Chironomus sancticaroli (Diptera: Chironomidae) in ecotoxicology: laboratory cultures and tests

Chironomus sancticaroli is a tropical species, easy to grow and to maintain in laboratory cultures. It has a fast reproduction cycle, under adequate conditions, around 30 days, allowing it to have many generations per year, an important criterion for selecting a test organism in ecotoxicology. Its life stages include: eggs, four larval instars (one planktonic and three benthic), pupa and adult (midges) This study aimed to: (1) review the methods for C. sancticaroli cultivation and its use in ecotoxicological tests, (2) establish a laboratory culture of C. sancticaroli, presenting the difficulties and discussing the ways to overcome them. Early 4th instar larvae was the most used in acute studies, while the 1st instar larvae (early 1st instar) was the most used in chronic studies; 96 h and 28 days were the most frequent durations in acute and chronic studies, respectively. The most common endpoints evaluated were organisms’ survival and development, and most of the ecotoxicological studies using C. sancticaroli were performed in laboratory. Most of the tested contaminants were pesticides and these had the most adverse effects on organisms. Most mesocosms with environmental contaminated samples did not show adverse effects on C. sancticaroli. Chronic and field studies as well as those testing the effects of the mixture contaminants on C. sancticaroli were still deficient. Keeping the laboratory environment and equipment effectively sanitized was important as well as maintaining stabilized conditions of temperature, photoperiod, physical, chemical and biological water quality in cultures.


Introduction
In the Chironomidae family, the genus Chironomus is the most diverse, encompassing about 2000 species in Europe, and 10000 only in Brazil (Callisto et al. (2007)). Some species have hemoglobin in the hemolymph, allowing individuals to tolerate environments with low oxygen levels (Richardi et al. 2015), and therefore, are useful as bioindicators of environmental quality (Al-Shami et al. 2011and López et al. 2018). Their life cycle is comprised of four phases: egg, larva (with four instars), pupa, and mosquito . The larva are aquatic sediment diggers and feed on detritus, which often expose them to chemicals via several uptake routes (Campagna et al. 2013). Chironomus comprise a proportion of the benthic biomass and stand out in the food chain due to its role in the cycling of residues into and from the sediments due to bioturbation (Gerould et al. 1983). They are a link between the sediment, the water column and the terrestrial environment, and also an important prey (at all life stages) for other invertebrates, fishes, amphibians, and insectivorous birds.
Among Chironomus species, Chironomus sancticaroli Strixino & Strixino, 1985(considered synonymous of Chironomus xanthus Rempel, 1939, but denominated through a consensus by sancticaroli (Trivinho- Strixino, 2011), stands out in ecotoxicological studies at tropical regions. C. sancticaroli is used as a bioindicator of sediment quality, mainly to evaluate contamination by xenobiotics in freshwater ecosystems (Richardi et al. 2015 andMacedo et al. 2020). It is restricted to Brazil and Argentina, has great ecological and regional relevance due to its abundance and role in the food chain (Janke et al. 2011). The use of C. sancticaroli in ecotoxicological studies is increasing, especially due to the fact it can be maintained in the laboratory and produce many generations per year (Corbi et al., (2014)). Those characteristics are important criteria when selecting a test organism for ecotoxicological tests, and to evaluate the quality of aquatic environments in tropical regions (Moreira-Santos et al. 2000 andBeghelli et al. (2018)). However, the best conditions for testing and cultivation of C. sancticaroli are still unknown and are necessary forthe establishment of robust protocols in ecotoxicology.
The first use of ecotoxicological studies with C. sancticaroli occurred in 1985 (Strixino and Strixino, 1985) and since then it has been increasing, reaching its peak in 2020 (Signorini-Souza et al. 2020;Morais et al. 2020;Macedo et al. 2020). This increase in the use of C. sancticaroli as a model organism may be due to greater acceptance of this species as a suitable test organism in tropical environments (Dornfeld et al. 2019). Additionally, there is a recent interest in studying the occurrence of contaminants in aquatic environments, especially when considering the emerging pollutants (i.e. contaminants, synthetic or natural, that have recently been introduced in the environment or with previously unrecognized negative effects on organisms; Wu et al. 2010).
Ecotoxicological tests with C. sancticaroli still miss important information such as their life cycle and development, their survival in laboratory and field conditions, and their responses when exposed in the tests (Janke et al. 2011;Morais et al. 2014;Rebechi et al. 2014). Furthermore, without standard protocols the use of C. sancticaroli in toxicological studies are based on varying conditions, limiting data comparisons (Raimondo et al. 2009). Therefore, this study aims to: (1) review the methods for C. sancticaroli cultivation and its use in ecotoxicological tests and (2) establish a laboratory culture of C. sancticaroli, presenting the difficulties and discussing the ways to overcome them.

Review of ecotoxicological data
To provide an overview of the ecotoxicological studies, we performed a systematic literature review. The review followed the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analyses) methodology using the Web of Science Core Collection (WoS), Scopus (Sco) and Scielo (Sci) as databases.
We found a total of 110 articles: 34 from Web of Science, 40 from Scopus, and 36 from Scielo. After applying the inclusion and exclusion criteria, we included 28 articles in the analysis ( Fig. 1; more details in supplementary material methods).

Larval instars
The most used larval instars were the 4 th and 1 st instars for acute and chronic tests, respectively (Table 1). The reason the 4 th instar was the most used in acute tests, is probably due to the unique characteristics of the larval stage (e.g. ventral length of head capsule, length of antennae, length of mandibles, length of mentum and length of ventromental plates; Richardi et al. 2013), and because during this stage it is easier to observe adverse effects during the tests. On the other hand, chronic tests need to assess the effects on part or all the organism's life cycle, which could be a reason for choosing the 1 st instar. Further, most studies used sediment in the test vessels, which can be associated with the fact that most of the organism's life cycle is benthic. Moreover, little sediment associated with a reduced surface area can lead to intraspecific predation, and an increase in the mortality of the larval stage, which can affect both acute and chronic tests.

Test duration
We found the studies that analyzed the 4 th instar generally monitored individual survival and were performed for 96 h (e.g. Printes et al. 2011;Novelli et al. 2012;Richardi et al. 2018; Table 1). The 96 h test duration is reported as a limit time to acute tests with this species. It is because a longer period allows the larvae to develop into the next instar (Fonseca and Rocha, 2004), mischaracterizing the acute test. All acute tests reported by the studies were carried out in very similar conditions (between 22°C and 29°C, photoperiod 12:12 h) and most of them used artificial sediments (i.e. sediments with uniform granulometry, similar composition, and fortified with specific contaminants) as substrate in test beakers. Artificial sediments are often used in acute tests because those tests are usually recommended when testing for water contamination rather than sediment itself.
Chronic tests with the 1 st instar were performed over 8 (Richardi et al. 2018 Table 1) at different conditions (between 22°C and 29°C, photoperiod 12:12 h). Ideally, chronic tests should analyze the effects on the organisms considering their entire life cycle, which can explain the most observed duration in the studies around 28 days. In addition, we found the tests duration in the studies also depended on the observed endpoints, especially considering emergence and sex ratios can only be observed in the final cycle of the organism, which is around 28 days.

Endpoints and biomarkers
Mortality and growth were the most used endpoints in acute and chronic tests, respectively (Table 1). These endpoints are also often evaluated in other ecotoxicological studies (Sánchez et al. (2004); Pepin (1991)), and one possible explanation can be related to the ease of these methods in comparison to others. However, other biomarkers were reported, such as enzymatic activity (Rebechi et al. 2014;Richardi et al. 2018

Laboratory and field studies
Most ecotoxicological studies were carried out in the laboratory (89%), while field experiments were used only few times (11%). Field studies can be more difficult, due to lack of accessibility, higher costs, and difficulty to control the external variables (Ramasundaram et al. 2005). However, many studies recognized the results from field experiments better represent the environmental conditions to which the organisms are subjected. Additionally, it is important to verify that the toxicity of the contaminant is maintained in the laboratory, and not modified (synergism or antagonism effect) by external factors typically found in the field.
It was reported by Macedo et al. (2020) that the active chlorine in calcium hypochlorite (Ca(ClO) 2 ) decreased C. sancticaroli emergence in 22% of the population and Ferreira-Junior et al. 2018 showed the percentage of emerged C. sancticaroli was reduced by 33.3% and 45.8% when exposed to 0.0016 and 0.0032 mg/L of a thiamethoxambased insecticide (whole compound).
All studies with heavy metals analyzed mortality and only one (Beghelli et al. (2018)) analyzed morphological alterations, reporting reduction in length and a higher occurrence of total damage on C. sancticaroli following the exposure to copper, nickel, and chrome in the sediment. Both studies showed that flame retardants can cause a delayed larval development and decreased number of emerging adults. In general, the main objectives of the studies with C. sancticaroli were having a benthic organism to evaluate water and sediment quality. Some of the studies had no effect on C. sancticaroli, however, they were carried out with environmental samples: sediment contaminated with heavy metals (Silvério et al. 2005) and a pure nanomaterial -graphene oxide (Castro et al. 2018), sludge contaminated with heavy metals (Sotero-Santo et al. 2007) and secondary effluent contaminated with disinfectant (Da Costa et al. 2014).
Besides C. sancticaroli, other species within the genus were also used as a model, for instance C. dilutes, C. riparius and C. piger (and others; Michailova et al. 2012;OECD, USEPA 2011). The genus Chironomus can be an option to assess both the sediment and the interstitial water, as it lives in the sediment, feeds on sediment particles and is in contact with the interstitial water through the skin.

Review of laboratory cultures methods
The review of laboratory cultures and methods of C. sancticaroli tests returned a total of 110 articles: 34 from Web of Science (WoS), 40 from Scopus (SCo), and 36 from Scielo (Sci). After applying the inclusion and exclusion criteria, only 1 article was selected ( Fig. 3; Fonseca and Rocha, 2004; more details in supplementary material methods).
The description of culturing methods of C. sancticaroli by Fonseca and Rocha (2004) was the most cited reference, being cited in 15% of the articles. However, their paper did not describe some difficulties and details (e.g., equipment clean, feeding, maintenance) regarding C. sancticaroli establishment. Another article by Maier et al. 1990 also described cultivation methods for C. decorus. When considering both studies, we noticed the two methods were similar. For instance, both studies used a tray or an aquarium filled with a sediment layer and water. The tray was covered with a screen. The screen had a mesh opening that prevented the mosquitoes from exiting, while also allowing them to copulate and spawn. The major differences between the studies were those regarding the species culturing requirements such as photoperiod, temperature, and water characteristics.

Establishment of new cultures of Chironomus sancticaroli: difficulties and pathways
Based on our personal experience and the literature reviewed we are going to summarize in this topic the main steps for the establishment of new cultures of C. sancticaroli.
Before starting a new culture of C. sancticaroli, it is necessary to prepare all the materials that are going to be used (Table 2) and to prepare a temperature and photoperiod-controlled environment, where the culture will be maintained. It is important that all the materials be free of contamination and are exclusively used for the culture, to avoid contamination. We recommend the use of new lab materials to prevent the species degeneration; more details will be given on a next topic -"cleaning the trays and the room".
It is also necessary to define the container typology where the experiments will be carried out. Usually The organisms for the new culture can be obtained either by field collection (it is necessary to correctly identify the species) or from an established culture from a research laboratory (the major advantage in using it is that the organisms have already been identified). If the first case is chosen, collecting the eggs in the field is preferable to collecting the larvae or adults, as suggested by Fonseca and Rocha (2004). According to them, the culture will consist of eggs from the same egg mass and belong to the same species. Once individuals have been collected in the field, it is not possible to guarantee the good health of organisms nor their sensitivity to chemicals due to previous exposures, therefore it is wise to wait for the second or third generation before using them in tests with reference substances.
In our personal experience, C. sancticaroli organisms were obtained from a research laboratory, the Ecotoxicology Laboratory of the Center for Water Resources and Applied Ecology, in São Carlos School of Engineering -University of São Paulo (São Carlos, Brazil). Five egg masses were brought to the Plankton Ecology Laboratory at the Federal University of Juiz de Fora (Juiz de Fora, Brazil) where the new cultures were established according to methods described in Fonseca and Rocha (2004). Each egg mass was kept in a 100 ml beaker and was fed with 10 ml of the green algae Raphidocelis subcapitata (2193916.67 cells/mL; Da Costa et al. 2014;Barbosa et al. 2019). The water used in the cultures was mineral water, which had the required characteristics for a good C. sancticaroli survival (the preferable water conditions are describe in Table 3). Ideally, the water used in colony maintenance should be reconstituted, adjusted to the hardness parameters of 16 mg/L; pH between 7.2 and 7.6 and conductivity of 160 µS/cm (OECD, USEPA, 2011). Fonseca and Rocha (2004) reported that their egg mass had around 500 to 600 eggs. In the beginning, the number of individuals hatching from the egg mass in our laboratory culture ranged from 150 to 250 individuals, which represents a low hatching rate (30% approximately). The low  (Moher et al. 2009) hatching rate was probably due to manipulation of the egg mass and its transport to the beakers, or possibly, due to the amount of algae solution added, as many algae became entangled and prevented larvae from hatching completely. We used tweezers to very carefully collect the egg mass, and the algae solution was only added to the beaker when the 1 st instar larvae hatched. With these small precautions we verified an increase in the survival of larvae. Unfortunately, no information was published about the hatching rate and the viability of the eggs for this species in the articles searched. The culturing water was kept static (with no aeration) for up to 48 h and until all the masses hatched. After 48 h, the hatched larvae (1st instar; 200 individuals) were transferred to 7-liter plastic trays (45.5 × 28 x 7.7 cm), containing 1/3 of sediment (river gravel granulometry 03) and 2/3 of water (about 4 liters; characteristics on Table 3). A cage was adapted in the top of the tray to avoid unwanted organisms and to keep the newly hatched adults in contact for mating and reproduction (Fonseca and Rocha, 2004). The cultivation water had a pH between 6.5 and 7.5, the culture room conditions had a temperature range of 22°C-25°C and a 12 h light: 12 h dark photoperiod. The culturing trays were continuously aerated with air pumps (Fig. 4). After 2 days of culture establishment (without food), the larvae were fed with 25 ml Tetramin® fish food solution (0.005 g/ml stock solution) three times a week. The same feeding frequency was used for all instar stages. We highlight that we have not tested different food concentrations in different stages. However, we recommend it for future studies. In our cultures, the water was frequently replenished (once/ week; approximately 34% renewal), until it reaches the initial volume of the tray.
In ecotoxicological tests, it is recommended to establish the number of larvae that will be used per treatment. Based on the articles searched, we found no consensus regarding the number of larvae used in either acute or chronic tests using C. sancticaroli. Although, we noticed that 6 larvae per treatment was commonly used in acute tests (Campagna et al. 2013;Colombo-Corbi et al. 2017;Dornfeld et al. 2019;Guimarães-Souto et al. (2018);Janke et al. 2011;Novelli et al. 2012;Silvério et al. 2005;Yamada et al. 2012).
The first generation of new egg masses was obtained 1 month after the establishment of the initial tray. After that, three masses from the main tray were removed and placed in a beaker filled with culture water and 10 ml of the Raphidocelis subcapitata algae solution. After hatching, the 1 st instar larvae were placed in a tray (300 organisms per tray). This procedure was repeated until we had enough trays for a test (approximately 4 or 5 trays).

General care
When food solution or water are added to the tray, care must be taken to not revolve the sediment, which could be stressing for the larvae (Fig. 5A). When removing the cages for maintenance, it is important to gently tap the sides of the cage, so that mosquitoes fly upwards and do not leave the cage (Fig. 5B). Additionally, the aeration must be gentle enough to not disturb the sediment.

Feeding the organisms
As commented above, we used a Tetramin® solution to feed the organisms. This was the solution used by most studies (e.g. Yamada et al. 2012;Sotero-Santos et al. (2007), Silva et al. 2019;Morais et al. 2020). Other alternatives to the use of Tetramin® solution are fish food such as Nutrafish® or Dog Chow® (Beghelli et al. (2018); Morais et al. 2014;Palacio-Cortes et al. 2017). However, information available from the articles on the methods for the preparation and storage of the fish food are insufficient. We mixed 2.5 g of Tetramin® solution and 500 ml of distilled water in a blender, which we called the stock solution. After preparation, the stock solution was stored in the refrigerator at 4°C to avoid fermentation. We recommend leaving the stock solution at room temperature for about 20 to 30 min before feeding the larvae. The stock solution can be used for about 1 month, when kept in the refrigerator at 4°C.

Temperature of the culturing room
Temperature is an important parameter for the development and survival of macroinvertebrate species in the laboratory (Strixino & Strixino, 1985). We have observed that, when the culture room was colder, the life cycle of the organisms was delayed, consequently delaying the start of tests. Therefore, any problem with controlling room

Cleaning trays and cultivating room
The culture and maintenance of the trays are extremely important and must be made with care to guarantee the larvae survival. We recommend the manipulation to be minimal, only to provide the essential, and all the materials used should be washed correctly to avoid contamination, for example by using alkaline detergent with a special brush, and then a rinsing it with distilled water multiple times.
In one of our experiments, we have observed the existence of fungus in some of the trays, which in our case could have come from the building wall where the experiment was performed or by overfeeding the trays, but brief suspension of feeding was not tested. Furthermore, we suggest that experiments take into account different amounts of food for each chironomid life stages.
In addition to the correct washing of materials and trays, it is important that the culturing room be also cleaned constantly, including walls and, even the number of people who enter the room should be limited. We recommend that everyone responsible for maintenance of the culturing be equipped with the correct laboratory I.P.E (Individual Protection Equipment) such as wearing coat, gloves, and keeping the hair up. Finally, it is necessary to prevent the entrance of other insects in the room, which could eventually contaminate the colony. By taking these precautions, one can ensure the health of the culture.

Conclusions and future perspectives
In summary, this study provides valuable recommendations regarding the culturing of C. sancticaroli, while also discussing some issues for the performance of toxicity tests. We have briefly described the most used test parameters and conditions in ecotoxicological studies that used C. sancticaroli as study model, and addressed the effect of contaminants such as heavy metals and emergent pollutants.
Based on the literature review, we recommend C. sancticaroli as a test organism for ecotoxicological studies. Some of the reasons are the recent increase in studies about this species, the lack of ecotoxicological data publications (only 28 studies) using the organism, and the number of contaminants present in the tropical aquatic The arrows show empty spaces, without sediments. B Method suggested to prevent mosquitoes from escaping the cage. Arrows show the direction in which the cages should be removed from top of the tray environment that can be better understood by using C. sancticaroli as a bioindicator.
For further ecotoxicological studies, we indicate the patterns that we found in this study (cultivation conditions and maintenance, most used instar for test, test duration, number of larvae, and endpoints). However, it is important to note that, unlike was tested here, we suggest glass containers to be used (or with other type of container, according to the pollutant to be tested), since plastic can be a source of contamination, and may have affected our results.
In addition, we suggest keeping the immature individuals in glass aquariums, with sediment, inside the cage greater than the size of the aquarium, with free space on the sides and above for flight and adult copulation, which may improve the reproduction and the survival of the culture. Additionally, the control of new generations through the crossing between mosquitoes should be better controlled and studied. Here, this part as well as survival rate and egg production were performed randomly. Finally, we acknowledge the need for more chronic and field studies, as well as effects of the mixture contaminants. This last statement is essential due to its relevancy to monitor aquatic and terrestrial environmental conditions.