrations, indicated
that the halogenated compounds were present in the influent and effluent [12,29].
The low-intensity bands in the region of 400–480 cm− 1 were attributed to metal-nitrogen (M − N) and metal-oxygen (M − O)
vibrations [30]. The LC-MS analysis suggested that both influent and effluent samples contained a very large number of transformation
products (Table 3). From the above discussion, it can be said that the majority of the transformation products (TPs) could be
metal-ligand complex compounds, since the pharmaceutical products are strong ligands that can combine with metal ions in varying
proportions, such as 1:1, 1:2, 1:3, 1:4, and mixed ligand complexes too [34]. These complexes are highly stable and toxic to aquatic
biota, and due to their inertness, they can manage to bypass removal mechanisms [34,35].

3.2.2. LC-MS analysis

Due to the presence of a large number of unknown TPs and the complex nature of influent and effluent, quantitative assessment was
not done. The LC-MS analysis result presented in Table 3 shows that the total number of compounds in the influent was 105 (70 in
influent-1 and 35 in influent-2) with an average m/z of 268.3, suggesting high molecular weight compounds were present in the
influent. However, the number of compounds was reduced to 28 in the effluent, with an average m/z of 238.6, suggesting there were
still a significant number of high molecular weight compounds in the discharged effluent. Mainly, three types of compounds, such as
chemicals used as reagents in manufacturing processes, manufactured products, and TPs produced before or during treatment pro­
cesses, can exist in the influent and effluent samples (Table 4).
For assigning a particular chemical for each m/z value, retention time (RT) and masses of (M − H)-, (M + H)+, (M+2H)2+, etc., were
matched (Table 4).
The assigned possible compounds, including organochlorine compounds, are polar, which have either replaceable protons to be
deprotonated (-ESI mode) or lone paired electrons (bases) to be protonated (+ESI mode) (Table 4). Some compounds have the ability
to be protonated or deprotonated depending on the conditions, so they can appear in both ESI modes. Moreover, some compounds have
the ability to accept/release more than one proton. Therefore, multiple signals were obtained for the same molecule. To avoid
overcounting or errors in the measurements, each possibility was carefully analyzed and reset. Each sample was scanned five times in
both ESI modes.
The number of TPs in the influent samples was 56 (38 in influent-1 and 18 in influent-2), while the number of TPs in the effluent
was 17 (Table 3). The most interesting thing was that only 4 TPs were common in both influents and effluent, suggesting TPs formed
before the treatment processes underwent some chemical changes (Table 3). Some of the compounds in the effluent characterized by
the FTIR and LC-MS analyses are presented in Table 4. Among the identified drugs, some cephalosporins or β-lactam antibiotics, such
as amoxicillin and flucoxacillin were present in the discharged effluent (Table 4). Other notable drugs in the effluent were nonsteroidal
anti-inflammatory drugs (ibuprofen) and antihistamine drugs (cetirizine), along with some other types of drugs (Table 4). These
pharmaceuticals are highly toxic to microorganisms and small fish along with other aquatic biota [36]. The data in Table 4 shows that
half of the identified chemicals in the discharged effluent were TPs, which could be the metal complexes of pharmaceutical ligands
(Fig. 2). Many reports suggest that these TPs are more toxic than their parent compounds [37,38,49].

3.3. Heavy metals in influent and effluent

The data presented in Table 5 show that the concentrations of all the heavy metals were within their respective permissible limits
set by both the ECR (2023) and DoE (2019) except for Cr and Cd. The value of Cr in the effluent was 0.0624 mg/L, which is within the
standard limit of the ECR (2023) for discharged effluent, whereas the value is higher than the standard limit set by the DoE (2019) for
surface water quality (Table 5).
Similarly, the Cd concentration was found within the permissible limit of the ECR (2023), whereas the value was somewhat higher
than the maximum allowable limit set by the DoE (2019) (Table 5).

3.4. Performance of the treatment plant

The pharmaceutical industry’s WWTP has mainly two sections; the aerobic biological treatment section for removing organic
compounds and a multi-layer filter followed by activated carbon filters for removing heavy metals (Fig. 1).

3.4.1. Biological process

It is difficult to track individual organic compounds, determine their concentrations, and evaluate their removal percentages due to
the very complex nature of influent and effluent and the presence of a large number of TPs. Therefore, the study assessed the per­
formance of the biological treatment quantitatively by COD and BOD5 reduction capacity and qualitatively by the analyses of FTIR and
LC-MS. The COD removal efficiency of the WWTP was found to be 72.29%, while the removal efficiency for BOD5 was 86.81%
(Table 1). The low COD removal efficiency as compared to BOD5, suggested that the treated water contained a significant amount of
non-biodegradable organic and metal-ligand complex compounds, which were discharged into the aquatic system. During the sample
digestion period in the COD determination process, these non-biodegradable compounds were oxidized on heating with an acidic
potassium dichromate solution, which increased the value of COD. The analyzed effluent contained 28 compounds, of which 11 were
manufactured and/or used as reagent compounds, the remaining 17 compounds were TPs (Table 4). The total number of compounds
found in the influents was 105 (Table 3). Therefore, the removal efficiency in terms of the number of compounds reduced 73.3%.

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M.A. Hossen et al. Heliyon 10 (2024) e29165

The term average m/z, which is introduced for the first time in this study, can be used to assess the performance of the biological
treatment process, since successful biodegradation of any large molecule produces smaller molecules.
The average m/z values for the influent and effluent were 268.3 (average of influent-1 and influent-2) and 238.6, respectively,
suggesting that there were still a significant number of high molecular weight compounds in the discharged effluent. However, only
four TPs were common in both influent and effluent samples, suggesting that TPs formed before the treatment processes underwent
some chemical changes during the treatment processes (Table 3). The removal efficiency of TSS was found to be 91.8%, indicating that
almost all suspended solids were removed during the activated sludge process in the biological treatment section (Table 1). The
removal percentage of EC was 65.6%, while the removal of TDS was found to be 72.2%. Since the nitrate ion (NO−3 ) is easily
biodegradable [43], the maximum removal rate for nitrate was found to be 92.3%. The removal efficiency for other anionic parameters
was as follows: Cl− (65.4%), PO3− 2−
4 (70.4%), and SO4 (58.8%) (Table 2).

3.4.2. Adsorption process

This study found that the removal efficiency of heavy metals in the WWTP was in the order of Cr (38%) < Pb (42.42%) < Cd
(53.85%) < Ni (54.12%) < Mg (54.6%) < Zn (60.58%) < Mn (63.3%) < Fe (65.04%) < Ca (73.3%) < Cu (84.62%) (Tables 2 and 5).
For the removal of heavy metals in the WWTP, two successive filters, such as multi-layer and activated carbon filters are used. These
filters are capable of removing heavy metals in varying percentages depending on the effect of pH, temperature, organic pollutants,
coexisting ions (anions/cations), and initial concentrations of the heavy metal ions [44]. The pH of the discharged effluent was 6.35,
which was acidic in nature (Table 1). Lower pH values reflect a high concentration of H+ in water, which competes with heavy metal
ions for ion exchange and electrostatic interaction on the adsorbent sites, decreasing the adsorption efficiency of heavy metals [44].
The high concentrations of cations, such as, K+, Na+, Ca2+, and Mg2+ in the influent can also compete with heavy metal cations,
decreasing heavy metal adsorption on the adsorbent surface. The concentrations of these cations in influent, especially Ca2+ and Mg2+
concentrations were found to be very high (Table 2), which might decrease the heavy metal removal efficiency. Moreover, anions
would interact with heavy metal cations to form coordination ions or precipitation, and compete with heavy metal anions for
adsorption sites [44]. The high concentrations of NO−3 , Cl− , PO3− 2−
4 , and SO4 in the influent (Table 2), suggested that the removal of
heavy metals might be affected by these anions. The presence of a strong ligand, especially chelating agents like EDTA, citrate, an­
tibiotics, etc., inhibits adsorption on adsorbents because of strong complex formation with the metal ions or by competing with the
surface for available adsorption sites. Complex formation occurs at a higher pH for the elimination of protons (H+) formed during the
reaction process [34,35]. In the WWTP, the influent was heated at a pH above 12 with sodium hydroxide (NaOH), which provided
favourable conditions to form metal complexes with pharmaceutical products. Metal complexes are thermodynamically very stable,
inert, less biodegradable, and toxic to microorganisms [48,54]. Valentina Uivarosi (2013) showed that pharmaceutical chelating
agents have a greater affinity for forming more stable chelates with hard Lewis’s acids like trivalent cations (Al3+, Fe3+, etc.) [34]. The
study also showed that the formation constant values for stable chelates of different pharmaceutical products, including antibiotics
were in the order of Al3+ > Fe3+ > Cu2+ > Fe2+ > Zn2+ > Mg2+ > Ca2+, suggesting that the stability of the chelates would follow the
same order [34]. However, the water solubility of the metal complexes is equally important since the insoluble compounds would be
separated during the sludge process. In the presence of high organic contents, Cr is expected to be in the Cr3+ form, which can form
very strong chelates with pharmaceutical chelating agents. The removal efficiency of Cr was found to be the lowest among the heavy
metals, suggesting the presence of water soluble chelates of the Cr3+ ion. This fact was also supported by the FTIR and LC-MS analyses.
The presence of absorption bands below 600 cm− 1 in the FTIR spectra of influent and effluent was clear evidence of M − N or M − O
bonds (i in Fig. 2). Further, the presence of a large number of TPs in the effluent suggested that these TPs could be stable chelates that
easily bypassed the adsorption processes [34,35]. Despite the borderline Lewis’s acid characteristic, Pb2+ was found to have a low
removal percentage (42.42%), suggesting that it formed mostly water-soluble stable complexes that could manage to escape being
absorbed. The removal percentages for other borderline Lewis’s acids were found between 54% and 73%, except for Cu, which is quite
an acceptable range described in the literature [44]. Cu2+ is a borderline Lewis’s acid that can be reduced to Cu+ in the presence of
reducing agents like iodide (I− ), which was highly expected to be in pharmaceutical wastewater (Fig. 2). Cu+ forms labile or less stable
complexes, which could be easily absorbed on the adsorbent surface, thereby increasing its removal percentage [45]. The average
removal percentage of alkali metals (Na, K) was 63.8%, which was in accordance with the EC removal percentage (Tables 1 and 2).
From the above discussion, it can be said that metal-ligand behaviors dominate the removal efficiency of heavy metals. In this way of
forming stable water soluble chelates, both organic compounds and heavy metals can manage to bypass the removal processes, thereby
lowering the removal efficiency of COD, BOD5, and some of the heavy metals.
To reach a concrete conclusion about metal-ligand interaction, which lowers the removal efficiency of WWTP, data on both lab­
oratory and different wastewater analyses are required. If the transformation mechanism of all pollutants is known accurately, their
successful removal process can be achieved by further research on this issue.

3.5. Environmental impact assessment

The CCME-WQI analysis suggested that the treated effluent water quality was poor, indicating various pollutants were present in
the discharged effluent, which is harmful to the aquatic biota.
The treated effluent contained β-Lactam antibiotics, nonsteroidal anti-inflammatory drugs, and TPs, along with toxic heavy metals
(Tables 4 and 5). The most widely used antibiotics in clinical medicine are β-lactam antibiotics. Resistance of microbes to β-Lactams is
increasing worldwide, which may become a severe threat because they have very low toxicity and are used to treat a broad range of
infections [46,47]. Antibiotics in high concentrations cause harmful effects on the environment in three ways. Firstly, the antibiotics

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M.A. Hossen et al. Heliyon 10 (2024) e29165

present in wastewater kill microorganisms by damaging their metabolic activities or causing toxicity. Those microorganisms actually
help impair the waste in the treatment process. Secondly, antibiotics contaminate microbial ecosystems. Thirdly, antibiotics present in
such a wide range in the environment lead to the development of antibiotic-resistant microorganisms [3]. Pollutants in pharmaceutical
effluents are emerging carcinogens and mutagens that are capable of increasing genome instability, altering blood cell indices, and
causing pathological lesions in fish tissues [4,55]. This is a serious threat to the normal functioning of aquatic ecosystems and the
survival of aquatic biota. Pharmaceutical effluent also contains varying concentrations of toxic heavy metals and metalloids. These
metals and metalloids are found to be extremely toxic to aquatic organisms, even at very low concentrations, which can cause sig­
nificant histopathological alterations in the tissues of aquatic organisms such as fish [6,7].
In many countries, even in Bangladesh, the river surface water is used for domestic and drinking purposes [51]. The treated effluent
of the WWTP is directly discharged into the river system. Therefore, the suitability of the discharged effluent for drinking was assessed
using the HPI index method, where the standards for domestic and drinking water supply were used.
The HPI analysis results showed that the discharged effluent was unsafe for human consumption for domestic and drinking pur­
poses (Table 6). However, the HEI analysis for the heavy metal pollution revealed that the treated water was in the low pollution
category considering the heavy metal concentrations. The RQs analysis for mixtures of heavy metals showed that they had a high
environmental risk (Table 6). Moreover, the presence of some antibiotics, nonsteroidal anti-inflammatory drugs, and a number of TPs
(Table 4) suggested that the discharged effluent had significant environmental impacts.
The WWTP managed to develop the CCME-WQI value by 52.10%, the HPI value by 53.16%, the HEI value by 53.25%, and RQMEC/
PNEC value considering only eight heavy metals and metalloids by 40.56%. Despite these developments, the discharged effluent fell
under poor water quality, which was unsafe for consumption and had a high environmental risk (Table 6).

3.6. Statistical analysis

To investigate the correlations between variables and the most significant variables contributing to the pollution of the discharged
effluent, Pearson correlation matrix and principal component analysis (PCA) were used.

3.6.1. Pearson correlation

A very strong negative linear relationship was found between DO and temperature (− 1.0), DO and TDS (− 0.98), and DO and EC
(− 0.97) at significant levels of 0.01 and 0.05 (2-tailed), respectively. A very strong positive correlation was also found between EC and
TDS (0.99), TDS and BOD (0.99), TDS and COD (0.99), BOD and COD (0.99) at significant levels of 0.05. Among the cationic pa­
rameters, a strong positive linear relationship was found between Ca2+ and Mg2+ (0.86), while among the anionic parameters, a strong
positive correlation was found between PO3− 2−
4 and SO4 (0.96) at significant levels of 0.05. Among the heavy metals, a very strong
positive linear relationship was also found between Mn and Fe (0.93) and Pb and Cr (0.99) at significant levels of 0.05. Each pair of the
positively correlated variables will change linearly, which means that if the concentration of one variable is increased, the concen­
tration of the other will also increase. Similarly, each pair of negatively correlated variables will change their concentrations in
opposite directions [6].

3.6.2. Principal component analysis (PCA)

Principal components (PCs) were extracted using eigenvalues greater than 1 [6]. Two principal components (PCs) were extracted
using a correlation matrix, which accounts for almost 100% of the total sample variance (Table 7). The first principal component (PC1)
is strongly positively correlated (>0.75) with Temperature, EC, TDS, BOD, COD, Ca2+, NO−3 , Cl− , PO3− 2−
4 , SO4 , Fe, and Ni, which has the
greatest variability (66.21%) (Table 7). The first principal component is also strongly negatively correlated with DO. However, the
second principal component (PC2) is strongly positively correlated with pH, TSS, K+, Cu, and Cd. A strong negative correlation was
also found in the PC2 with Cr and Pb (Table 7).
Positively correlated variables, either in PC1 or PC2, will vary together, that is, if one variable increases, the remaining ones tend to
increase as well. Negatively correlated variables will show the opposite trends. In the future, for any development of WWTP, these
trends must be considered for an effective solution.

4. Conclusion

The study results showed that the removal percentages for the major water quality parameters of the WWTP were in the order of
TSS (91.80%) > BOD5 (86.81%) > COD (72.29%) > TDS (72.20%) > EC (65.60%). The removal percentages of the cationic parameters
were in the order of Ca2+ (73.30%) > Na+ (72.20%) > K+ (55.60%) > Mg2+ (54.60%), while the removal percentages for the anionic
parameters were in the order of NO−3 (92.30%) > PO3− 2−
4 (70.40%) > Cl (65.40%) > SO4 (58.80%). The removal rates for the heavy
−

metals were in the order of Cu (84.62%) > Fe (65.04%) > Mn (63.3%) > Zn (60.58%) > Cd (53.85%) > Ni (54.12%) > Pb (42.42%) >
Cr (38%). The four vital parameters, such as BOD5, COD, NO−3 , and PO3− 4 failed to achieve the standard permissible limits of the ECR
(2023). The analysis of the treated effluent with different water quality indicators suggested that the discharged effluent was poor in
quality, and fell within the high environmental risk category in terms of heavy metals only without considering the threat associated
with EPs. The Pearson correlation and PCA analysis showed that EC, TDS, TSS, DO, BOD5, COD, and some other parameters were the
most correlating and contributing variables. This study argued that metal-ligand behaviors dominate the removal efficiency of heavy
metals as well as pharmaceutical products. Therefore, it is necessary to install more modern and efficient effluent treatment plants,
such as oxidation, for the removal of pharmaceutical products. It is highly recommended to carry out urgent research on the Korotoa

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Table 6
Water quality of the influent and discharged effluent assessed by different indices.
Index Value (before Water quality Value (after Water quality Remarks
method treatment) treatment)

CCME-WQI 24.17 Poor category 36.76 Poor category Considering all parameters, 52.10%
development
HPI 400.38 Unsafe for drinking 187.53 Unsafe for drinking Considering heavy metal pollution, 53.16%
development
HEI 8.92 Low pollution 4.17 Low pollution Considering heavy metal pollution, 53.25%
development
RQMEC/ 34.05 High environmental 20.24 High environmental Considering heavy metal pollution only,
a
PNEC risk risk 40.56% development
a
PNEC (ppb): Cr = 3.4; Mn = 209.88; Fe = 78; Ni = 44.3; Cu = 68.66; Zn = 87; Pb = 61.41; Cd = 57.33 [50,52,53].

Table 7
Rotated component matrix after Varimax Rotation.
PC1 PC2

Temp 0.98 NO−3 0.99 Temp 0.21 NO−3 − 0.09

pH − 0.11 Cl− 0.92 pH 0.99 Cl− − 0.39
EC 1 PO3-4 0.89 EC − 0.02 PO3-4 − 0.46
TSS 0.45 SO2-4 0.98 TSS 0.89 SO2-4 − 0.20
TDS 0.98 Cr 0.27 TDS − 0.19 Cr − 0.96
TH 0.73 Mn 0.67 TH − 0.69 Mn 0.75
DO − 0.98 Fe 0.88 DO − 0.21 Fe 0.46
BOD 0.97 Ni 0.98 BOD − 0.24 Ni − 0.18
COD 0.99 Cu 0.33 COD − 0.10 Cu 0.94
Na+ − 0.09 Zn 1 Na+ 0.99 Zn 0.02
K+ − 0.55 Pb 0.21 K+ 0.84 Pb − 0.98
Ca2+ 0.97 Cd − 0.58 Ca2+ − 0.24 Cd 0.82
Mg2+ 0.71 – – Mg2+ − 0.70 – –
Eigenvalues 16.55 8.45
% of Variance 66.21 33.79
Cumulative % 66.21 100

River and the river bank area for groundwater since antibiotics, other pharmaceuticals, and heavy metals may slowly accumulate in
the aquatic systems.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [M.G. Mostafa], upon reasonable
request.

CRediT authorship contribution statement

Md Anowar Hossen: Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis,
Data curation, Conceptualization. G.S. Sattar: Supervision, Resources, Project administration, Methodology, Funding acquisition,
Conceptualization. M.G. Mostafa: Writing – review & editing, Validation, Supervision, Software, Resources, Project administration,
Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing in­
terests: Md Anowar Hossen reports financial support, administrative support, and equipment, drugs, or supplies were provided by
University of Rajshahi. M.G. Mostafa reports a relationship with University of Rajshahi that includes: board membership and
employment. NA If there are other authors, they declare that they have no known competing financial interests or personal re­
lationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors would like to thank the authority of the pharmaceutical industry for their cooperation for this research. We would also
like to thank the authorities of the BCSIR and Central Science Laboratory of Rajshahi University.

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