Multi-taxon trends and the application of genotoxic biomarkers in the analysis of effluents from sewage treatment plant

Abstract

Sewage is a significant source of many contaminants, and the effectiveness of sewage treatment plants (STPs) is fundamental to ensure that the effluents produced by these plants have a minimal impact on aquatic environments and guarantee their long-term sustainability. The present study is based on a global scientometric survey of the published research on the application of genotoxicity biomarkers for the analysis of the effects of the contaminants found in the effluents and residues produced by STPs. The literature search focused on the Web of Science and Scopus databases. Research trends were investigated based on the year of publication of each study, the country in which it was developed, the type(s) of genotoxic assay applied, the model organism(s), the type of study (experimental or field study), the physicochemical parameters analyzed, and the principal findings of the genotoxic assays. A total of 134 papers, published between 1988 and April 2023, were selected for analysis. The studies were conducted in a total of 33 different countries, but primarily in Brazil, China, Germany. These studies employed 16 biomarkers to assess genotoxicity, of which, the micronucleus test was the most used. The studies reported on a number of genotoxic substances, such as pollutants, including pesticides, microplastics, metals, and drugs. The data produced by these studies provide important insights into the genotoxic effect of the xenobiotic agents found in STP effluents, which are capable of damaging the DNA of a range of different organisms.

Graphical abstract

Highlights

• •Genotoxic assays applied in sewage treatment plants (STPs) studies for 30+ years.
• •The micronucleus test and comet assay are the methodologies used most frequently.
• •Different organisms have been the focus of the genotoxic evaluation in STPs.
• •The biomarkers, model organism, and objectives of the studies tend to be related.

1. Introduction

In the anthropogenic landscape, bodies of water, such as rivers, lakes, and seas, typically receive large amounts of wastewater from industrial, agricultural, and domestic sources, including effluents from sewage treatment plants (STPs) [1]. Controlling the contamination of wastewater is critical to ensure the sustainability of water resources, and the effluents produced by sewage treatment plants must satisfy certain standards of quality in order to guarantee that these resources are not contaminated [2]. The effluents produced by STPs are a significant source of many contaminants, including pesticides [3], microplastics [4], heavy metals [5], and pharmaceutical residues [6,7], that can have severe impacts on both the environmental and human health. The effective removal of these contaminants during the treatment of sewage is a significant concern, worldwide, and a major driver of research on new treatment technologies [8,9].

The final destination of the residues produced by the sewage treatment process is also a major concern, due to their organic matter and nutrient content [10]. While these residues have been used in agriculture, they are often deposited in landfills or processed by incineration or pyrolysis [11]. In addition, the residues produced by STPs may also contain toxic substances [12], including genotoxic compounds, that can be transferred to plants and introduced into the food web [13], leading to biomagnification at higher trophic levels. Given all these concerns, there has been growing scientific interest in the evaluation of the effectiveness of STPs [9] and the environmental impact of dumping effluents into bodies of water [14,15]. In particular, genotoxicity assays have been considered to be an extremely valuable tool for the analysis of the safety of effluents [16,17].

Genotoxicity is defined as the potential of a compound to cause damage to the DNA of an organism, and, when not corrected, it can cause mutations, thus becoming a mutagenic agent [18]. Chemical substances, environmental pollutants, and radiation can all be considered to be genotoxic or mutagenic agents, depending on their potential to cause DNA damage [19]. These environmental stressors may impact the structure of the DNA, causing processes such as single or double-strand breaks, fusions, deletions, and the non-segregation of the chromosomes [20]. The damage caused to the DNA of somatic cells may thus accelerate ageing and cause diseases such as cardiovascular disorders and cancer [21], while damage to the germ cells can cause sterility and multifactorial disorders such as diabetes and other genetic problems [22]. Many studies have shown that wastewater is a source of both genotoxic and mutagenic agents [[23], [24], [25]].

Given this, the responses to contamination observed at the cellular level may not always reflect higher-level effects [26] however, cells are the structural and functional units of the entire organism and can generate responses in the population [27]. As STPs receive wastewater from a range of different sources, including domestic residences, industrial plants, and hospitals, their effluents and residues may contain xenobiotic substances that are potentially harmful to many different types of organism, including humans, at the cellular level. Considering the growing importance of these questions, the present study reviewed the scientific literature on the application of genotoxicity biomarkers in research on STPs, in order to define perspectives, trends, and the long-term potential of this types of research.

1.1. Tracking the scientific literature and selection criteria

Scientific references on the genotoxic impacts of sewage treatment plants were compiled from two databases, the Web of Science (www.isiknowledge.com) and Scopus (www.scopus.com). These databases were chosen for the present study because of their global scope and ample scientific content. The literature search was based on the identification of specific topics in the title, abstract, and keywords each paper, using the following combination of keywords and logical operators: “(wastewater treatment plant) OR (sewage treatment plant) OR (wastewater treatment plant waste) AND (genotoxicity)”. As the keywords were chosen to target the genotoxic effects of the treatment of sewage, studies of water treatment plants were not included.

The search period encompassed the interval between the date of the earliest study identified in the survey (1988) and April 2023. A total of 651 papers were identified initially during the literature search, although a majority were excluded during screening, based on the selection criteria (see Fig. 1), which required that the papers were reports of original research (no review studies or letters to the editor), were not duplicate reports or written in languages other than English and Portuguese, and present content directly relevant to the objectives of the present study. Following this screening, 134 papers were selected for further analysis. The following information was extracted from each paper: (i) key words (Fig. 2) (ii) the year of publication, (iii) the country in which the study was developed, (iv) the type(s) of genotoxic analysis applied, (v) the model organism, (vi) the type of study (experimental or field study), (vii) the physicochemical parameters analyzed, and (viii) the primary responses identified in the genotoxic assays (presence or absence of genotoxic effects). These data were presented in terms of both relative and absolute frequencies. A Montecarlo Randomization Test was performed considering the responses (positive or negative) of the model organisms to the main biomarkers used in the studies, in order to evaluate the efficiency of the biomarkers. The global geographic distribution of studies was mapped in geographic information system software (QGis).

Fig. 1.

Flowchart showing the steps followed in the present study for the selection and screening of the scientific papers on genotoxicity and sewage treatment plants identified during the literature search.

Fig. 2.

List of the most cited keywords in the selected studies.

1.2. Publication history and geographic distribution of the studies

We evaluated the number of publications on the genotoxic effects of effluents produced by the STPs per year in relation to the number of articles indexed in the databases in each year. The first study was published in 1988 (Fig. 3), although there was little progress over the subsequent 12 years, the number of publications only began to grow noticeably after 2008.

Fig. 3.

Timeline of the studies on the genotoxic effects of effluents from sewage treatment plants published between 1988 and April 2023 in relation to articles indexed in the databases in each year.

In 2015, the United Nations (UN) implemented the Sustainable Development Goals (SDGs), which included a specific objective to ensure universal access to clean drinking water and basic sanitation. This development sparked an increasing focus on wastewater reuse, technological advancements, and environmental concerns related to sewage treatment plants. This recent growth in research is a response to the increasing preoccupation of scientists and public authorities with urbanization and waste disposal. It is highly probable that this trend will persist in the foreseeable future, particularly considering the target of accomplishing the Sustainable Development Goals (SDGs) by 2030 [28]. However, despite the concerted research efforts in this domain, a decline in publications was observed in the years 2018 and 2019, followed by a subsequent upswing in the last four years.

The 134 papers selected for the present study reported on studies conducted in 33 different countries, of which, 17.27 % were conducted in Brazil (n = 24), followed by China (10,79 %, n = 15), Germany (8.63 %, n = 12), and Australia and India (both with 5.76 %, n = 8). The remaining studies were conducted primarily in countries from Europe and the Middle East (Fig. 4). Some of the studies were conducted in more than one country. Despite the number of studies conducted in Brazil, the continent with the greatest concentration of research was Europe, with 55 studies in 16 countries, followed by Asia, with 36 studies in eight countries, while the only other South American country recorded here was Argentina, with a total of 26 studies for the continent as a whole.

Fig. 4.

Geographic distribution of the published studies on the genotoxic effects of effluents from sewage treatment plants identified in the present study.

In 2020, only 55 % of the population of Brazil had access to public sanitation, according to the Basic Sanitation Ranking [29]. Poor sanitation is known to be linked to the transmission of diseases such as cholera, dysentery, typhoid fever, and polio, as well as parasitic infections [30], which drives research around the world toward the understanding of the risks of inadequate sanitation and sewage treatment. The problem of inadequate sanitation is further exacerbated by the fact that SARS coronaviruses have been detected in wastewater during outbreaks in China, Europe, and the United States [31].

In China and India, the rapid growth of population and urbanization is exerting pressure on sewage treatment plant infrastructure to meet the demands of increasing population capacity [32]. This situation has raised concerns regarding water pollution, human health risks, and environmental degradation. China has made significant investments in research aimed at enhancing sewage treatment technology and infrastructure [33,34]. On the other hand, India is focusing on the reuse and recovery of water resources as a strategic approach [35].

In Germany, sewage treatment regulations are stringent, reflecting the country's commitment to maintaining a high standard of water quality [36]. Consequently, invests in sewage treatment technology to enhance the efficiency of substance and pollutant removal [37,38]. In Australia, there is already a strong emphasis on environmental protection, with research efforts directed towards sewage treatment to minimize environmental impact [39,40]. Additionally, there is a focus on advancing technologies for the treatment and reuse of compounds, furthering the goal of sustainable wastewater management [9,41].

1.3. Genotoxic biomarkers and applications

The papers analyzed in the present study focused on a total of 9 different categories of model organisms, including humans, and applied 16 different analytical techniques for the assessment of the genotoxic impacts of effluents and residues produced by sewage treatment plant (Table 1). A summary of the main analytical techniques and their main findings can be found in Table 2.

Table 1.

List of studies according to model organism and biomarkers of genotoxicity.

Model organism	Genotoxic Biomarkers
Bacteria	UmuC test	Ames test	SOS chromotest	Rec assay	p53-CALUX	Vitotox	Testing with GMB bacteria
Bacillus subtilis				1
Escherichia coli	3	1	6				1
Salmonella typhimurium	25	32	9		1	2
Plant	Chromosomal aberrations	Micronucleus test	Anaphase aberration assay	Nuclear abnormalities	Comet assay	Mitotic index	Nuclear buds	PCR	DNA double-strand break assay	DNA-unwinding test	CEGA	Gamma-H2AX	p53-CALUX
Allium cepa	22	15	1	6	2	1
Tradescantia pallida	1	8
Vicia faba		6
Zea mays	1			1
Fish
Astyanax bimaculatus lacustris		1		1	1
Astyanax jacuhiensis		1
Cichla temensis		1		1	1
Cyprinus carpio		2		1	1
Danio rerio		2			5			1
Geophagus brasiliensis		2		1	1
Gobiocypris rarus		1			1			1
Hoplias malabaricus		1		1	1
Labeo bata		1		1
Lepomis macrochirus					1
Leuciscus cephalus		1
Onchorynchus mykiss		4		1	2		1			1
Oreochromis mossambicus					1
Oreochromis niloticus		4		2	1
Platichthys stellatus		1
Steindachnerina insculpita		1		1	1
Thymallus thymallus					1
Tilapia rendalli		2
Umbra limi	1
Mammal
Chinese hamster	1	3			1				1
Neoromicia nana (Banana Bat)		1			1
Rat		2			4
Crustacean
Daphnia magna					2
Gammarus fossarum					1
Eurynia dilatata					1
Bivalve
Dreissena polymorpha					1
Mytilus galloprovincialis		1			1
Bird cells
Eggs		1
Chicken											1
Protozoan
Tetrahymena thermophila					1
Human
HepG2 cells		3			6
Human leukocytes					3
Human lymphocyte cultures		1
LS 174T cell line												1
Epithelial colon cancer cells					1
Human hepato-cellular liver carcinoma cells (C3a)		2			3
TK6 de human lymphoblasts		1
HepaRG cell lines		1			1
Cellular tumor antigen p53													1

Table 2.

Summary of the papers identified in the present study that report on the genotoxic damage caused by the effluents and residues produced by sewage treatment plants.

Model organism	Test/Assay	Type of sample	Type of experiment	Findings	Reference
Bacteria
Salmonella typhimurium	UmuC test	Cell culture	Experimental study	Ø	[42]
	Ames test	Cell culture	Experimental study	↑ mutagenicity in some samples	[43]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity in the sediments of raw night soil	[44]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity in raw nightsoil	[45]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity in 3 of the 6 samples analyzed	[46]
	Ames test	Cell culture	Experimental study	Ø	[47]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[48]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity	[49]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[50]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity in some samples	[51]
	Ames test	Cell culture	Experimental study	↑ genotoxicity	[52]
	Ames test	Cell culture	Experimental study	↑ genotoxicity at the study site	[53]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[54]
	Ames test	Cell culture	Experimental study	↑ mutagenicity in the untreated sludge samples	[55]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[56]
	Ames test	Cell culture	Experimental study	↑ genotoxicity	[57]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity	[58]
Escherichia coli and Salmonella typhimurium	SOS chromotest and Ames test	Cell culture	Experimental study	Ø ↑ genotoxicity	[59]
	SOS chromotest and Ames test	Cell culture	Experimental study	↑ genotoxicity at sites 4 and 5	[60]
Salmonella typhimurium	UmuC test	Cell culture	Experimental study	Ø	[61]
	Ames and UmuC tests	Cell culture	Experimental study	↑ genotoxicity and mutagenicity	[62]
	SOS chromotest	Cell culture	Experimental study	Genotoxicity removed by treatment	[63]
	Ames test	Cell culture	Experimental study	Genotoxicity removed by treatment	[64]
	SOS chromotest	Cell culture	Experimental study	↑ genotoxicity in some samples	[65]
	UmuC test	Cell culture	Experimental study	Genotoxicity removed by treatment	[66]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity Genotoxicity removed by treatment	[67]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[68]
	Ames test	Cell culture	Experimental study	Ø	[69]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity	[70]
	UmuC test	Cell culture	Experimental study	Genotoxicity removed by treatment	[71]
	Ames test	Cell culture	Experimental study	↑ genotoxicity	[72]
Escherichia coli	SOS chromotest	Cell culture	Experimental study	↑ genotoxicity. Genotoxicity removed by treatment	[73]
Salmonella typhimurium	SOS chromotest	Cell culture	Experimental study	Genotoxicity removed by treatment	[16]
	SOS chromotest	Cell culture	Experimental study	↑ genotoxicity in some samples	[74]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[75]
	SOS chromotest	Cell culture	Experimental study	↑ genotoxicity. Genotoxicity removed by treatment	[76]
	UmuC test	Cell culture	Experimental study	Ø	[41]
	UmuC and Ames tests	Cell culture	Experimental study	Genotoxicity removed by treatment, but not mutagenicity	[77]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity. Genotoxicity removed by treatment	[78]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity	[79]
	SOS chromotest	Cell culture	Experimental study	Genotoxicity removed by treatment	[79]
Escherichia coli and Salmonella typhimurium	SOS chromotest, and UmuC and Ames tests	Cell culture	Experimental study	The SOS and umuC assays provided consistent results and were responsive at lower, but the Ames test was more variable and even provided false-positive responses	[26]
Bacillus subtilis	Rec assay	Cell culture	Experimental study	↑ genotoxicity in some samples	[80]
Escherichia coli	SOS chromotest	Cell culture	Field study	↑ genotoxic	[81]
Salmonella typhimurium	UmuC and Ames tests	Cell cultue	Experimental study	Treatment reduced genotoxic effects	[82]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[83]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[84]
	UmuC test	Cell culture	Experimental study	↑ genotoxic	[85]
	UmuC test p53-CALUX®	Cell culture	Experimental study	↑ genotoxicity in some samples	[86]
	Vitotox	Cell culture	Experimental study	↑ genotoxicity	[87]
	Ames test	Cell culture	Experimental study	Ø	[88]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[89]
	Vitotox	Cell culture	Experimental study	↑ genotoxicity	[90]
	UmuC and Ames tests	Cell culture	Experimental study	↑ genotoxicity and mutagenicity in some samples	[91]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity in some samples	[8]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[92]
Escherichia coli	Testing with GMB bacteria	Cell culture	Experimental study	↑ genotoxicity	[93]
Salmonella typhimurium	UmuC test	Cell culture	Experimental study	↑ genotoxicity	[94]
	UmuC test	Cell culture	Experimental study	↑ genotoxicity. Treatment reduced genotoxic effects	[9]
	Ames test	Cell culture	Experimental study	↑ genotoxicity and mutagenicity	[35]
	Ames test	Cell culture	Experimental study	Ø	[38]
	Ames test	Cell culture	Experimental study	Ø	[6]
	Ames test	Cell culture	Experimental study	Ø	[95]
	Ames test	Cell culture	Experimental study	↑ mutagenicity	[96]
	Ames test	Cell culture	Experimental study	Ø	[97]
Escherichia coli	SOS chromotest	Cell culture	Experimental study	Ø	[98]
Plant
Allium cepa	Chromosomal aberrations	Root cells	Experimental study	↑ frequency of aberrant cells	[99]
	Chromosomal aberrations	Root cells	Experimental study	↑ frequency of aberrant cells	[47]
	Chromosomal aberrations	Root cells	Experimental study	↑ frequency of aberrant cells	[100]
Vicia faba	Micronucleus test	Root cells	Experimental study	↑ frequency of micronuclei	[3]
Tradescantia pallida	Micronucleus test	Inflorescences	Experimental study	↑ mutagenicity	[56]
Allium cepa	Anaphase aberration assay	Root cells	Experimental study	Ø	[56]
	Chromosomal aberrations	Root cells	Field study	Ø	[101]
Tradescantia pallida	Micronucleus test	Inflorescences	Experimental study	Ø	[12]
Zea mays	Chromosomal aberrations, nuclear abnormalities	Pollen mother cells	Experimental study	↑ chromosomal and nuclear abnormalities	[102]
Tradescantia pallida	Micronucleus test	Inflorescence	Experimental study	↑ frequency of micronuclei	[23]
Vicia faba	Micronucleus test	Root cells	Experimental study	↑ frequency of micronuclei	[103]
Allium cepa	Micronucleus test, chromosomal aberrations	Root cells	Experimental study	↑ micronuclei and chromosome breaks	[104]
	Chromosomal, nuclear abnormalities	Root cells	Experimental study	↑ total nuclear abnormalities	[105]
	Micronucleus test	Root cells	Experimental study	Ø	[87]
	Chromosomal aberrations, micronucleus test	Root cells	Experimental study	Ø	[106]
	Chromosomal aberrations	Root cells	Experimental study	↑ chromosomal aberrations in most samples	[107]
	Micronucleus test	Root cells	Experimental study	↑ frequency of micronuclei in 11 of 20 samples	[107]
Tradescantia pallida	Micronucleus test	Root cells	Experimental study	↑ frequency of micronuclei	[108]
Allium cepa	Chromosomal and nuclear abnormalities, micronucleus test	Root cells	Experimental study	↑ genotoxicity and mutagenicity	[109]
	Chromosomal aberrations	Root cells	Experimental study	↑ mutagenicity	[83]
Vicia faba	Micronucleus test	Root cells	Experimental study	Treatment responsible for reduced genotoxicity	[33]
	Micronucleus test	Root cells	Experimental study	↑ frequency of micronuclei	[110]
Allium cepa	Micronucleus test, chromosomal aberrations	Root cells	Experimental study	The solubilized sewage sludge was genotoxic; Solubilized sludge and solubilized biosolids induced an increase in the frequency of mitotic and chromosomal abnormalities ↑ genotoxicity and mutagenicity in the raw sludge	[111]
	Micronucleus test	Root cells	Experimental study	Ø	[84]
	Micronucleus test	Root cells	Experimental study	Ø	[112]
	Comet assay	Root cells	Experimental study	↑ genotoxicity, which was removed by treatment	[113]
	Chromosomal and nuclear abnormalities, micronucleus test	Root cells	Experimental study	↑ genotoxicity	[114]
	Micronucleus test	Root cells	Experimental study	Ø	[72]
	Chromosomal aberrations	Root cells	Experimental study	↑ chromosomal aberrations	[115]
	Chromosomal and nuclear abnormalities, micronucleus test	Root cells	Experimental study	↑ genotoxicity	[116]
Tradescantia pallida	Micronucleus test	Inflorescences	Experimental study	↑ frequency of micronuclei	[112]
	Micronucleus test	Stem inflorescences	Experimental study	↑ frequency of micronuclei	[117]
Allium cepa	Chromosomal and nuclear abnormalities, micronucleus test	Meristematic cells	Experimental study	↑ genotoxicity ↑ mutagenicity in some samples	[10]
	Comet assay	Root celss	Experimental study	↑ genotoxicity	[118]
Vicia faba	Micronucleus test	Root cells	Experimental study	↑ genotoxicity. Some treatments reduced the genotoxic effects	[2]
	Micronucleus test	Root cells	Experimental study	↑ genotoxicity, which was removed by treatment	[119]
Allium cepa and Tradescantia pallida	Micronucleus test Chromosomal aberrations	Pollen and root cells	Experimental study	Ø ↑ mutagenicity in some samples	[48]
Allium cepa	Chromosomal aberrations	Root cells	Experimental study	↑ genotoxicity. Treatment reduced genotoxic effects	[120]
	Chromosomal aberrations	Root cells	Experimental study	Ø	[95]
	Micronucleus test and chromosomal aberration	Root cells	Experimental study	↑ genotoxicity. Treatment reduced genotoxic effects	[121]
	Chromosomal and nuclear abnormalities, micronucleus test	Root cells	Experimental study	↑ genotoxicity and mutagenicity in some samples	[122]
	Chromosomal aberrations	Root cells	Experimental study	Ø	[123]
	Chromosomal aberrations	Root cells	Experimental study	↑ genotoxicity	[124]
Fish
Umbra limi	Chromosomal aberrations	Heart cell culture	Experimental study	↑ Increase frequency of chromosomal aberrations	[125]
Platichthys stellatus	Micronucleus test	Buccal epithelium, blood and liver cells	Experimental study	Ø	[125]
Tilapia rendalli, Oreochromis niloticus, and Cyprinus carpio	Micronucleus test	Peripheral blood (gills)	Field study	Ø	[126]
Onchorynchus mykiss	DNA-unwinding test	Liver	Experimental study	↑ genotoxicity	[51]
RTL-W1 cells of Onchorynchus mykiss	Comet assay			↑ genotoxicity in 3 of 8 reunited fractions	[55]
Oreochromis niloticus Tilapia rendalli	Micronucleus test	Peripheral erythrocytes	Field study	Ø	[101]
Thymallus thymallus	Comet assay	RTL-W1 cells	Experimental study	↑ genotoxicity	[57]
Labeo bata	Micronucleus test, nuclear abnormalities	Gills and kidney blood	Field study	↑ frequency of micronuclei, and necrotic, apoptotic, and binucleated cells	[127]
Geophagus brasiliensis Cichla temensis Hopliasma labaricus Astyanax bimaculatus lacustris Oreochromis niloticus Cyprinus carpio and Steindachnerina insculpita	Micronucleus test Comet assay Nuclear abnormality	Peripheral blood	Field study	↑ frequency of micronuclei in Cichla temensis and Hoplias malabaricus ↑ genotoxicity in Steindachnerina insculpita and Cichla temensis ↑ frequency of nuclear abnormalities in Oreochromis niloticus and Hoplias malaricus	[14]
Oncorhynchus mykiss	Micronucleus test, nuclear buds	Blood	Experimental study	Ø	[128]
Oncorhynchus mykiss larvae and juveniles	Micronucleus test, nuclear abnormality	Blood	Experimental study	↑ frequency of micronuclei	[129]
Danio rerio	Comet assay	Embryos	Experimental study	Ranking of biofilm genotoxicity: B1<B2 B6<B5<B4<B3Ranking of sediment genotoxicity: S2<S3 S1	[130]
	Comet assay, micronucleus test	Liver cell suspensions, peripheral erythrocytes	Experimental study	↑ genotoxicity. Wastewater treatment not effective in removing genotoxicity	[131]
	Micronucleus test Comet assay	Blood Liver	Experimental study	↑ frequency of micronuclei ↑ genotoxicity	[132]
	Comet assay	Liver	Experimental study	↑ genotoxicity	[112]
Lepomis macrochirus	Comet assay	Blood	Experimental study	↑ genotoxicity	[133]
Oncorhynchus mykiss	Micronucleus test	Cell culture	Experimental study	↑ frequency of micronuclei	[24]
	Comet assay	Blood	Experimental study	↑ genotoxicity after ozonation	[77]
Astyanax jacuhiensis	Micronucleus test	Peripheral blood	Experimental study	Ø ↑ genotoxicity	[106]
Leuciscus cephalus	Micronucleus test	Blood	Field study	↑ frequency of micronuclei	[81]
Geophagus brasiliensis	Micronucleus test	Blood	Field study	↑ frequency of micronuclei	[15]
Danio rerio	Comet assay	Embryos	Experimental study	↑ genotoxicity	[134]
Oreochromis niloticus	Micronucleus test and Nuclear abnormalities	Blood	Experimental study	↑ genotoxicity and mutagenicity in raw wastewater. Treatment reduced genotoxic and mutagenic effects	[135]
Oncorhynchus mykiss	Micronucleus test	Blood	Field study	↑ genotoxicity in some samples	[37]
Oreochromis mossambicus	Comet assay	Fish liver	Experimental study	↑ genotoxicity	[89]
Danio rerio	PCR of the genes involved in the biotransformation of xenobiotics	Total RNA - liver	Experimental study	↑ potential genotoxicity	[136]
Gobiocypris rarus	Comet assay, micronucleus test and PCR	Fish liver and blood	Experimental study	↑ potential DNA damage and frequency of micronuclei	[137]
Human cells
HepG2 cells	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[65]
	Comet assay, micronucleus test	Urinary extracts	Experimental study	↑ genotoxicity	[138]
Human leukocytes	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[75]
Human lymphocyte cultures	Micronucleus test	Cell culture	Experimental study	↑ frequency of micronucleus in some samples	[26]
Human leukocytes	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[84]
LS 174T cell line	Gamma-H2AX	Cell culture	Experimental study	Ø	[139]
Epithelial colon cancer cells	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[140]
HepG2 cells	Comet assay, micronucleus test	Cell culture	Experimental study	Ø	[141]
	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[142]
Human hepato-cellular liver carcinoma cells (C3a)	Comet assay, micronucleus test	Cell culture	Experimental study	↑ genotoxicity ↑ frequency of micronuclei	[87]
	Comet assay and micronucleus test	Cell culture	Experimental study	↑ genotoxicity ↑ frequency of micronuclei	[90]
TK6 de human lymphoblasts	Micronucleus test	Cell culture	Experimental study	↑ frequency of micronuclei in some samples	[143]
HepG2 and HepaRG cell lines	Comet assay, micronucleus test	Cell culture	Experimental study	↑ genotoxicity in some samples. Treatment reduced genotoxic effects ↑ frequency of micronuclei in some samples	[38]
Human leukocytes	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[95]
HepG2 cell	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[25]
Human hepato-cellular liver carcinoma cells (C3a)	Comet assay	Cell culture	Experimental study	↑ genotoxicity	[17]
Cellular tumor antigen p53	p53-CALUX	Cell culture	Experimental study	Ø	[97]
Mammal
Chinese hamster	Chromosomal aberrations	Ovary cells culture	Experimental study	↑ genotoxicity in some sampled sites	[125]
	Micronucleus test	Lung fibroblast cells	Experimental study	↑ frequency of micronuclei	[62]
Rat	Micronucleus test, comet assay	Bone marrow and peripheral blood	Experimental study	Ø	[144]
	Comet assay and Micronucleus test	Peripheral blood, liver and kidney cells, bone marrow	Experimental study	↑ genotoxicity at higher effluent concentrations	[145]
Neoromicia nana Banana Bat	Comet assay, micronucleus test	Peripheral blood	Field study	↑ genotoxicity Ø micronucleus frequency	[146]
Rat	Comet assay	Hepatocyte cells	Experimental study	↑ genotoxicity Genotoxicity was removed by treatments	[72]
Chinese hamster	Micronucleus test	Ovarian cell culture	Experimental study	↑ genotoxicity	[147]
	Comet assay, micronucleus test	Ovarian cell culture	Experimental study	↑ genotoxicity in some samples	[141]
	DNA double-strand break assay	Ovarian cell culture	Experimental study	↑ genotoxicity Genotoxicity removed by treatment	[34]
Rat	Comet assay	Fibroblasts	Experimental study	↑ genotoxicity	[6]
Crustacean
Gammarus fossarum	Comet assay	Hemocytes Spermatozoa Oocytes	Field study	Ø ↑ genotoxicity Ø	[148]
Daphnia magna	Comet assay	Homogeneized total	Experimental study	↑ genotoxicity Genotoxicity removed by treatment	[113]
	Comet assay	Homogeneized total	Experimental study	↑ genotoxicity Genotoxicity removed by treatment	[149]
Bivalve
Dreissena polymorpha	Comet assay	Hemolymph	Experimental study	↑ genotoxicity. Genotoxicity removed by treatment	[150]
Mytilus galloprovincialis	Micronucleus test, comet assay	Hemocytes	Field study	↓ genotoxic damage in the study area after 6 years	[151]
Eurynia dilatata	Comet assay	Hemolymph	Experimental study	↑ genotoxicity	[152]
Bird cells
Eggs	Micronucleus test	Erythrocytes	Experimental study	↑ frequency of micronuclei in non-processed samples	[6]
Chicken	CEGA	Embryo liver	Experimental study	↑ potential genotoxicity in non-processed samples	[6]
Protozoan
Tetrahymena thermophila	Comet assay	Cell culture	Experimental study	↑ genotoxicity Genotoxicity removed by treatment	[153]

A range of experimental procedures can be used to assess the genotoxicity of effluents (Fig. 5; Table 1). In the papers selected or analysis in the present study, the principal techniques applied for the assessment of genotoxicity were the micronucleus test (n = 54 studies; 25.82 % of the total), the comet assay (n = 39; 18.31 %), Ames test (n = 33; 15.49 %), umuC test and the observation of chromosomal aberrations (n = 25; 11.74 %). It is important to note here that many of the studies applied more than one procedure for the assessment of genotoxicity in relation to the effluents produced by STPs, and 213 different procedures were reported in the 134 studies.

Fig. 5.

The genotoxicity biomarkers mentioned for the assessment of sewage treatment plant effluents in the papers analyzed in the present study.

The micronucleus test is the most prominent of these procedures. Micronuclei are whole chromosomes or fragments of chromosomes that are excluded from the daughter nucleus following cell division [154]. Micronuclei are considered to be good indicators of chromosomal instability and DNA damage [155,156], and can be induced by a range of different environmental factors, such as pollution, exposure to xenobiotics, radiation, and diseases [157,158]. This test has many potential advantages, in particular the fact that it can be applied in many different types of organism, but also because it is fast, reliable, and low-cost. In the papers analyzed in the present study, this biomarker was applied to the analysis of samples of mussels, fish, rats, bats, humans, and plants (Table 1).

The comet assay was the second most frequent procedure applied in the analysis of genotoxicity in STPs and was often applied together with the micronucleus test. The two procedures are complementary, given that the micronucleus test identifies permanent DNA damage that is fixed as a mutation, while the comet assay detects recent damage that can be repaired by cellular mechanisms [159]. A number of advantages of the comet assay, including (i) the detection of damage at the level of the single cell; (ii) most types of eukaryotic cell are suitable for the procedure; (iii) only a small sample of cells is required; (iv) it is typically faster and more sensitive than the other methods available for the assessment of DNA strand breaks, and (v) the strand breaks form rapidly after exposure to genotoxic agents, which permits the early assessment of the response of the biota [159].

Many of the studies also used procedures such as the umuC and Ames test, which were applied in vitro in bacteria. As STPs are required to operate within the parameters established by the local legislation on water quality, these tests are widely used to determine efficiency of the treatment for the removal of genotoxic agents at each stage of the process until the release of the effluents into bodies of water [44,71]. These tests are also applied in studies of new sewage treatment technologies [8,9].

The umuC test detects the induction of umuC gene expression by substances that damage the DNA. This gene activates the bacterial SOS response to molecular DNA damage through the activation of repair mechanisms [160]. The umuC test is simple, fast, low-cost, sensitive, and easily replicated, and since its development, it has been used to detect the presence of genotoxic compounds in various types of material and environments. Japan, Germany, and Malaysia have adopted the umuC test as an official method for the analysis of drinking water, sewage, and wastewater [160]. Up to now, this is the only test that has satisfied the standards of the International Organization for Standardization for the analysis of the genotoxicity of water and wastewater (ISO/CD 13829), which accounts for its prominence in the papers analyzed in the present study.

By contrast, the Ames test is a procedure that identifies compounds with the potential to induce gene mutations, such as a shift in the reading frame or the substitution of base pairs [161,162]. It can be used to detect the mutagenic potential of chemicals, environmental substrates, body fluids, foods, drugs, and physical agents, and has been widely used to assess water quality, and it is also a simple, rapid, and low-cost test, which also offers the possibility of establishing a dose-response relationship [1].

The chromosomal aberration (CA) test is used to detect damage to the chromosomal DNA caused by mutagenic agents or during the repair of DNA damage [163]. The CA test has been widely applied in the biomonitoring of human genotoxicity, given that it is a biomarker of cancer risk [164], and can be used to assess the genotoxic potential of environments (in particular aquatic habitats) and plants [165]. All these methods permit the evaluation of the genotoxicity of effluents in a range of different model organisms and cells (Table 1).

The most-used model organisms (Fig. 6B) were bacteria (n = 70; 37.63 % of the total), plants (n = 43; 23.12 %), and fish (n = 36; 19.35 %). This distribution is reflected in the choice of genotoxic assays employed as shown in Table 1. To assess the effectiveness of the biomarkers, we specifically focused on these model organisms and evaluated the nature of their responses (Table 3), categorizing them as either positive or negative (Table 4).

Fig. 6.

The types of experiment (A) and model organisms (B) used in the genotoxicity studies of sewage treatment plants identified in the present study.

Table 3.

Efficiency of the applied biomarker in relation to the model organism.

	Bacteria	Fish	Plant	Human cells
Ames test	88,24 %
umuC test	91,67 %
Chromosome aberration		100,00 %	79,17 %
Comet assay		100,00 %	100,00 %	100,00 %
Micronuclei test		70,59 %	81,48 %	100,00 %

Table 4.

Model organism responses to the used biomarker.

	Effecta	Bacteria	Fish	Plant	Human cells
Ames test	Negative	4
	Positive	30
	Total	34
	Negative	2
umuC test	Positive	22
	Total	24
Chromosome aberrations	Negative		0	5
	Positive		1	19
	Total		1	24
Comet assay	Negative		0	0	0
	Positive		13	2	13
	Total		13	2	13
Micronuclei test	Negative		5	5	0
	Positive		12	22	7
	Total		17	27	7

^aEffect Negative – there was no response to the biomarker; Effect positive – there was response to the biomarker.

In the case of bacteria, the umuC and Ames tests emerged as the most frequently employed methods [35,58,69,94]. Notably, our observations indicate that the umuC test demonstrated greater efficiency compared to the Ames test. Typically, the Ames test is utilized for detecting point mutations, while the umuC test provides a more comprehensive assessment by detecting various forms of DNA damage, such as strand breakage and DNA adducts [166]. Therefore, the umuC test offers a broader scope for genotoxicity evaluation in bacterial systems.

For fish and plants, the micronucleus test, the comet test and the CA test were the main procedures [3,102,106,107,126,131] (Table 1). Among these, the comet test and CA test have proven to be more efficient in evaluating genotoxicity in fish. The micronucleus test, while primarily associated with point mutations and exhibiting a positive response only in the presence of mutations, is often employed in conjunction with other biomarkers that are more sensitive to damage and in fish may suffer from variability according to biological factors in the animal or the environment [158]. This complementary use reinforces the reliability and comprehensiveness of the obtained results. On the other hand, in plants, the comet assay has efficiency compared to other genotoxicity tests. In this organism, the comet assay has proven to be versatile and easy to apply and has been widely used to help understand the impact of genotoxic agents [167,168].

In human cells, both the comet assay and the micronucleus assay were efficient in tracking genotoxic impacts of sewage treatment plants. In human biomonitoring, these techniques are characterized by their ease of use, low cost, and applicability to various cell types in vitro. Moreover, they provide valuable results for assessing lifestyle factors and the risk of exposure to environmental pollutants [[169], [170], [171]].

It is interesting to note here that the testing of bacteria is related primarily to the monitoring of water quality and the assessment of effluent treatment, as are most of the studies of plants [56,84,122]. The studies that used fish as the model organism tended to focus on the environmental impact, in particular, the effects of the discharge of effluents into bodies of water, for which, the comet assay and micronucleus test are the preferred procedures [15,81,126]. These findings emphasize the importance of employing different model organisms and biomarkers in a study, to guarantee a more integrated and multi-taxon perspective that ensures the quality of the study, and the reliability of the results.

The understanding of the type of study is essential to the identification of potential knowledge gaps in the analysis of the genotoxicity of sewage effluents (Fig. 6A). The vast majority of studies analyzed here (n = 124 studies; 92.54 % of the total) on experimental study, involving the collection of effluent samples for laboratory experimentation. Relatively few of the studies examined here adopted a field study approach (n = 10; 7.46 %), in which samples were collected from organisms in their natural habitat (STP or watercourse), or organisms were exposed to a contaminated environment outside the laboratory, both under ambient conditions [81].

Experimental study in laboratory provide the potential for the application of controlled and standardized procedures, ensuring scientifically more reliable results, which is essential for the systematic analysis of the efficacy sewage treatment, the assessment of the compliance of a plant with the local legislation, and the evaluation of new technologies for the treatment of sewage. Despite these controls, laboratory studies may not provide a realistic perspective on the actual impacts in the field. By contrast, field studies are essential for the more reliable assessment of the impacts of sewage effluents, given the natural mixture of compounds and contaminants in the environment. These studies are important because they provide insights into the organism's biology and its capacity to adapt to environmental impacts, which are fundamental to effective environmental monitoring. In general, the most effective approach is to combine experimental and field study, to provide the most integrated perspective on the problem.

1.4. Physical-chemical parameters of the water

A total of 128 different physical parameters and chemical substances were analyzed in the studies reviewed here (Table 5), with the most frequently analyzed being the pH of the water (n = 34 studies; 5.17 % of the total of citations), followed by the chemical oxygen demand (COD) (n = 31; 4.71 %) and the measurement of the concentrations of heavy metal, such as Lead, Copper, Cadmium, Chromium, Nickel, Mercury, Arsenic, Silver, and Molybdenum (n = 29; 4.41 %). Other important parameters include ammonia (n = 24; 3.65 %), total phosphorus (n = 23; 3.50 %), dissolved organic carbon and electrical conductivity (both n = 20; 3.04 %), and zinc, total nitrogen, drugs, and the biochemical oxygen demand (BOD) (each n = 19; 2.89 %). To a lesser extent, some of the studies also quantified pesticides, including heptachlor, aldrin, DDT, toxaphene, mirex, atrazine, carbendazim, imidacloprid, tebuthiuron, metolachlor, ametryn, simazine, terbutryn, prometryn, diuron, and fluometuron.

Table 5.

Physical-chemical parameters analyzed in the studies of genotoxicity in sewage treatment plant effluents identified in the present review.

Parameter	Frequencya
pH	34 (5.17 %)
Heavy metals	29 (4.41 %)
Chemical Oxygen Demand (COD)	31 (4.71 %)
Ammonia	24 (3.65 %)
Total Phosphorus	23 (3.50 %)
Dissolved Organic Carbon (DOC), Electrical conductivity	20 (3.04 %)
Zinc (Zn), Biochemical Oxygen Demand (BOD), Total Nitrogen (N), Drugs	19 (2.89 %)
Total Suspended Solids (TSS), Nitrate	18 (2.74 %)
Nitrite	16 (2.43 %)
Temperature, Dissolved Oxygen (%)	14 (2.13 %)
PAHs	13 (1.98 %)
Iron (Fe)	12 (1.82 %)
Magnesium (Mg), Potassium (K), Calcium (Ca)	11 (1.67 %)
Pesticides, Phosphate, Total Organic Carbon (TOC)	10 (1.52 %)
Total Dissolved Solids (TDS), Sodium (Na)	9 (1.37 %)
Chloride	8 (1.22 %)
Fecal coliforms (E. coli), Aluminum (Al), Manganese (Mn)	7 (1.06 %)
Barium (Ba), Selenium (Se), Color	6 (0.91 %)
17β-estradiol (E2), Cobalt (Co), Nonylphenol (NP), Turbidity (UNT), Polychlorinated Biphenyls (PCBs), Total alkalinity	5 (0.76 %)
Sulfur (S), Estriol (E3), Estrone (E1), Semivolatile organic compounds	4 (0.61 %)
Bisphenol A (BPA), Salinity, Redox potential (Eh), Sulphate, Bromide, Carbonate hardness, Alkyl-phenols, Total hardness	3 (0.46 %)
Acidity, Adsorbable Organic Halogens (AOX), Ortho-phosphate, Free chlorine, Hexazinone, Organic Matter, Oxygen saturation, Methyl Triclosan (mTCS), Total chlorine, Volatile Organic Compounds (VOCs), 17α-estradiol, Acids and esters, Androgen androstenedione, Chlorophylla, Testosterone, 4-octylphenol, Octylphenol (OP), Polybrominated Diphenyl Ethers (PBDEs), Naphthalene- and benzene-sulfonates, Polychlorinated dibenzo-p-dioxins and -furans	2 (0.30 %)
Oil products, 17α-Ethinylestradiol (EE2), UV254, 17β-trenbolone, Alkylbenzensulphonates (LASs), Ammonium molybdate, Iodine (I), Trichloroacetic Acid (TCAA), Androsterone, Ash, Base saturation percentage, BPK-5, Brix degree, 5α-dihydrotestosterone, Bromodichloromethane (CHCl2Br), Bromoform (CHBr3), C/N ratio, Cation Exchange Capacity (CEC), Chloroform (CHCl3), Clophosphamide, Di-(NP2EO) ethoxylate, Dibromochloromethane (CHClBr2), Dibromoroacetic Acid (DBAA), Dichloroacetic Acid (DCAA), Di-ethylhexylphthalate (DEHP), Diethylstilbestrol (DES), Dioxins and furans, Dissolved substances, Etiocholanolone, Free CO2, KPKCr (MgO2 L−1), KPKMn (MgO2 L−1), Moisture, Mono-(NP1EO) ethoxylated, Monobromoacetic Acid (MBAA), Monochloroacetic Acid (MCAA), N-(1-naphthyl) ethylene diamine dihydrochloride, Nonylphenolethoxylates (NPE), Oxygen content, PBTA-type mutagens, Platinum (Pt), Polyethoxylated non-ionic surfactants, p-t-octylphenol, Resistivity (ORP), Salicylic Acid, Specific gravity, Sterol, Sulfamethoxazol, Surface-active substances, Surfactant, Testosterone propionate, Total gadolinium, Total sulphates, UV absorbance, Total cyanide, Corrosion inhibitors, Food additives	1 (0.15 %)

^aData showed in absolute and relative frequency. Number of citations of each parameter/total number of.

Another major concern reported in some studies was the problem of emerging contaminants and unregulated manmade substances, which often persist in the environment and are capable of disturbing physiological parameters [172]. These substances include drugs, including 17α-ethinyl estradiol, triclosan, triclocarban, methyl triclosan, Naproxen, Ketoprofen, Ibuprofen, Diclofenac, and Carbamazepine, as well as chemical compounds, such as dioxins, furans, PAHs, and bisphenol A.

While the legislation varies considerably among different countries, there is a general consensus on the use of physical, chemical, and biological parameters to measure water quality and effluent loads [173]. The quality of the water in sewage effluents is only tested rigorously in developed countries [174], which reflects a fundamental problem in developing countries. The findings of the present study indicate that the parameters investigated most frequently include the contamination of the water by metals, the amount of organic matter, the corrosive potential of the water, and the velocity of chemical reactions.

cited parameters (658).

The effective analysis of the effluents produced by STPs and their potential for the contamination of the environment depends on the adequate measurement of these parameters [14,15]. As the pollution of water involves the mixing of compounds that can modify the toxicity of substances by synergism or antagonism [24], understanding both the individual action and the interaction of these compounds is fundamental to the development of more effective research on the genotoxic potential of these effluents [175]. highlight the need to use multiple biomarkers of genotoxicity in conjunction with physiological and biochemical variables to guarantee the most complete possible perspective on this type of environmental pollution.

Most of the studies presented here detected potential genotoxic agents in the effluents produced by the STPs or in the bodies of water into which these effluents are discharged [3,25,47,83,127,149]. This reflects the response of different organisms to the genotoxic potential of these environments. In the three groups of organisms featured in the largest number of studies, significant genotoxic responses were recorded in more than 80 % of the papers, reaching 89.65 % in bacteria, 81.13 % in plants, 83.87 % in fish, and 100 % human cells. This emphasizes the importance of this type of research for the monitoring and eventual correction of the production and release of genotoxic agents by STPs. The absence of a significant genotoxic response is almost nil among the studies.

2. Conclusions and future perspectives

The results of the present study show that the genotoxicity of the effluents of sewage treatment plants has been the focus of scientific research for more than 30 years. Most of these studies have evaluated the effectiveness of treatments and the destination of the residues, such as the sludge. In the specific case of the sludge, one of the primary products of the treatment of sewage, caution is required for the disposal of this residue in agricultural soils or urban landscaping to avoid potential contamination and environmental damage [10,13,94,116]. The discharge of effluents into water courses constitutes a more complex scenario, and even when the legislation on the permitted concentrations of contaminants is respected, many studies have revealed negative impacts, in particular on fish [81,101,126,127].

A range of different biomarkers are used to assess the genotoxic potential of effluents produced by sewage treatment plants. The effectiveness of these biomarkers, when considering the most utilized model organisms, is notably high, underscoring their efficiency in research investigations. There is a clear correlation between the biomarker type, the model organism, and the specific objectives of the study. While a large number of different organisms have been studied, many studies tend to concentrate on a single species, with a focus on a few groups such as bacteria, fish, and plants. Consequently, it is important to encourage studies that encompass multiple species, particularly organisms from diverse taxonomic groups.

Field studies with vertebrates and invertebrates are still scarce. Some groups of animals, such as birds and bats, may represent especially valuable research models, given their dispersal capacity and exploitation of a wide range of prey, as well as the use of different water sources, which may include sewage treatment tanks. Insects that take advantage of the treatment process or exploit the areas of effluent discharge may also be important indicators of environmental quality. In addition to free-ranging animals, both vertebrates and invertebrates, studies of humans, in particular workers that may be exposed to genotoxic substances, should be encouraged and supported. These studies will be essential for the better understanding of the environmental risks associated with sewage treatment plants, and the development of management practices that ensure environmental sustainability over the long term.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article. Data included in article and supplementary material.

Funding

Brazilian National Council for Scientific and Technological Development (CNPq), Brazil, 161325/2021-1.

CRediT authorship contribution statement

Renata Maria Pereira de Freitas: Writing – review & editing, Writing – original draft, Methodology, Investigation. Marcelino Benvindo-Souza: Writing – original draft, Methodology, Investigation. Thiago Bernardi Vieira: Validation, Methodology, Formal analysis. Klebber Teodomiro Martins Formiga: Visualization, Validation, Supervision, Methodology, Conceptualization. Daniela de Melo e Silva: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflicts of interest related to this study.