Using freshwater snail Biomphalaria glabrata (Say, 1818) as a biological model for ecotoxicology studies: a systematic review

Over time, a growing increase in human pollutants in the aquatic environment has been observed. The global presence of residues in water bodies reinforces the need to develop improved methods to detect them and evaluate their ecotoxicological effects in aquatic environments. Thus, this study aimed to present the main assays using Biomphalaria glabrata as a biological model for ecotoxicological studies. We performed a systematic literature review with data published up to June 2022 on the Web of Science, SCOPUS, Science Direct, PubMed, and SciELO databases. Thirty studies were selected for this review after screening. Biomphalaria glabrata has been studied as an ecotoxicological model for different substances through toxicity, embryotoxicity, cytotoxicity, genotoxicity, and bioaccumulation assays. Studies evaluating the impact of B. glabrata exposure to several substances have reported effects on their offspring, as well as toxicity and behavioral and reproductive effects. This review presents various assays using B. glabrata as a biological model for ecotoxicological studies. The use of a representative species of ecosystems from tropical regions is a necessary tool for tropical environmental monitoring. It was observed that the freshwater snail B. glabrata was effective for the evaluation of the ecotoxicity of several types of chemical substances, but further studies are needed to standardize the model.


Introduction
Recently, many human micro pollutants have been detected in aquatic environment (Reid et al. 2019), often due to inefficient removal during conventional wastewater treatment (Golovko et al. 2021). Among many anthropogenic substances, pharmaceutical waste is a clear example of the human contamination of the environment (Wood et al. 2015;Ben et al. 2020;Wilkinson et al. 2022). The presence of pharmaceutical, industrial, textile, and mining residues, among many others, in the aquatic environment, reinforces the need to develop improved methods to detect and evaluate the ecotoxicological effects of substances in the aquatic environment (Tkaczyk et al. 2020).
It is known that only the detection of residues in the environment does not necessarily mean that they are bioavailable, nor that they will have harmful effects on systems. The use of biological models in ecotoxicological studies directly contributes to assessing the risk of the presence of these residues in aquatic environments, since chemical monitoring is increasingly less informative about ecological effects .
In general, ecotoxicological tests are efficient, fast, and low cost; being considered an important tool to assess the harmful effects of human activities in aquatic environments, these tests can be defined as test systems that expose certain substances to organisms or cells, and subsequently, assess biological effects at different levels of biological organization (Schuijt et al. 2021). Thus, the use of bioassays to assess Responsible Editor: Philippe Garrigues * Gabriel Souza-Silva silva_gs@yahoo.com water quality is essential to obtain an estimate of the effects of residues in aquatic environments that may pose a risk to life (Wilkinson et al. 2022).
One of the advantages of these tests is the ability to simulate controlled conditions in the laboratory, contributing to the accuracy, analysis, and interpretation of results, as there is a growing concern regarding ecotoxicological monitoring (Campana and Wlodkowic 2018;Altenburger et al. 2019), The implementation of new biological models for ecotoxicological studies, such as freshwater snail Biomphalaria glabrata, directly contributes to the approximation of laboratory simulation to environment on a global scale.
Classified as a benthic macroinvertebrate, B. glabrata has a limited migration pattern in the ecosystem, being suitable for evaluating specific ecotoxicological impacts in the tropical region. The early stages of the life cycle respond quickly to stress, and the adult stages are more resistant to some substances (such as iron nanoparticles) (Caixeta et al. 2021), which allows us to interpret the cumulative effects of pollutants, making them good indicators of local ecosystem conditions (Barbour et al. 1999;Tallarico 2016).
Bimphalaria glabrata is a species of freshwater herbivorous snail belonging to the Planorbidae family. When adults, they usually have a shell with a maximum diameter of 40 mm and 11 mm in width, wound in a left-handed spiral. In 2001, the American Society of Parasitologists presented a proposal for sequencing its genome, but only in 2017 its genome was characterized (Adema et al. 2017). This characterization provided important information about the biology of the freshwater snail and allowed us to better understand their immune systems (Castillo et al. 2020). B. glabrata has been detected in North, Central, and South America, with high prevalence throughout Brazil. It is also present in Southern Africa and some countries in Asia (GBIF.org 2022).
This freshwater snail has an innate defense response similar to vertebrates, being able to recognize and destroy different pathogens (Abaza et al. 2016). Due to the growing scientific knowledge of its immune system, this snail has been targeted by ecotoxicity research (Lima et al. 2018(Lima et al. , 2019Siqueira et al. 2020Siqueira et al. , 2021Caixeta et al. 2021). Its internal defense system is mainly composed of immune cells (hemocytes) and soluble factors in the hemolymph (Monte et al. 2019). Vasquez and Sullivan (2001) presented the importance of hemolymph in the snail's immunological process, suggesting that soluble elements of its hemolymph, such as lectins, play a direct role in the recognition and immunefighting mechanism (Vasquez and Sullivan 2001).
Hemocytes, cells produced in the hematopoietic tissues, are found in the snail's hemolymph and their main function is the internal defense (Abaza et al. 2016). Its morphology and enzyme content are variable, being classified into two types: hyalinocytes and granulocytes (Cavalcanti et al. 2012). Considering the vast knowledge about the immune system and its embryonic development, snails have been used as biological models to evaluate the ecotoxicity of several substances, including emerging pollutants, showing great potential .
Among the numerous characteristics that favor the use of B. glabrata as a biological model, the sensitivity to environmental changes stands out, allowing the evaluation of cellular, tissue, and behavioral responses, unlike other biological models such as cyanobacteria, microalgae, and some microcrustaceans. This is one of the few biological models that allows the ecotoxicological study of water-insoluble residues, because through its scraping characteristic, it absorbs substances present at the bottom of the water (Pena et al. 2022;Wang et al. 2022;Caixeta et al. 2021;Lima et al. 2019;Oliveira-Filho et al. 2019).
In addition, B. glabrata has been used as suitable model system to evaluate the mechanism of action of several chemical compounds (such as nanomaterials, drugs, heavy metals, pesticides, and wastewater) and have been shown to be important in ecotoxicological analyses due to the possibility of exploring different biomarkers, such as embryo, adult snail, immune cells, and tissues (such as cardiac, nervous, reproductive) in different environmental compartments to assess the toxicity of traditional and emerging pollutants . Considering the potential of the snail as a biological model, the objective of this study was to present the main tests performed using B. glabrata as a biological model for ecotoxicological assessments.

Search strategy
A systematic literature review was performed using the Web of Science, SCOPUS, ScienceDirect, PubMed, and SciELO databases. Keywords "Biomphalaria glabrata," "ecotoxicology," "toxicity," "genotoxicity," "embryotoxicity," and "cytotoxicity" were combined for composing the search descriptor and used to search interest publications in the databases, until June 2022. The systematic review followed the guidelines recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Moher et al. 2009).

Eligibility criteria
The inclusion criteria for the papers were (i) using B. glabrata as a biological model; (ii) presenting data on possible ecotoxicological effects (toxicity, embryotoxicity, or cytotoxicity); (iii) studies performed in vivo or in vitro; (iii) having at least one control group; (iv) exposure to organic or inorganic substance, and (v) English language. The exclusion criteria were (i) scientific review papers; (ii) abstracts in scientific events; (iii) technical reports; (iv) parasitological studies; (v) another species of snail; (vi) molluscicidal activity; and (vii) duplicate papers. Any discrepancies between the decisions, whether inclusion or exclusion of articles, were resolved by a consensus among the reviewers.
Rayyan software was used to assist in the article screening process (Ouzzani et al. 2016). All results found in the databases (Web of Science, SCOPUS, ScienceDirect, Pub-Med, and SciELO) were converted into comma-separated values (CSV) format and transferred to Rayyan software. After the transfer, the duplicates were removed one by one. For the first stage of the screening process, the keywords: (i) Biomphalaria glabrata; (ii) ecotoxicity; (iii) mollusk; (iv) emerging pollutants; and (v) toxicity were selected as criteria for inclusion of articles, while the keywords (i) review; (ii) parasitology infection; (iii) molluscicides; and (iv) molluscidal activity were selected as criteria for exclusion of articles.
During the first stage, all the titles and abstracts of the articles found were read and those with the inclusion words were selected, while those with the exclusion words were discarded. The second stage of the screening process consisted of the complete reading of the articles selected in the first stage. At this stage, articles that used the mollusk B. glabrata as a biological model for ecotoxicity studies were selected for the composition of this systematic review. ƛ

Data extraction
Before data extraction, a list of information to be extracted from each work included in this review was determined among the authors. The information extracted from each study were (i) year of publication; (ii) main assays; (iii) sample analyzed; (iv) sample size; (v) matrix analyzed; (vi) matrix concentrations; (vii) exposure time; (viii) mortality; (ix) lethal concentration; (x) main results; (xi) study place; and (xi) type of study (in vivo and/or in vitro).

Study type classification
Ecotoxicological studies were classified as: studies that showed the interaction between the mollusk and the test substance, evaluating its toxicity, under different endpoints (mortality, cytotoxicity, embryotoxicity, and bioaccumulation). For this, some criteria should be met, such as (i) assessing the toxicity of a given substance of environmental risk (such as persistent organic pollutants and metals); (ii) use of concentration ranges compatible with ecotoxicity tests (such as 10 g/L); (iii) studies previously self-classified as ecotoxicological (followed by a review of the classification by the authors of this work); (iv) having as objective of the work the evaluation of the aquatic environmental toxicity of a determined substance; and (v) preferably present EC50% and/or LC50% data (Martinez-Haro et al. 2015;Kase et al. 2016;Stiernström et al. 2016;Rudén et al. 2017;Kienzler et al. 2017;Moermond et al. 2017;Sales et al. 2022). Studies whose objectives were (i) evaluating the molluscidal activity (such as pesticides), (ii) the standardization of testing (such as comet assay), and/or (iii) studies that used the mollusk (B. glabrata) infected by a parasite (such as Schistosoma mansoni) were not classified as ecotoxicological studies.

Article search
A total of 1117 studies were found in the first screening stage, and 730 studies remained after excluding duplicates. Then, all titles and abstracts were read to find ecotoxicological studies using B. glabrata as a biological model. At the stage of reading the titles and and abstracts, articles that presented information on molluscidal activity, parasitological infection, and non-exposure to organic or inorganic substance or review studies, were excluded. After this second stage, 57 studies remained for full-text reading of the text. All the inclusion requirements presented had to be fulfilled to be selected. During the complete reading of the articles, those that did not use the freshwater snail B. glabrata as a biological model for ecotoxicological studies were excluded; review studies were also excluded. Finally, 30 studies published between 1973 and 2022 (Estevam et al. 2006;Baynes et al. 2019a, b;Oliveira-Filho and Paumgartten 2000;Bellavere and Gorbi 1981;Lima et al. 2018;Lima et al. 2019;Oliveira-Filho et al. 2019;Oliveira-Filho et al. 2016;Douglas and Sullivan 1992;Mona et al. 2013;Siqueira et al. 2020;Siqueira et al. 2021;Sullivan and Castro 2005;Santos et al. 2018;Salice and Roesijadi 2002;Ansaldo et al. 2009;de Albuquerque et al. 2014;Garcia et al. 2021;Tallarico et al. 2014;Nakano et al. 2003;Caixeta et al. 2021;Cheng and Sullivan 1973;Cheng and Sullivan 1977;Gustafson et al. 2015;Kaur et al. 2016;Münzinger and Guarducci 1988;Rivero-Wendt et al. 2014;Sullivan and Cheng 1876;Pena et al. 2022;Wang et al. 2022) were eligible for this systematic review (Fig. 1).

Progresses of the freshwater snail in ecotoxicological studies
Figure 2 is a brief history of the use of the B. glabrata as a biological model. It has been used as a biological model since 1973, where it was used to evaluate the ecotoxicological effects of chemical substances at different concentrations (Cheng and Sullivan 1973). This work showed that copper sulfate was able to increase the heart rate of snail even at low concentrations (2.5 mg/L). However, only concentrations of 875 mg/L and 2500 mg/L were lethal for 50% and 100% of snails, respectively (Cheng and Sullivan 1973). In addition, it was possible to observe that copper sulfate causes a reduction in hemolymph osmolarity (Cheng and Sullivan 1977).
In 1988, B. glabrata was used to assess the ecotoxicity of a chemical substance (zinc chloride) at different stages of its life, both adult and embryos (Münzinger and Guarducci 1988). In 1992, an in vitro study was carried out using B. glabrata hemocytes. In this study, the snail immune cells were exposed for 15 min to ammonium chloride, and the rate of phagocytosis was evaluated at  (Douglas and Sullivan 1992).
In 2002, different strains of B. glabrata were used to evaluate the ecotoxicity of cadmium on different stages of the snail's life, like embryo, newborns, and adults (Salice and Roesijadi 2002). As of 2005, different snail's systems (nervous, reproductive, and immune) began to be used for ecotoxicological assessments (Sullivan and Castro 2005;Mona et al. 2013), however, only in recent years, after the sequencing of its genome (Adema et al. 2017), an increasing number of ecotoxicity studies using it as a biological model were observed.
In general, these studies assess potential emerging pollutants in the environment, such as nanomaterials (Caixeta et al. 2021;Oliveira-Filho et al. 2016;Lima et al. 2019), domestic and industrial sewage sludge (Siqueira et al. 2020(Siqueira et al. , 2021, herbicides (Lima et al. 2018), textile dye (Garcia et al. 2021), and pharmaceutical products (Baynes et al. 2019a, b;Kaur et al. 2016;Melo et al. 2019). In addition, a wide variety of possibilities for cytotoxic, genotoxic, histotoxic, and embryotoxic assays have been developed, but without a standardization among them. The main advantages and disadvantages of using B. glabrata as a biological model for ecotoxicological studies are presented in Table 1. Table 2 shows the studies selected to compose this systematic review, with the principal results obtained after exposure of B. glabrata to different substances. The main sampled biomarkers used in the studies included were (i) snails (toxicity and bioaccumulation), (ii) embryos (embryotoxicity), and (iii) hemocytes or other cells (cytotoxicity). In the studies, toxicity was evaluated through exposure to different concentrations of the exogenous substance (pollutant), for a period, with mortality as the main outcome. The classification of bioaccumulation of a study can be defined as the verification of the increase in the concentration of a substance, present in the environment, in the tissues of the snail through its absorption. Embryotoxicity tests, on the other hand, were classified as tests to be used in embryos to evaluate exposure in embryonic development, after a specific substance. Finally, cytotoxicity assays are those that evaluated the impact of exposing B. glabrata hemocytes or other tissues to a specific chemical substance, mainly observing morphological and functional changes on the cells.

Location of study
Studies using the freshwater snail B. glabrata as biological model for ecotoxicological studies were carried out in six countries. Among them, Brazil (n = 16; 53.4%) stands out with the highest number of studies, followed by the USA (n = 7; 23.4%), the UK (n = 3; 10.0%), Italy (n = 2; 6.6%), Egypt, and Argentina (n = 1; 3.3%, each). The high number of studies carried out in Brazil can be explained by the high distribution of B. glabrata throughout the country. Unlike Lymnaea stagnalis, which is widely distributed in temperate zones and requires lower temperatures for the development of ecotoxicological tests, B. glabrata is widely distributed in tropic and subtropical zones and does not require lower temperatures for ecotoxicological tests, demonstrating with greater fidelity the possible impacts from the presence of aquatic contaminants in local ecosystems. Furthermore, it has several biomarkers (embryos, tissues, cells, enzymes, and DNA) that allow monitoring of the water courses quality affected by the presence of pollutants (Barbosa and Barbosa 1994;Southerland and Stribling 1995). In addition, this specific snail has great relevance to public health, as it is an intermediate host of the parasite that causes schistosomiasis; and consequently, a considerable number of studies with the objective of developing new molluscicides have been done, as well as their use as an of biological model in ecotoxicology (Tallarico 2016).

Snail size
The average size of the snails used in the included studies was 12 ± 2 mm. The selection of snail size must be exclusive to B. glabrata. This exclusivity must be considered at the time of the experiment because the adult size differs between the standardized model of Lymnaea stagnalis (Linnaeus, 1758) (OECD 2016) and Biomphalaria glabrata (Say 1818). For this reason, the use of 12 ± 2-mm snails is recommended for ecotoxicological studies. However, the choice of size must also consider the objective of the study. In studies with the goal of studying ecotoxicological effects on young snails or newborns, the size of the shell is smaller (Salice and Roesijadi 2002;Pena et al. 2022).
The standardization of snail size is essential to ensure the reproducibility of the experiments. For genotoxicity and/or cytotoxicity assays involving B. glabrata hemocytes (Douglas and Sullivan 1992; Sullivan and Castro 2005;Mona et al. 2013;Rivero-Wendt et al. 2014;Kaur et al. 2016;Siqueira et al. 2020Siqueira et al. , 2021, the size of the snail directly influences the amount of hemocytes present in the hemolymph (Gérard and Théron 1997). However, some studies did not report the size of the snail used in the experiments (Rivero-Wendt et al. 2014;Kaur et al. 2016), reinforcing the lack  Tallarico et al. 2014;Nakano et al. 2003;Caixeta et al. 2021;Cheng and Sullivan 1973;Cheng and Sullivan 1977;Gustafson et al. 2015;Kaur et al. 2016;Münzinger and Guarducci 1988   Although in B. glabrata, the shell size of the snail can be easily measured using a caliper, its correlation with the age of the snail is very complex and variable. Usually, snails with a shell between 3 and 4 mm can be classified as juveniles, while those with a shell between 11.5 and 13.5 mm are adults (Gérard and Théron 1997). However, some factors such as water, feeding, and calcium supplementation can directly influence the development and consequently its size of the snail. Thus, the correlation between size and age is not simple; and for its standardization in ecotoxicological studies, it is not enough to consider only the size, but also the age of the snail as two variables, even if there is a correlation between them (Barbosa et al. 1992).
In the studies included in this systematic review (Douglas and Sullivan 1992;Sullivan and Castro 2005;Mona et al. 2013;Siqueira et al. 2020;Siqueira et al. 2021), it can be observed that the size of the snail used in cytotoxicity or genotoxicity assays ranged from 8 (Mona et al. 2013) to 19 mm (Douglas and Sullivan 1992). This variation in size makes it difficult to compare results between articles, since in addition to the influence of size on the concentration of hemocytes in the hemolymph B. glabrata, there is an influence on the age of the cells, cell activity, and hemocyte response to the test substance (Gérard and Théron 1997).

Maintenance and creation
Biomphalaria glabrata are generally kept and bred in plastic aquariums with a volume of approximately 100 ml per snail. This freshwater snail creation temperature ranged from 20 to 30 °C, and most studies recorded an average water temperature of 25 °C (46.7%). The photoperiod was poorly informed; only 40.0% (n = 12) of the studies reported it, with seven (58.4%) using the photoperiod of 12/12 h (light/dark), four (33.3%) studies 16/8 h (light/dark), and one (3.3%) study 0:24 h (light/dark). In most studies, the water used to raise snails is filtered, with pH 7 and chlorine-free. However, some variations in the maintenance of the snails could be observed, such as the variation of the pH of the water, reaching up to 8, and the type of water, such as filtered water without chlorine (n = 17; 56.6%), purified with Nolan-Carrik saline solution (n = 3; 10.0%), and natural (n = 1; 3.3%). In addition, several studies did not present information regarding the maintenance and creation of snails (Fig. 3).
Some parameters related to water quality can be analyzed during maintenance and creation of snails. These parameters are hardness, alkalinity, oxygen saturation, conductivity, ammonia, nitrate, nitrite, and dissolved oxygen in the water. Monitoring of these parameters, especially water hardness, must be carried out to ensure good results during ecotoxicity tests. Water hardness is directly linked to the ability of snails to reproduce and develop in the aquarium. In addition, water with high hardness values can cause a reduction in the toxicity of some test substances, such as metals .
Lettuce leaves (Lactuca sativa) are the main source of food used to maintain B. glabrata. However, some differences related to the feeding of the snails were found, such as feeding frequency, food supplementation, and amount of food available. Some studies provided food daily without deprivation (n = 8; 26.7%), while others had some dietary restrictions such as 1 cm 2 of lettuce per snail per day (n = 3; .0%), feeding on alternate days (n = 2; 6.6%), every 2 days (n = 1; 3.3%), or three times a week (n = 1; 3.3%). In some cases, fish food (TetraMin) was used as a supplement (n = 4; 13.2%) or as the main food (n = 2; 6.6%).

Experimental design
The data reviewed showed a lack of standardization in the experimental design and protocols while using the snail B. glabrata in ecotoxicological assays. The main variables that showed the lack of standardization were (i) exposure time; (ii) number of replicates; (iii) sample size; and (iv) number of concentrations.

Exposure time
Exposure time is a factor that, in many works, is divided into two segments, acute and chronic exposure. Acute toxicity derives from the short-term exposure of individuals to a substance in a short period of time, between 24 and 96 h. Chronic toxicity is crucial to complement the acute toxicity test and obtain adequate information about the studied agents. In general, chronic toxicity tests are performed for 7 to 21 days (Souza et al. 2022). A total of 23 (76.6%) studies evaluated the toxicity of a given substance on B. glabrata. Among them, 13 (56.2%) studies only evaluated acute toxicity, 7 (30.4%) chronic toxicity, and 4 (13.4%) both toxicities, on the snail. The results showed that the exposure time used to assess acute toxicity was mainly 24 h (n = 8; 34.8%), followed by 48 h (n = 6; 26.1%), 96 h (n = 4; 17.4%), 72 h (n = 1; 4.3%), and 144 h (n = 1; 4.3%). For chronic exposure, 28 days were used more frequently (n = 4; 17.3%), followed by 15 days (n = 3; 13.0%), 30 days (n = 2; 8.6%), 14, and 21 days (n = 1; 4.3%, each). The main factors that directly influence the exposure time of the studies, whether acute or chronic, were the concentrations used and the properties of the test substance. In most studies that evaluated acute toxicity, snails' exposure for 24 h was sufficient to observe changes, such as behavioral changes, mortality, and infertility. In chronic exposure, the use of a 28-day exposure period, can be explained through the OECD guideline No. 243, elaborated with the objective of evaluating the effects of prolonged exposure to chemicals on the reproduction and survival of the freshwater snail Lymnaea stagnalis, for 28 days (OECD 2016). Furthermore, by extracting data from the articles included, it was observed that all studies that evaluated chronic toxicity after 28 days of exposure also performed embryotoxicity tests, corroborating the hypothesis.
Eighteen (60.0%) studies performed assays for embryotoxicological assessments. Regarding embryo exposure time, the results showed that the main exposure time was 24 h (n = 8; 44.4%), followed by > 192 h (n = 7; 38.9%), 48 and 96 h (n = 3; 16.7%, each), 144 and 192 h (n = 2; 11.1%; each), 72, 120, and 168 (n = 1; 5.5%, each). Only one article (1/18) did not report the exposure time used for the test (Kaur et al. 2016). B. glabrata has an average duration of its embryonic period of 8 days (Camey and Verdonk 1970;Kawano et al. 1992;Kawazoe 1976;Tallarico et al. 2014). Thus, the exposure time of the snail in periods longer than 192 h is sufficient to analyze all stages of embryonic development in terms of hatching rate. However, some factors, such as temperature, can influence the development time (Kawazoe 1976). Considering the embryology described in the literature (Camey and Verdonk 1970;Kawano et al. 1992), the blastula embryonic stage (15 h post fertilization) is most frequently used for embryotoxicity studies with B. glabrata. In addition, it should be considered that depending on the embryonic stage, there is a difference in sensitivity to a particular pollutant (Garcia et al. 2021).
Among the 30 studies included, seven (23.3%) performed cytotoxicity assays. Compared with the toxicity and embryotoxicity assays, the cytotoxicity assay showed the greatest variations in terms of the exposure time of the snails to the test substance. This exposure, as well as toxicity, can be divided into two segments, acute and chronic exposure. Of the seven studies, one (14.3%) performed only acute exposure to snails, three (42.9%) only chronic exposure, and two (28.6%) performed both exposures. For acute exposure, the most frequent exposure time used was 48 h (28.6%), while chronic exposure was 15 days (28.6%). Among the studies, only one (14.3%) (Douglas and Sullivan 1992) performed the cytotoxicity assay in vitro, using an exposure time of 15 min.

Replicates
The replicates among the selected studies were quite heterogeneous. In studies with toxicity tests (acute or chronic), the average of replicates was 2.2, with a higher prevalence of triplicate tests (n = 11; 36.7%), like embryotoxicity studies, with an average of 2.0 and a higher prevalence of triplicate (n = 7; 23.3%). As for the studies that carried out cytotoxicity and bioaccumulation assays, only half showed data on the number of replicates used (6/12), with the sum of the average replicates having a value of 2.3 per concentration.
Considering the recommendations of OECD No. 243 (OECD 2016) on the number of replicates per concentration, only one study (3.3%) used some 6 replicates in the experiments. Consequently, the low number of studies exposes the need to develop studies to determine the number of replicates in B. glabrata, considering not only the number of replicates and concentrations, but also the number of snails used in the assay. Therefore, based on the data of the selected studies, the use of at least 3 replicates of the experiment must be performed.

Sample size
From the data collected from the included articles, an average of 20 snails per replicate were obtained, with most studies using 10 snails per replicate of the toxicity experiment (n = 8; 26.6%). However, the sample size of the studies was quite variable and without standardization. The number of freshwater snails used in each replica ranged from 5 to 166 individuals. This high variation depends on the test methodology performed and the amount of test concentrations used during the toxicity test. For studies with a greater number of concentrations, a smaller number of snails used were observed. On the other hand, studies with smaller amounts of concentrations of the test substance, presented a larger sample size. Considering the recommended by OECD no. 243 (OECD 2016), a sample group of at least 30 individuals (5 snails × 6 replicates) should be used for each concentration. Hence, we observed that 43.5% (10/23) of the studies used at least 30 snails per concentration.
For embryotoxicity studies, in addition to the number of embryos used in the experiments, the sample is also not standardized. Some studies used embryos (average = 125), egg masses (average = 7), and snail (average = 25). As in the toxicity test, a high variation in the number of embryos used for embryotoxicological evaluation was observed, ranging from 100 to 300 embryos, with a higher prevalence of 100 embryos per replicate of the experiment (n = 7; 38.9%). Few studies used egg masses and snail (n = 4; 22.2%, each) to describe the sample size.
In the cytotoxicity assays, a sample average of 30 snails per concentration, ranging from 5 to 50, was observed in the studies. This number of snails used was sufficient to observe the effects caused by exposure to the test substance in all studies. A smaller number of snails were observed in studies that evaluated data on cytotoxicity to hemocytes after exposure to a given substance. This low sample number can be explained by the focus of the analysis being on the cells present in the snail hemolymph, which allows us to evaluate thousands of cells after extracting them. Thus, for cytotoxicity studies on B. glabrata hemocytes, we recommend the use of at least 5 snails per concentration, with at least 3 replicates.
On the other hand, studies that evaluate a set of cells, such as cells of the snail reproductive system, tend to present a greater number of snails per treatment group. Excluding the studies that evaluated hemocytes, the smallest number of snails used per concentration was 15. Therefore, a greater number of snails are recommended (minimum of 10 snails and 3 replicates) for studies that evaluate different systems, reproductive, nervous, digestive, and cardiac.
As can be seen, a varying concentrations number was used in all test classifications. A prevalence of the number of concentrations used lower than 5 can be observed in the evaluated studies (n = 20; 66.7%). This number contradicts the recommendation of the OECD guidelines. According to OECD, the use of at least five concentrations is recommended to obtain a concentration-response curve, no observed effect concentration, lethal concentrations, and lowest observed effect concentration (OECD 2016). On the other hand, considering the WHO recommendation, for the first chemical toxicity screening, at least three concentrations (10, 100, and 1000 mg/L) should be used. A greater number of studies (n = 22; 73.3%) meet this recommendation (WHO 1965).

Freshwater snail and pollutants interaction
The interaction between chemical compounds (pollutants) and the freshwater snail basically depends on the physical properties, metabolism, exposure medium, exposure time, and substance concentration. When it comes to B. glabrata, the interaction between foreign objects and hemocytes occurs via patterns of lectin recognition and production (Martins- Pereira et al. 2008;Pila et al. 2016;Humphries and Deneckere 2018). After recognition and activation, hemocytes produce reactive oxygen species (ROS) ) and inflammatory mediators such as BgMIF, a protein homologous to the mammalian macrophage migration inhibitory factor (MIF) (Garcia et al. 2010). In addition, other proteins functionally homologous to mammalian cytokines TNFα, IL-1β, and IL-17 have already had their participation described in the internal defense system of B. glabrata (Castillo et al. 2020). Thus, quantification of the concentration of these cytokines may be a potential biomarker to be explored and elucidated.
In the embryos, which have a gelatinous membrane composed mainly of an outer membrane, a colloidal substance, an ovigerous capsule, and a perivitelline substance (Kawano et al. 2008), the interaction with pollutants can be influenced, since this membrane constitutes a barrier which reduces the absorption of chemical products Duarte et al. 2015). However, the difference in response according to the type of pollutant studied is still limited . Thus, further studies should be carried out to elucidate the interaction between the embryo and different types of pollutants.
Overall, this freshwater snail integrates the effects of short-term environmental variations, responding quickly to stress. In addition, considering its 8-day embryonic cycle (Tallarico et al. 2014), it can be easily monitored and analyzed throughout the period, when compared to other organisms such as zebrafish (Danio rerio) that have an embryonic cycle of 35 days (Adrian et al. 2005).
The absorption of chemical compounds in B. glabrata depends on several factors, such as compound properties, biodegradability, water solubility (filtration), water insolubility (scraping), exposure medium, exposure time, and concentration. In addition, the behavior of the snail, such as escaping from the water, can contribute to a reduction in the exposure time and, consequently, the absorption of the substance (Oliveira-Filho et al. 2016;Caixeta et al. 2020Caixeta et al. , 2022. While there are not many ecotoxicological studies done with B. glabrata, bioaccumulation is another parameter with potential relevance in these studies, considering the snail's ability to bioaccumulate various substances in the environment through human contamination . Through nitroperchloric chemical digestion, it is possible to evaluate the bioaccumulation of nanoparticles of cadmium (CdTe Quantum Dots) on the hepatopancreas after 24 h of exposure (Lima et al. 2019), silver (AgNPs) on the snail's body after 15 and 35 days of exposure (Oliveira-Filho et al. 2019), iron (GLA-IONPs), and ferric chloride (FeCl3) in the visceral mass after 28 days of exposure (Caixeta et al. 2021). Another study (Sullivan and Cheng 1876) evaluated the accumulation of copper sulfate (CuSO4) on hemolymph after 24 h of exposure.
The absorption of some substances by snails, such as metals, occurs mainly through ingestion. Once ingested, these substances are processed in their digestive gland, called hepatopancreas, an organ with great potential for bioaccumulation. Besides hepatopancreas, bioaccumulation has been demonstrated in other sites of the snail, such as the body, mantle, oocytes, hemocytes, and shell . Recently, Caixeta et al. (2020) demonstrated through a systematic review that absorption and bioaccumulation of nanomaterials (NMs) in snails such as B. glabrata depend on (i) physical properties of NMs; (ii) environmental transformations of NMs; (iii) snail species, and (iv) exposure conditions. Chemical digestion is commonly used to assess and determine bioaccumulation in snails (Lima et al. 2019;Oliveira-Filho et al. 2019).
After exposure, B. glabrata can be washed or transferred to another medium with filtered, chlorine-free water to remove traces of the substance of interest to assess their recovery and ability to eliminate the test substance (Tallarico et al. 2014;Lima et al. 2018Lima et al. , 2019. This recovery time ranged from 24 h (Tallarico et al. 2014) to 30 days (Oliveira-Filho et al. 2016).
In a study with γ-Fe2O3 nanoparticles, after removing the remaining snails from the exposure medium, through observation by X-ray microtomography, it was possible to evaluate the elimination of the nanoparticles, where after 30 days of recovery, it was feasible to observe a considerable reduction in the organisms (Oliveira-Filho et al. 2016). In another study (Sullivan and Castro 2005), after exposure to colchicine for 72 h, snails were monitored for 4 weeks. This observation made it possible to assess B. glabrata intoxication, such as the difficulty of fixation and immobility, even with low mortality during exposure (10%) (Sullivan and Castro 2005).

Toxicity assay
Within the toxicity tests, it is possible to evaluate some parameters related to the freshwater snail after exposure, such as mortality and abnormal responses (Cheng and Sullivan 1973;Tallarico et al. 2014;Lima et al. 2019). Parameters such as mortality were evaluated during exposing B. glabrata, often with the aim of determining the lethal concentration for 50% of the organisms (LC50) (Estevam et al. 2006;Oliveira-Filho and Paumgartten 2000;Bellavere and Gorbi 1981;Sullivan and Cheng 1876;Münzinger and Guarducci 1988;Sullivan and Castro 2005;Tallarico et al. 2014;Kaur et al. 2016;Lima et al. 2019;Oliveira-Filho et al. 2019;Siqueira et al. 2020;Siqueira et al. 2021;Caixeta et al. 2021;Pena et al. 2022). The main characteristics that were considered for classifying dead snails are (i) loss of hemolymph; (ii) shell depigmentation; (iii) immobility; and (iv) lack of heartbeat. Moreover, different from other models used for ecotoxicological studies, such as cyanobacteria, Daphnia spp., and brine shrimp, the freshwater snail B. glabrata allows for a greater ease of evaluation of recovery after exposure, as they can be easily washed with filtered and chlorine-free water to remove traces of the exposure substance (Tallarico et al. 2014;Lima et al. 2018Lima et al. , 2019. In addition to assessing snail mortality, there is the possibility of performing behavioral analysis with said organism. This analysis is based on the observation of behavioral changes caused by exposure to a particular substance, such as frequency of going to the surface and leaving the water, distribution by the aquarium (Pieri and Jurberg 1981), reclusion in the shell, lethargy, and water evasion (Caixeta et al. 2021). The existence of albino B. glabrata aids the analysis of heart rate during and after exposure. This feature is advantageous for assessing heartbeats, as the shell of this snail is nearly transparent, and the heart can be seen through the shell under a microscope (Cheng and Sullivan 1973).

Embryotoxicity assay
The embryotoxicity assay with B. glabrata was proposed about 60 years ago to assess the toxicity of the pesticide pentachlorophenol (Olivier et al. 1962) and recently explored on Biomphalaria through a systematic review . However, only after the 1970s, when the snail embryonic stages were described in detail (Camey and Verdonk 1970), a greater number of embryotoxicity studies could be observed . The freshwater snail B. glabrata has an average embryonic period of 8 days (Camey and Verdonk 1970;Kawano et al. 1992;Kawazoe 1976;Tallarico et al. 2014). For ecotoxicological studies, this parameter is commonly used to evaluate the embryonic development of the snail in the blastula (0-15 h after the first cleavage), gastrula (24-39 h), trochophore (48-87 h), and veliger stages (96-111 h) (Camey and Verdonk 1970).
Mortality rate is one of the main parameters evaluated in embryotoxicity studies. Various criteria are used for classifying dead newborn snails, such as absence of heartbeat, immobility, and shell discoloration (Münzinger and Guarducci 1988;Salice and Roesijadi 2002;Nakano et al. 2003;Ansaldo et al. 2009;Ansaldo et al. 2009;de Albuquerque et al. 2014;Tallarico et al. 2014;Oliveira-Filho et al. 2016;Santos et al. 2018;Lima et al. 2019;Siqueira et al. 2020Siqueira et al. , 2021Caixeta et al. 2021;Baynes et al. 2019a, b;Garcia et al. 2021;Pena et al. 2022). Among the various causes associated with embryo mortality, such as oxidative damage and ROS production, morphological changes are the most explored. These malformations can occur hydropic, cephalic, in the shell, and foot. Recently, a review presented the main causes that lead to these changes in Biomphalaria snails .
The development time of B. glabrata embryos is another biomarker used in ecotoxicity studies. Normally, the development of the embryo is monitored and evaluated daily to observe possible changes. In some studies, it was possible to observe that the substances used were able to inhibit the development of the snail in the early (de Albuquerque et al. 2014;Tallarico et al. 2014;Oliveira-Filho et al. 2016;Santos et al. 2018;Siqueira et al. 2020Siqueira et al. , 2021Garcia et al. 2021;Pena et al. 2022) and late stages of development (Salice and Roesijadi 2002;Tallarico et al. 2014;Caixeta et al. 2021;Pena et al. 2022).
After a period of approximately 168 h after fertilization, B. glabrata embryos start to hatch. This is another important biomarker used in embryotoxicity assays, since the inhibition of hatching caused by the exposure of the embryo to a certain substance can induce its death. The reduction in the hatching rate of snails could reduce their population and, consequently, worsen environmental damage (Kristoff et al. 2011). This reduction may be a consequence of developmental delay or embryo malformation caused by exposure to the test substance, as already reported in several studies (Caixeta et al. 2021;Lima et al. 2019;Ansaldo et al. 2009;Münzinger and Guarducci 1988;Tallarico et al. 2014).

Cytotoxicity analyses
The cytotoxicity assay is based on the ability of a given substance to promote metabolic or structural changes, resulting or not in cell death. Although not a very sensitive test, the trypan blue dye assay is a simple method to assess plasma membrane integrity. Dead cells that have lost plasma membrane selectivity take up the dye and become nonviable, while cells with intact membranes do not stain with the dye, and remain viable (Avelar-Freitas et al. 2014). This assay has been used in hemocytes of B. glabrata with the objective of evaluating its viability, before and after exposure to a certain substance (Douglas and Sullivan 1992).
However, this method does not allow differentiation of the type of cell death, such as apoptosis. Thus, using Giemsa staining, it was possible to overcome this limitation as well as allowing the evaluation of cellular changes, such as micronuclei and binuclei (Lima et al. 2019). Therefore, hemocytes from B. glabrata have been used as biomarkers in ecotoxicological studies for decades. However, like the other biomarkers presented in this study, there is a lack of standardization in this assay.
Some authors (Lima et al. 2018(Lima et al. , 2019Siqueira et al. 2020Siqueira et al. , 2021 use criteria developed to classify micronuclei in human lymphocytes (HUman MicronNucleus Project-HUMN) or mussel hemocytes (Mussel MN cytome-MUMNcyt Project) as a scoring method for genotoxicity (Fenech et al. 2003;Bolognesi and Fenech 2012). However, there are no specific criteria for scoring cellular changes such as micronuclei in B. glabrata hemocytes. Considering the similarity between the two classification criteria for micronuclei in human lymphocytes and B. glabrata hemocytes, the use of both criteria is not technically wrong, as long as all points are fully met.
Several methodologies were used to evaluate the genotoxic effects on the B. glabrata. Among them, electrophoresis is used to assess the toxicity after acute and chronic exposure of snails to specific substances, such as industrial and domestic sludge (Siqueira et al. 2020(Siqueira et al. , 2021, herbicides (atrazine and glyphosate), proteins and isoenzymes, and RNA and DNA (Mona et al. 2013).
The concentration of circulating hemocytes in the hemoglyph of B. glabrata can be used to determine cytotoxicity. This assay allows the quantitative assessment of the circulating hemocyte population before and after exposure using a neubauer hemocytometer (Sullivan and Castro 2005). However, the size of the shell must be considered in the count, as it is related to the age of the snail and, consequently, to its sexual maturity, which influences the concentration of hemocytes in the hemylphon (Sminia et al. 1974;Borges et al. 2006). Similar to human macrophage, hemocytes are cells that can carry out phagocytosis by engaging and destroying exogenous substances such as bacteria. Based on this characteristic, the phagocytosis assay can assess the cytotoxicity of a given substance by quantitatively or qualitatively measuring the phagocytic rate of the hemocyte after exposure (Douglas and Sullivan 1992).
In addition to using the dye to assess nuclear damage, the alkaline comet assay has been used in B. glabrata hemocytes to assess these effects after exposure to the test substance (Siqueira et al. 2020(Siqueira et al. , 2021, as described by Singh et al. (1988) and Grazeffe et al. (2008). Different criteria can be used to interpret the result, such as (i) measuring the length of the comet's tail; (ii) classification of comets by visual inspection, typically into five categories; (iii) image analysis, with a load-coupled device camera connected to a computer with appropriate software, commercially available or from the Internet; and (iv) automated systems, which search for comets and perform the analysis with minimal human intervention (Collins et al. 2008).
Ethical issues.
One of the goals of sustainable human development is the search for a solution that is adequate and in accordance with the three R's (replacement, refinement, and reduction) of animal research. For this, the use of invertebrate organisms can offer a simplified and alternative model for research, in addition to being faster, cheaper, and ethically acceptable when compared to tests on mammals (Matthiessen 2008;Schiffelers et al. 2014). Some measures can be taken with respect to the animal, such as searching for methods that cause less suffering to animals; reduction of external sources of stress, pain, and discomfort; not using more animals than necessary to produce reliable scientific data.

Compiled of results
The results of this study show the wide variety of possible assays using B. glabrata, with responses to soluble and insoluble substances in water, which makes it a biological model with potential for ecotoxicological studies. This response to insoluble substances derives from the foraging habit of the snail, which can contribute to the bioaccumulation of substances precipitated at the bottom of the aquarium. Finally, the main information about B. glabrata, as ecotoxicological model, regarding the toxicity, embryotoxicity, and cytotoxicity tests, was summarized and presented in Table 3.

Final considerations and future perspectives
From the data obtained in this study, it was observed that the freshwater snail B. glabrata was effective for the evaluation of the ecotoxicity of several types of chemical substances. Although there is a standardized protocol for snails L. stagnalis (OECD No. 243), its use in tropical regions, like in Brazil, is not recommended, as it would lead to the risk of introducing yet another exotic species. In addition, the use of a representative species of ecosystems from tropical regions is a necessary tool for tropical environmental monitoring, since Biomphalaria has a wide distribution in tropical regions. For this reason, the use of B. glabrata as a biological model for ecotoxicological studies will help to increase the representativeness of the aquatic environment in laboratory studies.
Increasingly, freshwater snail B. glabrata has been being used as a biological model for ecotoxicological studies. This review considered three main points: first, the feasibility of using B. glabrata as a biological model; finally, the evaluation of possible toxic effects caused by these substances on the snail's organism or cells. The studies included in this review provided a preview of the potential of this freshwater snail as a biological model, as it has a diverse immune system, complex interactions, and possibilities between the snail and substances.
There is also the possibility of carrying out tests (toxicity, cytotoxicity, and embryotoxicity) at different stages of the snail's life in ecotoxicological studies. This possibility allows the evaluation of a certain substance from several perspectives, whether in the snail (mortality, lethal concentration, bioaccumulation, behavioral changes, abnormal responses), embryo (mortality, lethal concentration, bioaccumulation, malformations, developmental time, and hatching rate), and/or hemocyte (viability, metabolic activity, phagocytic activity, structural changes, and DNA damage). This characteristic reinforces the potential of its use as a biological model, since traditionally used models, such as cyanobacteria (Microcystis spp.) and microcrustaceans (Artemia spp. and Daphina spp.) do not allow for evaluations as broad as this snail.
We found that freshwater snail B. glabrata has been used in toxicity tests for decades. Despite the growing use of the snail as a biological model, and based on the data of this review, several gaps deserve attention and research, such as the following: 1) Development of studies with the objective of methodological standardization, using the B. glabrata as a biological model, in ecotoxicological studies. 2) Development of guides for standardization of experimental conditions such as water (pH, temperature, hardness, conductivity, alkalinity, and nitrate), photoperiod, and feeding before and during the experiment, as well as exposure time, exposure method, snail size and quantity, minimum number of replicates, and concentrations. 3) Establishment of robust specifications that allow the interpretation of test results, through the description of the main changes in the snail (behavior, toxicity, cytotoxicity, and embryotoxicity) 4) Carrying out studies that evaluate the ecotoxicology of the test substance not only on the B. glabrata adult, but also at different stages of life, from embryonic development to sexual maturity. 5) Conducting studies using the same test substance to compare data between B. glabrata and Lymnaea stagnalis. 6) Conducting genotoxicity studies, although the sequencing of the mollusk B. glabrata has been described in the literature, is still little explored. 7) Development of specific reviews for different endpoints, such as cytotoxicity and genotoxicity, on B. glabrata. 8) Production of new ecotoxicological studies using different biological models, including B. glabrata, exposed to the same substance.

Data availability
The datasets used during the current study are available from the corresponding author on reasonable request.

Declarations
Ethical approval Not applicable.

Consent to participate
All authors agree with their participation in the composition of this manuscript.

Consent for publication
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