A R T I C L E  I N F O		ABSTRACT
REVIEW ARTICLE		Introduction: Macrolides are a group of antibacterial agents. Given their clinical importance, and the consistent rise in resistance among pathogenic bacteria, macrolides have been the targets of extensive research. Materials and Methods: This review considered the number of macrolides in different wastewater and the removal of these drugs. The antibiotics were frequently detected in influents and effluents, ranged from ng/L up to lower μg/L. In influent, the highest concentrations of clarithromycin (6080 ng/L), roxithromycin (>103 ng/L), erythromycin (3900 ng/L), and azithromycin (1949 ng/L) were detected in Croatia, Chinese, USA, and Singapore municipal wastewater treatment plants, respectively. Results: The removal efficiency of macrolides during wastewater treatment processes varies and is essentially dependent on a combination of macrolides physicochemical properties, location of municipal wastewater, and the operating conditions of the treatment systems. The application of alternative techniques, including membrane separation, activated carbon adsorption, advanced oxidation processes, biodegradation, and disinfection were the dominant removal routes for macrolides in different wastewater treatment processes. A combination of these techniques can also be used, leading to higher removals, which may be necessary before the final disposal of the effluents or their reuse for irrigation or groundwater recharge. Conclusion: Many antibiotics cannot be removed completely in wastewater treatment processes and would enter into the environment via effluent and sludge. The molecular structure of macrolides and their load-bearing capacity has led to the advantage of biological treatment over other treatments. However, the main part of the treatment has been done using biological treatment.
Article History: Received: 19 September 2021 Accepted: 20 November  2021
*Corresponding Author: Mehdi Ahmadi Email: Ahmadi241@gmail.com Tel: +989126779273
Keywords: Wastewater Treatment, Macrolide, Environment.

Introduction
Pharmaceutical compounds which are widely used for different purposes today are detected in natural surface water, groundwater, and wastewater ¹. Antibiotics, beta-blockers, anti-inflammatories, lipid regulators, beta-agonists, hormones, antineoplastic, and iodinated contrast media are some of the several usually administered remedial and diagnosis groups of drugs ^2,3. The wide use of antibiotics has contributed to spreading these compounds in the wastewater. Antibiotics are usually classified as bactericidal when they remove the infecting bacteria or as bacteriostatic when they inhibit the growth without killing bacteria. They are classified to different groups according to their chemical structure and mode of action, such as aminoglycosides, β-lactams, tetracycline, and quinolones ^4-6. A trace volume of antibiotics has been known in natural water systems worldwide, frequently relating their occurrence to wastewaters and livestock operations ^{4, 7}. Extensive use of these drugs may cause many biological hazards; since, in addition to their direct presence in the environment, they prevent the effective treatment of wastewater. Most antibiotics are poorly absorbed by humans or animals and consequently, after prescribing antibiotics, some of them are metabolized (usually 55-80%). A mixture of metabolites and conjugates of raw materials is excreted unaltered through urine and faeces, and along with sanitary wastewater, reaching municipal wastewater treatment plants ^8-10. Another route to enter the environment is to discharge expired drugs into toilets and household waste. However, the concentration of antibiotics residue in the environment is low, ordinarily at ng/L to μg/L level in natural water ¹¹ and wastewater ^12,13, and μg/kg to mg/kg level in soil ¹⁴ and sludge ¹⁵. The occurrence and removal of antibiotics in the environment, including wastewater, groundwater, and surface water have drawn great attention of researchers in recent years ^{16, 17}. Critical and persistent effects of antibiotics on ecosystems, the resistance of bacteria to antibiotics, and increasing tolerance of antibiotics by humans and livestock have not been well known which are at the source of increasing global concern ¹⁸. Municipal wastewater is an important source of organic contaminants in the aquatic environment ¹⁹. Municipal wastewater treatment is the process of removing contaminants from effluents, especially domestic wastewater, which includes chemical, physical, and biological processes ^{20, 21}. This process removes these pollutants and provides treated wastewater that is safe for the environment. The wastewater characteristics play an important role in the selection of treatment types. Antibiotics are one of the most important drugs for controlling dangerous diseases, and high amounts of these compounds are released into municipal wastewater due to extreme waste ²². This study focused on macrolide antibiotics, which are among the most famous antibacterial. Among several kinds of resistant antibiotics, macrolides recently came under special scrutiny. Macrolides are composed of a macrocyclic lactone of different ring sizes, to which one or more deoxy sugar or amino sugar residues are attached. Macrolides act as antibiotics by binding to bacterial 50S ribosomal subunits and interfering with protein synthesis. They bind at the nascent peptide exit tunnel and partially occlude it. Thus, macrolides have been viewed as 'tunnel plugs' that stop the synthesis of every protein. The persistence of macrolides in water is defined based on their half-life value.  The half-life value for Azithromycin is < 5 h ²³, Tylosin is 9.5–54 days, and for Erythromycin is < 17 days ²⁴. The given half-life values refer to surface water. These values can be much higher (longer half-life) in the case of groundwater or soil/sediments due to the scarcity or lack of sunlight and aerobic conditions ²⁵. The half-life of macrolides makes them stable in the environment. This matter disrupts the microbial ecology of surface water. The ecotoxicity of the macrolides is shown in Table 1. This Table shows macrolides high toxicity to aquatic organisms.

 

Compound	Organism	Ecotoxicity indicator, (mg/L)	Ref.
Azithromycin	Daphnia magna (crustacean)	120 (IC50)	26
	Pseudokirchneriella subcapitata (green algae)	0.5 (IC50)	27
	Skeletonema marinoi (diatom)	0.214 (IC50)	27
Clarithromycin	Vibrio fischeri (luminescent bacteria)	no effect	28
	Daphnia magna (crustacean)	25.72 (EC50)	28
	Pseudokirchneriella subcapitata (green algae)	0.002 (IC50)	28
Tylosin	Selenastrum capricornutum (green algae)	0.95 (EC50)	29
	Lemna gibba (duckweed)	0.3 (LOEC)	30
	Pseudokirchneriella subcapitata (green algae)	3.8 (EC50)	31
Erythromycin	Vibrio fischeri (luminescent bacteria)	no effect	28
	Daphnia magna (crustacean)	22.45 (EC50)	28
	Pseudokirchneriella subcapitata (green algae)	0.02 (IC50)	28

This review began with a summary of the treatment and removal of macrolides in municipal wastewater treatment plants from various countries in the world. Their main representatives, ERY, CLA, AZI, and ROX have been included in the EU Watch List of potentially hazardous compounds for the aquatic environment. The widespread occurrence of macrolide antibiotics in municipal wastewater, as well as their incomplete removal during wastewater treatment, has been frequently reported. This review examined conventional and advanced treatment methods, including anoxic, aerobic and anaerobic biological processes, activated carbon, ozonation, chlorination, and advanced oxidation processes. The review also contained an extensive list of tables showing the removal percentage of macrolides using different treatment methods.
Materials and Methods
Search strategy
Considering that English articles published during 2004-2020, which included occurrence and removal efficiency in different treatment plants in different countries, international databases were searched, including Thomson Reuters–Web of Science, Scopus, and Science Direct. Searching was done employing relevant keywords, such as macrolides occurrence in “municipal wastewater”, “macrolides removal”, “macrolides physicochemical properties”, and “wastewater treatment plants”. Prisma protocol principles were used in the articles screening process. Finally, 266 articles were found; only 96 were cited in this review, as the most relevant and essential for this study.
Inclusion criteria of the study
Articles that met the following criteria were included in the study; 1- Studies conducted on the occurrence of macrolides in municipal wastewater 2- Studies conducted on the removal of macrolides in municipal wastewater, 3- Studies conducted on different strategies for removal of macrolides, 4- Original studies and 5- Existence of full text. The authors used the information of articles, including the city/country of municipal wastewater treatment plant, the abundance of macrolides in the wastewater, and the methods used to remove macrolides. According to the reviewed articles, the classification of different removal methods was shown.
Data extraction and analysis
The data structure included the number of macrolides in different wastewater, number of macrolides in influents and effluents, name of authors, municipal wastewater treatment plants of study, province, urban and country, year, and removal management method. Finally, the extracted data included treatment processes, removal efficiency, and location of municipal wastewater plant (city/country). The results were classified into eight groups, as follows: physical treatment, biological treatment, a combined of biological process techniques, advanced oxidation processes, physicochemical treatment, natural treatment, advanced treatment, and combination of treatment processes. The study examined these groups and their effects on the removal of macrolides reported in municipal wastewater.
Molecular structure
Macrolides were introduced to the world in 1952 by Mc Guire et al. with the introduction
of erythromycin derived from the fungus Streptomyces Erythreus. Macrolides are characterized by a large highly substituted macrocyclic lactone ring which can vary from 12-16 atoms with one or more chains of deoxy sugars (mainly cladinose and desosamine) attached to a hydroxyl group. They contain a dimethylamino group which makes them basic. They are sparingly soluble in water but dissolve relatively well in polar organic solvents ^32-35 (Table 2).

Compound	Acronym	CAS number	Molecular weight (g/mol)
Erythromycin	ERY	114-07-8	733.93
Clarithromycin	CLA	81103-11-9	747.95
Azithromycin	AZI	83905-01-5	748.98
Fidaxomicin	FID	873857-62-6	1058.04
Carbomycin A	CAR	4564-87-8	841.97848
Josamycin	JOS	16846-24-5	828.006
Kitasamycin	KIT	39405-35-1	701.84
Midecamycin	MID	35457-80-8	813.968
Oleandomycin	OLE	3922-90-5	687.858
Solithromycin	SOL	760981-83-7	845.01
Spiramycin	SPI	8025-81-8	843.053
Troleandomycin	TRO	2751-09-9	813.968
Tylosin	TLY	1401-69-0	916.10
Roxithromycin	ROX	80214-83-1	837.047

Macrolides, environmental occurrence, and removal efficiency
There are three main stages of the wastewater treatment process, aptly identified as primary, secondary, and tertiary treatment. In some wastewater, more advanced treatment is required; this stage uses a combination of primary, secondary, and tertiary treatments ¹⁷. In this review, the performance of currently applied methods for the removal of macrolides in municipal wastewaters was analyzed. In this review, the occurrence and removal of macrolide antibiotics were investigated at municipal wastewater in many countries. The most frequently detected macrolide antibiotics in the present study were AZI, ERY, CLA, ROX, and TLY. The concentrations of these compounds ranged from 28 to 5500 ng/L, 20 to 3900 ng/L, 25 to 6080 ng/L, 10 to 1500 ng/L, and 1 to 1500 ng/L, respectively. The other macrolide antibiotics have been reported in small amounts from municipal wastewater. Previous studies have revealed that several treatment techniques are available to remove macrolides in municipal wastewater treatment plants, including coagulation, flocculation, sedimentation, filtration, biological treatments, such as activated sludge (AS); sequencing batch reactors (SBR); membrane bioreactor (MBR); physio-chemical treatment, such as UV irradiation; reverse osmosis (RO); chlorination; ultrasonication (US); an advanced oxidation processes (AOPs), such as ozonation; UV/TiO₂; UV/H₂O₂; and Fenton/photo-Fenton.^{22, 36-37}.
Result
Physical treatment
When physical and mechanical properties are used to separate and remove external dissolved solids, it is called physical wastewater treatment. These processes include coagulation, flocculation, sedimentation, filtration, grain collection, grit chamber, sand filtration, etc. Most of these methods are performed before wastewater treatment, which is also called primary treatment. With physical treatment, the amount of macrolides removal has been rarely reported ^{38, 39}. The range percentage removal of macrolides by the physical method was reported to be between 0 and 33%. The physical treatment method is not a good way to remove the drug but to explain the reported 33% removal; it can be attributed to the structure of hydrophilic macrolides. This might be affected by the removal of the fine suspended particles adsorbing these hydrophilic compounds. The physical treatment is effective in combination with other treatments. The percentage removal of macrolides from Kloten-Opfikon in Switzerland using physical treatment has been reported 0-4% for ERY, 11%-14% for CLA, and 10%-33% for AZI ⁴⁰. The GC method was applied for removing Clarithromycin from municipal wastewater in Guangdong, in China, with influent of 125 ng/L, zero reported (Table 3). Nakada et al. ³⁸ discussed macrolides removal in terms of chemical structure. They reported that removal of the CLA, ERY, ROX, and AZI during sand filtration was generally inefficient. They concluded that the reason for inefficient sand filtration is lack of hydrophobicity. Table 3 shows that physical treatment has not provided any notable removal for the investigated macrolides. The removal percentage range is 0 to 31%; it has been observed that clarification has a higher efficiency method in removing macrolides than other physical methods.

	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	SF	0	228	Tokyo/Japan	38
	S + GC + Sed	11-14	330–600	Kloten–Opfikon/Switzerland	40
	GC	0	125	Guangdong/China	39
	GC	5.67	50	Guangdong/China	39
ERY-H2O	SF	0	150	Tokyo/Japan	38
	GC + Sed	0-4	60-190	Kloten–Opfikon/Switzerland	40
	GC + Sed	0	810 ± 11	Wan Chai/China	41
	GC	0	~900	Guangdong/China	39
	GC	13.8	~700	Guangdong/China	39
ROX	SF	5.36	27.2	Tokyo/Japan	38
	GC + Sed	3-9	10-40	Kloten–Opfikon/Switzerland	40
	GC	3.04	70	Guangdong/China	39
	GC	2.42	40	Guangdong/China	39
	Sed	31	108 ± 3.3	Dalian/China	42
AZI	SF	0	-	Tokyo/Japan	38
	S + GC + Sed	10-33	90–380	Kloten–Opfikon/Switzerland	40
	Sed	29.8	345 ± 21	Dalian/China	42

Biological treatments
The physical treatment will only be able to separate a part of the suspended solids (which can be separated) and finally a small amount of macrolides matter. Thus, to separate and remove soluble materials, as well as colloidal and non-sedimentary materials, another step of treatment is required. In secondary treatment, biological agents are often used to convert and decompose pollutants⁴³. Removal is usually performed by biological processes in which microorganisms utilize the organic impurities as food, reducing them into carbon dioxide, water, and energy for their growth and replication⁴⁴. Biological treatment methods have traditionally been used for the management of pharmaceutical wastewater. Biological treatment processes are divided into three main groups, including aerobic, anaerobic, and anoxic processes. Aerobic applications include activated sludge, membrane batch reactors, and sequence batch reactors. Anaerobic methods include anaerobic sludge reactors, anaerobic film reactors, and anaerobic filters and anoxic method include the process by which nitrate NO₃ nitrogen is converted to molecular nitrogen gas in the absence of oxygen⁴⁵.
Aerobic treatment
Variations on aerobic treatment, including SBR, MBR, moving bed biofilm reactor (MBBR), and AS were shown to have added advantages for the treatment of wastewater ⁴⁶. However, aerobic process was discussed in detail regarding the subject of this article.
Activated sludge modification process
The activated sludge process is used for the reduction of organic matter present in the wastewater. Conventional activated sludge (CAS) with a long hydraulic retention time (HRT) has historically been the method of selection for the treatment of wastewater. Extended activated sludge is another kind of activated sludge that has been widely used in many countries ³⁷. The SBR is an activated sludge method of treatment in which separate tanks for aeration and sedimentation are not required and there is no sludge return ⁴⁷. This system is ordinarily used to treat wastewater from small communities and can accept periodic loadings without becoming disturbed ⁴⁸. Macrolide antibiotics, including AZT, CLA, ROX, ERY, and ERY-H₂O, indicated different results suggesting a difference with the activated sludge process. High variability was observed in the removal efficiencies, Table 4 shows removal efficiency macrolides significantly ranged between 0 to 100 %. Earlier studies have reported that macrolide antibiotics are often moderately removed by activated sludge processes for municipal wastewater ^{40, 49-51}. Nakada et al. ³⁸ applied a combination of ozon and SF with activated sludge treatment and the removal efficiency was above 80%. They observed that by using activated sludge with a hydraulic retention time of 9 h, removal efficiencies of 0, 38.9%, 40.9%, and 18.6% were observed for AZI, ERY, CLA, and ROX, respectively. Göbel et al. ⁴⁰ investigated two conventional activated sludge (CAS) systems, including the CAS system at the municipal wastewater treatment plant Kloten–Opfikon, Switzerland (CAS-K) and CAS system at the municipal wastewater treatment plant Altenrhein, Switzerland (CAS-A). The results were discussed based on temperature, hydraulic retention time, and solids retention time. In CAS-K, hydraulic retention time was ~15 h; solid retention time was 10–12 d, and wastewater temperature was 12-16°C. In CAS-A hydraulic retention time was ~31 h, solid retention time was 21–25 d, and wastewater temperature was 12-19°C. The removal efficiencies of AZI, ERY-H₂O, CLA, and ROX using CAS-K and CAS-A were reported 0 and 22%-55%; 0-6% and 0-7%; 0-9% and 4%-20%, and 0-38% and 5%-38%, respectively. Dong et al. ⁵² studied CW, SP, AS, and MP for occurrence and removal of 19 antibiotics (including four macrolides) in a county of eastern China. Their review analysis demonstrated that AS and CW outperformed the MP and SP processes and AS performed better than the CW process in terms of antibiotics removal. Bing and Zhang ⁵³ investigated the mass flows and the removal of ROX and ERY-H₂O in two wastewater treatment plants (WWTPs) of Hong Kong. The mean removal efficiencies using activated sludge process for ROX and ERY-H₂O were 46% and 15% in Shatin and 40% and 26% in Stanley. Valiparambil et al. ⁵⁴ investigated four STPs in South India. They studied the seasonal effects on the occurrence and removal efficiency during pre-monsoon, monsoon, and post-monsoon seasons. They found that effluents received
in the monsoon season had the highest concentration range versus other seasons which may be due to the higher incidence of flu/infections. The performance of activated sludge systems depends on the type of macrolide and the location of the wastewater. Generally, higher rates of removal have been reported for CLA than for other macrolides. Efficiency of reported removals may depend on whether the effluents have been sampled after aeration and sludge separation or after sedimentation following activated sludge treatment ⁵⁴.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (city/country)		Ref.
CLA	CAS	40.9	228	Tokyo/Japan		38
	CAS	0-9	330–600	Kloten–Opfikon/Switzerland		40
	CAS	4-20	-	Altenrhein/Switzerland		40
	CAS	83.4 ± 25.7	173	Hikkaduwa/Sri Lanka		55
	CAS	87	230	Castellon/Spain		56
	CAS	90	-	Germany		57
	CAS	91	2200	Eastern China		52
	CAS	62	71	Zagreb/Croatia		58
	SBR	50	850	Gyeonggi/South Korea		59
ERY	CAS	38.9	150	Tokyo/Japan		38
	CAS	0-6	60–190	Kloten–Opfikon/Switzerland		40
	CAS	0-7	-	Altenrhein/Switzerland		40
ERY-H2O	CAS	-	261	Hikkaduwa/Sri Lanka		55
	CAS	26	-	Stanley/Hong Kong		53
	CAS	15	-	Shatin/Hong Kong		53
	CAS	-	280	Eastern China		52
	CAS	15	36	Zagreb/Croatia		58
	SBR	24	290	Gyeonggi /South Korea		59
	EA	65	24	STP1	Karnataka/ India	54
	EA	0	59
	EA	31	6
	EA	0	28	STP2
	EA	0	26
	EA	100	7
ROX	CAS	18.6	27.2	Tokyo/Japan		38
	CAS	0-38	10–40	Kloten–Opfikon/Switzerland		40
	CAS	5-38	-	Altenrhein, Switzerland		40
	CAS	69.8 ± 38.4	108	Hikkaduwa/Sri Lanka		55
	CAS	40	-	Stanley/Hong Kong		53
	CAS	46	-	Shatin/Hong Kong		53
	CAS	100	-	Germany		57
	CASS	50	500	Harbin/China		60
	CAS	-	280	Eastern China		52
	SBR	24	290	Gyeonggi/South Korea		59
AZI	CAS	0	-	Tokyo/Japan		38
	CAS	0	90–380	Kloten Opfikon/Switzerland		40
	CAS	22-55	-	Altenrhein/Switzerland		40
	CASS	0	28	Harbin/China		60
	CAST	0	28	Harbin/China		60
	CAS	78	1949	Singapore		61
	CAS	100	-	Germany		57
	CAS	19	350	Zagreb/Croatia		58
LIN	CAS	42.1	65.5	Singapore		61

Moving bed bioreactor (MBBR) treatment
MBBR technology is an advancement over the CAS technology and is a biological process of attached growth type ⁶². This method consists of an activated sludge aeration system where the sludge is collected on recycled plastic carriers. These carriers have an internal large surface for optimal contact with water, air, and bacteria. MBBR technology is more efficient than ASP and SBR and requires less area. The data of macrolides removal using MBBR are shown in Table 5. Xiangjuan Yuan et al. ⁴¹ studied the occurrence, fate, and environmental impact of CLA, ERY-H₂O, ROX, and AZI in two municipal wastewater treatment plants located in Wuxi City, East China. The analysis showed that a maximum concentration of CLA, ERY- H₂O, ROX, and AZI in influent was > 100, 10, > 103, and 232.5-876.9 ng/L, respectively. The removal percentage range was 20% to 76.2%. The removal range of the macrolides was almost identical with MBBR treatment.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (city/country)	Ref.
CLA	MBBR	20	850	Gyeonggi/South Korea	59
	MBBR	59.9	> 100	Wuxi/china	41
ERY- H2O	MBBR	60.8	10	Wuxi/china	41
ROX	MBBR	53.7	> 103	Wuxi/china	41
AZI	MBBR	76.2	232.5-876.9	Wuxi/china	41

MBR process
The MBR is a combined of conventional biological treatment processes with membrane filtration to provide an advanced level of organic and suspended solids removal and in some cases nutrient removal. The MBR is one of the most modern methods of wastewater treatment. Removal efficiencies of macrolides from the municipal wastewater using MBR are shown in Table 6. According to the results of the studied wastewater Gyeonggi-province, South Korea using MBR exhibited better performance over MBBR and SBR for most macrolides ⁵⁹. Wang et al. ³⁶ investigated the use of MBR linked with RO and NF to remove drugs from municipal wastewater. In this study, MBR was operating with HRT of 3.2 h, mean pH 7.8, and from texture polyvinylidene fluoride and polyethylene terephthalate with an effective area of 0.8 m². The results showed that for macrolide antibiotics, MBR removal efficiency was 74% to 82% (Table 6). By comparing MBR and CAS methods (Table 3 and 5), it can be concluded that MBR has a better effect on most macrolides (CLA 91.4%, ERY- H₂O 90%, ROX 74%, AZI 91.4%, and LIN 62.1%) than CAS.

	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	MBR	60	850	Gyeonggi/South Korea	59
	MBR	82	368	China	36
	MBR	71.3	1497	Singapore	61
	Aerobic	16.8	125	Guangdong/China	39
	MBR	52	2020	Castell- Platjad’Aro/Spain	63
	FBR	5.6-14		Altenrhein/Switzerland	40
	MBR	71.87-74.06	6080	Croatia	64
ERY- H2O	MBR	77	20	China	36
	MBR	0.71	-	Zagreb, Croatia	65
	MBR	40	44	Jeolla/South Korea	66
	MBR	42	44	Jeolla/South Korea	66
	MBR	64.8	652	Singapore	61
	Aerobic	13.7	~900	Guangdong/China	39
	Aerobic	21	221	Beijing/China	67
	MBR	81	49	Castell-Platja d’Aro/Spain	63
ROX	MBR	59	290	Gyeonggi/South Korea	59
	MBR	74	79	China	36
	MBR	0.36	-	Zagreb/Croatia	65
	FBR	35±6	-	Altenrhein/Switzerland	40
	Aerobic	9.91	70	Guangdong/China	39
	Aerobic	29	129	Beijing/China	67
AZI	MBR	80	1410	China	36
	MBR	91.4	1949	Singapore	61
	MBR	77	118	Castell-Platja d’Aro/Spain	63
	MBR	23.25-52.62	-	Croatia	64
	FBR	30±6	-	Altenrhein/Switzerland	40
TYL	Aerobic	2	6.42	Beijing/China	67
SPI	Aerobic	0	7.46	Beijing/China	67
JOS	Aerobic	0	0.86	Beijing/China	67
LIN	MBR	62.1	65.5	Singapore	61

Anaerobic treatment
Anaerobic treatment processes consist of several methods in which microorganisms break down organic components of the wastewater in the lack of oxygen. Configurations of anaerobic reactors include up-flow anaerobic reactors, anaerobic film reactors, and up-flow anaerobic filters ⁶⁸. The performance of an anaerobic condition was evaluated for the removal of macrolides from municipal wastewater ^{39, 67, 69}. Kasturi Dutta et al. ⁶⁹ investigated a two-stage AFMBR and AFBR followed by AFMBR and used GAC as a carrier medium in both stages. They found that the
two-stage AFMBR was able to treat municipal wastewater at a minimum HRT of 1.28 h. Using AFBR, the effluent was obtained by 132 ± 19.1 ng/L and 140 ± 4.9 ng/L for ERY-H₂O and CLA, respectively, and using AFMBR, it was obtained 43.9 ± 2.1 ng/L and 35.5 ± 2.1 ng/L for these two macrolides, respectively. Li et al. ⁶⁷ investigated the occurrences and fates of five macrolides in a wastewater reclamation plant in Beijing, China. The concentrations of TYL, SPI, and JOS in the influent were low, obtained 6.42 ng/L, 7.46, and 0.86 ng/L for TYL, SPI, and JOS, respectively. This study indicated that macrolides were mainly removed from the wastewater with anaerobic treatment. The removal percentage range of TYL, SPI, and JOS were reported to be between 23% and 68% (Table 7).

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (city/country)	Ref.
CLA	AFBR	56.9	324 ± 6.4	Taiwan	69
	AFMBR	74.6	324 ± 6.4	Taiwan	69
	Anaerobic	2.99	125	Guangdong/China	39
ERY	Anaerobic	6.45	~900	Guangdong/China	39
	Anaerobic	31	221	Beijing/China	67
ERY-H2O	AFBR	56.9	319 ± 42.4	Taiwan	69
	AFMBR	74.6	319 ± 42.4	Taiwan	69
ROX	Anaerobic	17.6	70	Guangdong/China	39
	Anaerobic	39	129	Beijing/China	67
TYL	Anaerobic	68	6.42	Beijing/China	67
SPI	Anaerobic	55	7.46	Beijing/China	67
JOS	Anaerobic	23	0.86	Beijing/China	67

Anoxic treatment
Anoxic treatment is the chemical and biological treatment of wastewater that decreases nitrate, phosphorus, and other residual organics and solids in wastewater effluent ⁷⁰. Zhou et al. ³⁹ chose a municipal wastewater treatment plant in Guangdong Province in China. They reported that using anoxic treatment the removal percentage of macrolides for CLA, ERY, and ROX was obtained 49.2%, 10.2%, and 11.1%, respectively. Wenhui Li ⁶⁷ investigated wastewater reclamation plants in Beijing-China. The anoxic treatment parameters in the studied wastewater plant: water flow, sludge flow, and hydraulic residence time were 10 × 10⁵ m³/d, 44.7 × 10⁵ kg/d, and 3, respectively. Wenhui Li ⁶⁷ reported that mean influent concentrations of JOS, TYL, ROX, and ERY were 0.86 ng/L, 6.42 ng/L, 129, and 221 ng/L, respectively. The mean concentrations of JOS, TYL, ROX, and ERY after anoxic treatment were obtained 0.13 ng/L, 2.11 ng/L, 100 ng/L, and 172 ng/L, respectively. The removal efficiency of different macrolides ranged from 0 (ROX, ERY and TYL) to 62% (JOS). The three macrolides, SPI, JOS, and TYL, detected with low frequencies and at relatively low concentrations, were removed effectively during the Anoxic treatment (Table 8).

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	Anoxic	42.9	125	Guangdong/China	39
ERY	Anoxic	10.2	~900	Guangdong/China	39
	Anoxic	0	221	Beijing/China	67
ROX	Anoxic	11.1	70	Guangdong/China	39
	Anoxic	0	129	Beijing/China	67
TYL	Anoxic	0	6.42	Beijing/China	67
SPI	Anoxic	4	7.46	Beijing/China	67
JOS	Anoxic	62	0.86	Beijing/China	67

Biological combined processes
This section describes a combination of different biological processes utilized for the treatment of several macrolides’ antibiotics. Table 9 reveals removal efficiency using AO and A₂O treatment. Aerobic tanks may be coupled with anoxic or anaerobic tanks to give biological nutrient removal. The A₂O process is a patented two-stage biological process. In the first stage, under anaerobic conditions, a three-series chamber anaerobic baffled reactor (ABR) was used, while in the second stage, an AS with a settler was utilized. Park et al. ⁵⁹ evaluated the removal efficiency of CLA and ROX in a municipal WWTP in South Korea using the A₂O process. The removal efficiency of 15% (CLA) and 7% (ROX) indicated low removal efficiency of this treatment. Xiangjuan Yuan et al. ⁷¹ presented the concentrations of macrolides in various sludge samples along with the A₂O treatment process. The results indicated that the mean concentrations of anaerobic sludge, anoxic sludge, oxic sludge, and return sludge for ERY-H₂O were 4.06, 9.75, 6.45, and 3.30 μg/kg; for CLA were 8.85, 28.31, 7.76, and 7.19 μg/kg; for ROX were 13.06, 23.57, 12.17 μg/kg, and 11.21 μg/kg; and for TYL were 0.25, 0.28, 0.28, and 0.28 μg/kg, respectively.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	A2O	15	850	Gyeonggi/South Korea	59
	A2O	51	35.8	Harbin/China	60
	AO	8.7	35.8	Harbin/China	60
	A2O	55	~1750	Kyoto/Japan	72
	A2O	56	~650	Beijing/China	72
	A2O	95	550	China	71
	AO	85	~5000	Shiga/Japan	72
	A2O	3.6	> 100	Wuxi/china	41
ERY-H2O	A2O	80	500	China	71
	A2O	13	10	Wuxi/china	41
	A2O	53.58	66.3-159.5	Tehran/Iran	73
	A2O	67.8	159.5	Tehran/Iran	74
ROX	A2O	7	290	Gyeonggi/South Korea	59
	A2O	25	~100	Kyoto/Japan	72
	A2O	13.6	> 103	Wuxi/China	41
	AO	69	500	Harbin/China	60
	A2O	72	500	China	71
	AO	73	~213	Shiga/Japan	72
	A2O	0	500	Harbin/China	60
	A2O	27	~800	Beijing/China	72
AZI	A2O	60	28	Harbin/China	60
	AO	0	28	Harbin/China	60
	A2O	40	~250	Kyoto/Japan	72
	A2O	13	~280	Beijing/China	72
	AO	95	~5500	Shiga/Japan	72
	A2O	17.5	232.5-876.9	Wuxi/China	41
	A2O	66.6	43.3	Tehran/Iran	73

Advanced Oxidation Processes (AOP_s)
 AOPs include photo Fenton (UV/H₂O₂/Fe²⁺), UV/H₂O₂, solar photo Fenton, UV- Photolysis, Oz, US, Oxidation, and Fenton Processes (H₂O₂/Fe²⁺). Many researchers have used advanced oxidation methods to investigate the removal of macrolides in municipal wastewater^{38, 39, 67, 72, 75}. Table 10 presents the removal efficiency of macrolides in municipal wastewater by AOPs. Sousa et al. ⁷⁵ reported full removal of 19% out of 22% pharmaceuticals with ca. 32 kJ/L solar UV energy. The Beijing wastewater in China was investigated⁶⁷ using CAS system, coupled with subsequent ultrafiltration and ozone oxidation system. They observed that removal contribution of ozone oxidation system for JOC, TYL, ROX, and ERY was 27%, 27%, 100%, and 83%, respectively. Among various technologies that have been developed and applied to remove macrolides, Oz and photocatalysis with TiO₂ have both shown encouraging results. The Oz has shown good removal efficiencies on a wide range of different macrolides, both at the laboratory and full scales. In order to achieve the desired removal of macrolides, the technology can be improved with additional features, such as photocatalytic enhancement⁷⁶.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	Oz	84.6	228	Tokyo/Japan	38
	OD	77	~950	Beijing/China	72
	Photocatalysis	40	24-676	Portugal	76
	Photocatalytic + Oz	> 94	24-676	Portugal	76
	OD	70.1	50	Guangdong/China	39
	OD	1.1	>100	Wuxi/china	41
ERY	Oz	88.7	150	Tokyo/Japan	38
	Photocatalysis	35	-	Portugal	76
	Photocatalytic + Oz	100	-	Portugal	76
	OD	0	60	Wuxi/china	41
	OD	55.3	~700	Guangdong/China	39
	Oz	83	221	Beijing/China	67
	Oz	63	2600	Gwinnett/USA	77
ROX	Oz	90.9	27.2	Tokyo/Japan	38
	OD	43	~775	Beijing/China	72
	OD	52.1	40	Guangdong/China	39
	OD	0	>103	Wuxi/china	41
	Oz	92.3	129	Beijing/China	67
AZI	Oz	92.6	-	Tokyo/Japan	38
	OD	10	~60	Beijing/China	72
	Photocatalysis	100	631	Portugal	75
	Photocatalysis	50	-	Portugal	76
	Photocatalytic + Oz	> 95		Portugal	76
	OD	7.1	232.5-876.9	Wuxi/China	41
TYL	Oz	27	6.42	Beijing/China	67
SPI	Oz	48	7.46	Beijing/China	67
JOS	Oz	27	0.86	Beijing/China	67

Physio-chemical treatment
Physicochemical treatments are very important within the wastewater treatment systems and before any biological and advanced treatment technologies. This treatment of wastewater focuses primarily on the separation of colloidal particles^{78, 79}. Physiochemical treatment options for this review were divided into four main topics, including membrane processes, reverse osmosis, and activated carbon. The removal of macrolides with RO, MF, and UF during the drinking water and wastewater treatment processes at full- and pilot-scale have also been investigated^{50, 63, 65-67}. Macrolide antibiotics can be removed by physicochemical treatment (Table 11). Membrane filtration processes using RO and NF showed excellent removal (> 95%) for ERY⁶⁶. The removal of ERY in wastewater by RO and NF was <1.0. Li et al. ⁶⁷ studied Beijing municipal wastewater in China. Based on their study concentrations of TLY, ROX, ERY, and JOS after UF treatment were 0.23, 1.7, 143, and 186 ng/L, respectively. The removal efficiency of individual macrolide ranged from 0 (ERY) to 23% (ROX). Removal of macrolides by physio-chemical treatment is defined by multiple synergies of electrostatic and other physical forces acting between a special solute, the solution, and the membrane itself ⁶³.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	RO	100	-	Zagreb/Croatia	65
	NF	97		Wuxi/China	65
	RO	48	2020	Castell-Platjad’Aro/Spain	63
	RO	100	-	Zagreb/Croatia	65
	NF	< 1.0	44	Jeolla/South Korea	66
	RO	< 1.0	44	Jeolla/South Korea	66
	UF	0	221	Beijing/China	67
	RO	19	49	Castell-Platja d’Aro/Spain	63
ROX	RO	100	-	Zagreb/Croatia	65
	UF	23	129	Beijing/China	67
AZI	RO	100	-	Zagreb/Croatia	65
	RO	23	118	Castell-Platja d’Aro/Spain	63
TYL	UF	2	6.42	Beijing/China	67
SPI	UF	0	7.46	Beijing/China	67
JOS	UF	6	0.86	Beijing/China	67

 

Natural wastewater treatment
Natural treatment systems, such as CW and SP are used for wastewater treatment. This treatment is an alternative wastewater treatment system that reproduces the processes of removing contaminants which occur in natural wetlands and ponds. Removal efficiencies of the natural wastewater treatment related to macrolides are presented in Table 12. Studies have shown that natural treatment of antibiotics has shown a strong dependency on the specific wastewater treatment process and was higher in summer than in winter. It indicates the vital role of biological degradation, removal efficiency, and associated ecological risk assessment ^{52, 80, 81}. The results showed that the removal efficiencies of AZI, CIP, and SMZ were 78.8%, 23%, and 17.6% in winter and 80.9%,1.5%, and -30.6 in summer, respectively (Tezmant WWTP-Egypt). The reason for the negative removal percentage of SMZ in summer was transmutation of N₄–acetyl sulfamethoxazole (SMZ metabolite, 43% in the excreted urine) to the parent compound of sulfamethoxazole.
Among the numerous important factors, temperature may play an important role in the removal of antibiotics in WWTFs, which is closely related to microbial activity and growth rate. However, studies have shown inconsistent results. In brief, higher and more stable removals of the macrolides have been achieved in summer in both AS and CW processes. Considering the significant change in the influent concentrations of ROX, its removals by CW in summer ranged from 60% to 98%, while some negative removals have been observed in winter. The removal of micropollutants by CW is a result of complex Physico-chemical and microbial interactions, including substrate sorption, plant uptake, and biological degradation ⁸². Apart from the poor biological degradation activity in winter, both desorption of substrate-bound compounds and the potential cleavage of conjugates in winter can cause negative removals ^{83, 84}. Therefore, better removals have been achieved in the AS process in summer and winter seasons and the CW process in summer.

Macrolides	Treatment processes			Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	CW1		Typha-FM-SF	22	250 ± 84	Leon/Spain	80
	CW2		Typha-FW-SF	32
	CW3		Typha-FW-SSF	39
	CW4	Unplanted-FW-SSF		50
	CW5	Phragmites-FM-SF		11
	CW6	Phragmites-SSF		31
	CW7	Unplanted-SSF		32
	CW			81	650	Eastern China	52
	SP			78	700	Eastern China	52
ERY-H2O	CW1	Typha-FM-SF		0	56 ± 26	Leon/Spain	80
	CW2	Typha-FW-SF		0
	CW3	Typha-FW-SSF		0
	CW4	Unplanted-FW-SSF		0
	CW5	Phragmites-FM-SF		0
	CW6	Phragmites-SSF		64
	CW7	Unplanted-SSF		0
ERY	CW				340	Eastern China	52
	SP				190	Eastern China	52
ROX	CW				250	Eastern China	52
	SP				280	Eastern China	52

Advanced treatment
Advanced wastewater treatment is any process that decreases the level of pollutants in wastewater that is available through conventional secondary or biological treatment. Table 13 shows the removal of macrolides in municipal wastewater using advanced treatment. According to research studies, chlorination treatment has shown the highest efficiency ^{41, 85}. On the other hand, studies have shown different results for UV treatment to remove macrolides in municipal wastewater. The removals by UV treatment for CLA (63%), ERY-H₂O (52.5%), TLY (60%), ROX (20.8%), and AZI (29.7%) were reported with high efficiency^{53, 68, 86}. UV treatment showed low efficiency for OLE in municipal wastewater^{85, 86}. It seems that the removal efficiency of UV treatment depends on the structure of the macrolide and the amount of them in municipal wastewater.

Macrolides	Treatment processes	Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	pre-UV	63	319	Varese/‏Italy	85
	post-UV	0
	UV	86.9	> 100	Wuxi/China	41
	UV	9	775	Japan	86
ERY-H2O	CLO	63.7	-	Stanley/Hong Kong	53
	pre-UV	0	12	Varese/Italy	85
	post-UV	0
	DI	24	-	Stanley/Hong Kong	53
	UV	52.5	60	Wuxi/China	41
	UV	9	275	Japan	86
ROX	CLO	55.3	-	Stanley/Hong Kong	53
	DI	18	-	Stanley/Hong Kong	53
	UV	15	40	Guangdong/China	39
	UV	20.8	> 103	Wuxi/China	41
AZI	UV	29.7	232.5-876.9	Wuxi/China	41
	UV	5	102	Japan	86
TYL	UV	60	4.0 ± 3.0	Milan/Italy	85
SPI	pre-UV	25	603	Varese/Italy	85
SPI	post-UV	17	603	Varese/Italy	85
LIN	pre-UV	37	9.7	Varese/‏Italy	85
	post-UV	0
OLE	pre-UV	0	2.2	Varese/‏Italy	85
	post-UV	0

Combined processes of treatment
One of the great challenges of researchers is to use solutions to improve the performance of wastewater treatment plants to remove residual pharmaceuticals in the wastewater, especially antibiotics. The presence of antibiotics in wastewater over time causes microorganisms to become resistant to these drugs. The most important step in the development of a wastewater treatment plant is to choose a process that, in addition to having economic and proper efficiency is appropriate with the environmental and climatic conditions. The results indicated that in combined processes a high efficiency of removal was obtained; therefore, researchers use a combination of various treatments. Many studies have used primary, secondary, and tertiary processes to remove macrolides from municipal wastewater^87-90. In the first stage, under anaerobic conditions, a three-series chamber ABR was used, while in the second stage, an aerobic activated sludge with a settler was applied. Lin et al.⁸⁷ demonstrated that there were many pharmaceuticals in influents of WWTPs, and the ST processes applied by the WWTPs are variably and inadequately effective in removing numerous pharmaceutical contaminants from influent wastewater. Researchers have shown that synergistic effects were in the in situ O₃, CMF, and BAC processes which were effective in removing almost all kinds of pollutants⁵⁵. Other studies indicated removal rates of above 95% for most of the macrolides using MBR with RO/NF ³⁶. Table 14 presents several combined methods for removing macrolides from municipal wastewater plants in different countries.

Macrolides	Treatment processes		Removal (%)	Influent (ng/L)	Location (City/Country)	Ref.
CLA	O3+CMF			173	Hikkaduwa/Sri Lanka	55
	S+ Sed+ G+ AS		44.5	~2200	4 WWTPs in Taipei/Taiwan	87
	MBR+ RO		100	368	China	36
	MBR+ NF		100
	ST+ GAC+ UV		98	-	Eastern United States	88
	ST+ MBBR +TR + SF		91
	ST+ Sed+ Oz/ BAF/ GAC+UV		99
	PT+ SBR+ UV		96
	AABR+ MBR+UV		100
	PT +SBR+ CLO		18
	PT+ AS+NAS+ CLO		24
	SC + NaClO		0	50	Guangdong/China	39
	SC+ UV		15
	RFDFs		66.2	>100	Wuxi/china	41
	RFDFs		85.2
	SBR+A2O+OD+MBR		52	~25	12 WWTPs/China	89
	GC + A2O+ OD+ CAS+ MBR+ UV+ RFDF+ ClO2+ UF+ Oz+ CS		75	550.3	14 WWTPs/China	90
	NF/RO	NF90	> 99.9	6080	Croatia	64
		RO XLE	> 99.9
		NF270	75.88
	MBR+RO		100	-	Zagreb/Croatia	65
ERY	Pre- O3 + CMF+ BAC		97	390	Jindawanxiang/North China	91
	MBR+RO		100	20	China	36
	MBR-NF		98
	S+Sed+ G+AS		43.8–100	~3000	4 WWTPs in Taipei/Taiwan	87
	ST+ GAC+UV		97	-	Eastern United States	88
	ST+ MBBR+ TS+ SF		97
	ST + Sed+ Oz+ BAF, GAC + UV		98
	PT+ SBR + UV		0
	AABR+ GAC+UV		0
	PT + SBR+ CLO		0
	PT+ AS+ NAS+ CLO		0
	PT + AS		39.11	254.24 ± 15.36	Southwest/China	51
	SBR + A2O +  OD + MBR		53	~250	12 WWTPs/China	89
	PT + AS + Anaerobic		0	470 ± 2.5	Tai Po/China	92
	PT+ BT		19	740 ± 14	Shatin/China	92
			9	590 ± 0.7	Stonecutter’s island/China	92
	AS + OD + AL		43.8-100	3900	Wisconsin/USA	93
	CAS + MF		10	2600	Gwinnett/USA	77
	BT+ phosphorus precipitation		-	200	Nancy/France	94
	GC + A2O/MBBR+ OD +A2O/CAS+CAS/MBR+ UV+ RFDF + ClO2 + UF + O3 + CS		78	1151.6	14 WWTPs/China	90
	Primary +AS		39	200	China	50
	PT + CAS		39.11	254.24 ± 15.36	Southwest/china	51
ROX
	Pre- O3 + CMF + BAC		96	-	Altenrhein, Switzerland	40
	O3+CMF		95	175	Jindawanxiang/North China	91
	MBR-RO		100	79	China	36
	MBR-NF		97
	PT + CAS		11.7	404.0 ± 34.2	Southwest/China	51
	SBR+ A2O + OD+ A2O /MBR		48	~80	12 WWTPs/China	89
	AS+ OD+ AL		67	1500	Wisconsin/USA	93
	GC+ A2O /MBBR+ OD+ A2O /CAS+ CAS/MBR+ UV+ RFDF+ ClO2+ UF+ O3+ CS		67	1035.7	14 WWTPs/China	90
	MF/RO			10	Brisbane/Australia	13
AZI	Pre- O3 + CMF+ BAC		99	40	Jindawanxiang/North China	91
	O3+CMF		99
	MBR-RO		98	1410	China	36
	MBR-NF		97
	ST + GAC + UV		100	-	Eastern United States	88
	ST + MBBR+ TS + SF		96
	ST + Sed+ O3, BAF+ GAC + UV		100
	PT + SBR+ UV		0
	AABR + MBR+ UV		0
	PT + SBR + CLO		60
	PT +AS + NAS + CLO		45
	PT + CAS		50.55	362.5 ± 21.7	Southwest/China	51
	SBR + A2O +OD+ A2O /MBR		45	~450	12 WWTPs/China	89
	GC + A2O /MBBR + OD + A2O /CAS + CAS/MBR + UV +  RFDF + ClO2 + UF + O3 + CS		51	1687.2	14 WWTPs/China	90
	NF/RO	NF90	> 99.9	-	Croatia	64
		RO XLE	> 99.9
		NF270	80.08
TYL	AS + OD + AL		50	1500	Wisconsin/USA	93
	MF/RO			1	Brisbane/Australia	13
	PT + AS		100	65	China	50
SPI	AS+ AOPs		91	Up to 30000	Campania/Italy	95
	RE-PST		0.3	380	Al Ain /United Arab Emirates	96
	RE-SST		24.7
	RE-FE		24.7

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