Chironomus Sancticaroli Research Articles

Anaerobic treatment removing tetrabromobisphenol A and biota safety: How do tropical aquatic species respond to effluent toxicity over short- and long-term exposures?

Wastewater containing tetrabromobisphenol A (TBBPA), a commonly used flame retardant found in wastewater, can present significant toxic effects on biota, yet its impact on tropical freshwater environments is not well understood. This study explores the effectiveness of two independent anaerobic treatment systems, the acidogenic reactor (AR) and the methanogenic reactor (MR), for the ecotoxicity reduction of TBBPA-rich wastewater in four tropical freshwater species. Despite presenting good physicochemical performance and reduced toxicity of the influent for most species, AR and MR treatments remain acute and chronic toxicity. Overall, MR exhibited greater efficacy in reducing influent toxicity compared with AR. TBBPA bioaccumulation was observed in Chironomus sancticaroli after short-term exposure to 100% MR effluent. Multigenerational exposures highlighted changes in the wing length of C.sancticaroli, showing decreases after influent and AR exposures and increases after MR exposures. These findings underscore the need for ecotoxicological tools in studies of new treatment technologies, combining the removal of emerging contaminants with safeguarding aquatic biota. PRACTITIONER POINTS: Acidogenic and methanogenic reactors reduced the acute and chronic toxicity of wastewater containing tetrabromobisphenol A. Both treatments still exhibit toxicity, inducing short- and long-term toxic effects on four native tropical species. The aquatic species Pristina longiseta was most sensitive to effluents from acidogenic and methanogenic reactors. TBBPA concentrations recovered from Chironomus sancticaroli bioaccumulation analysis ranged from 1.07 to 1.35 μg g-1. Evaluating new treatment technologies with multiple species bioassays is essential for a comprehensive effluent toxicity assessment and ensuring aquatic safety.

Biofragmentation of Polystyrene Microplastics: A Silent Process Performed by Chironomus sancticaroli Larvae.

Polystyrene (PS) is one of the main synthetic polymers produced around the world, and it is present in the composition of a wide variety of single-use objects. When released into the environment, these materials are degraded by environmental factors, resulting in microplastics. We investigated the ability of Chironomus sancticaroli (Diptera, Chironomidae) to promote the fragmentation of PS microspheres (24.5 ± 2.9 μm) and the toxic effects associated with exposure to this polymer. C. sancticaroli larvae were exposed to 3 different concentrations of PS (67.5, 135, and 270 particles g-1 of dry sediment) for 144 h. Significant lethality was observed only at the highest concentration. A significant reduction in PS particle size as well as evidence of deterioration on the surface of the spheres, such as grooves and cracks, was observed. In addition, changes in oxidative stress biomarkers (SOD, CAT, MDA, and GST) were also observed. This is the first study to report the ability of Chironomus sp. to promote the biofragmentation of microplastics. The information obtained demonstrates that the macroinvertebrate community can play a key role in the degradation of plastic particles present in the sediment of freshwater environments and can also be threatened by such particle pollution.

Sugarcane vinasse provokes acute and chronic responses and bioaccumulation of metals in benthic macroinvertebrates.

Brazil is a major producer of sugarcane bioethanol, which has raised concerns about its environmental impact. The industrial process for obtaining ethanol generates a by-product with a high pollution potential called vinasse. If vinasse reaches watercourses, it may affect the biological communities, such as the aquatic macroinvertebrates, which include species sensitive to environmental contamination. Thus, this study evaluated the ecotoxicological effects of sugarcane vinasse on tropical benthic macroinvertebrates (Allonais inaequalis, Chironomus sancticaroli, Strandesia trispinosa, and Hyalella meinerti). The study was divided into three phases. First, acute toxicity tests were carried out with the four species. The species A. inaequalis (average LC50 = 0.460% confidence interval, CI 0.380-0.540%) was more sensitive to vinasse than C. sancticaroli (LC50 0.721%, CI 0.565-0.878%), H. meinerti (EC50 0.781%, CI 0.637-0.925%), and S. trispinosa (EC50 1.283%, CI 1.045-1.522%). In the second phase, the consequences of chronic exposure to vinasse were assessed in the two more sensitive species. Impairments in reproduction and population growth rates for A. inaequalis and on the development, metamorphosis, and body growth of C. sancticaroli larvae occurred. Finally, the bioaccumulation of metals after chronic exposure was determined in the third phase. Vinasse provoked decreases in the body residue of the essential metals Zn and Mn and the accumulation of Cd, Pb, and Cr with the potential for biomagnification throughout the food webs. Low concentrations of vinasse (below 1%) provoked lethal and sublethal effects on benthic organisms, with several cascade effects on aquatic environments, given the ecological importance of this group in freshwater and terrestrial ecosystems.

Has the Rio Doce "time bomb" been defused? Using a weight-of-evidence approach to determine sediment quality.

The Fundão mine tailings dam rupture of 2015, in the Rio Doce basin, Brazil, resulted in the deposition of tailings downstream of the dam. It has yet to be determined if metals associated with the tailings have contributed toxicity to organisms, burying a time bomb that could be ticking. Currently the data on toxicity to benthic and aquatic organisms have not been assessed sufficiently to allow an informed assessment using an approach based on weight-of-evidence. This study was conducted to ascertain if sediments at "hot spots" that received Fundão tailings reflected elevated concentrations of metals and if these concentrations were sufficient to result in toxicity to freshwater organisms. The lines-of-evidence considered included assessing metals concentrations in relation to sediment quality criteria, establishing biogeochemical characterizations, completing an evaluation of potential metal release upon resuspension to provide information on bioavailability, and identifying acute and chronic toxicity effects using sensitive native species for waters (water flea, Daphnia similis) and sediments (burrowing midge larvae, Chironomus sancticaroli). Only porewater concentrations of iron and manganese exceeded Brazilian surface water criteria, whereas most trace elements exhibited no enrichment or elevated environmental indexes. The concentrations of bioavailable metals were assessed to be low, and metal concentrations did not increase in the overlying water upon resuspension; rather, they decreased through time. Toxicity testing in resuspended waters and bulk sediments resulted in no acute or chronic toxicity to either benthic or aquatic species. The low metal bioavailability and absence of toxicity of the tailings-enriched sediments was attributed to the strong binding and rapid removal of potentially toxic metal ions caused by oxyhydroxides and particles in the presence of iron-rich particulates. The findings of these sediment hot-spot studies indicate the Fundão dam release of tailings more than six years ago is not causing the current release of toxic concentrations of metals into the freshwaters of the Rio Doce. Integr Environ Assess Manag 2024;20:148-158. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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.

Toxicological effects caused by environmental relevant concentrations of ketoconazole in Chironomus sancticaroli (Diptera, Chironomidae) larvae evaluated by oxidative stress biomarkers

Ketoconazole (KTZ), a broad-spectrum fungicidal drug, has been a significant problem in recent decades due to its toxic action on non-target aquatic organisms. Thus, the present study aimed to evaluate determine the effects that environmental relevant concentration of the commercial formulation of KTZ can exert on benthic macroinvertebrates, more specifically on larvae of the insect Chironomus sancticaroli. Acute toxicity tests with KTZ indicated lethal concentration (LC50) of 9.9 μg/L. Analyses of prolonged exposure to KTZ (chronic toxicity) indicated an increase in the rate of mentum deformity by approximately 3 times at concentrations of 0.6 and 2.4 μg/L. All biomarkers analyzed showed an increase after exposure to KTZ (0.6 and 2.4 μg/L), with average values of 115 % for superoxide dismutase (SOD), 63 % for catalase (CAT), 111 % for glutathione S-transferase (GST) and 59 % for malonaldehyde (MDA) in C. sancticaroli larvae. Thus, the toxic effects on survival, development (length and weight), mentum and redox responses caused by commercial KTZ in low concentrations were observed on C. sancticaroli larvae. In addition, the results suggest that biochemical biomarkers can be used for studies involving environmental disturbances.

Adsorption of Metals by Chitosan Beads in Sugarcane Cultivation Streams: Implications for Chironomus sancticaroli Insect Larvae (Diptera: Chironomidae)

Adsorption of Metals by Chitosan Beads in Sugarcane Cultivation Streams: Implications for Chironomus sancticaroli Insect Larvae (Diptera: Chironomidae)

The silent harm of polyethylene microplastics: Invertebrates growth inhibition as a warning of the microplastic pollution in continental waters

Microplastics are now widespread throughout the environment, but there is still scarce knowledge about their impact on freshwater invertebrates, especially from tropical environments. In this study, we carried out experiments to investigate the harmful effects of polyethylene microplastics (MPs 40–48 µm in size) on Chironomus sancticaroli and Daphnia magna, which are bioindicators from different climate zones. In short-term exposures, the MPs presented no significant effect on the survival and mobility of the species, although D. magna showed 51 ± 10% of the intestinal tube filled by MPs after 96 h of exposure to 320 mg MP L−1. Chronic exposures showed that C. sancticaroli has its larval length reduced in the 320 mg MP L−1 treatment (p ≤ 0.05), presenting EC10 of 302.36 mg MP L−1 for larval growth. Similarly, D. magna had its body length reduced after 21 days of exposure to 160 and 320 mg MP L−1 treatments (p ≤ 0.05). On the other hand, the dynamics of the molting cycle was not affected by the MPs in chronic exposures. Our findings support the recent discussion regarding the variable and complex effects of microplastics on the biota, which depend on the model species and exposure time, showing that species growth can be potentially impacted by microplastic pollution. The prediction of this biological endpoint in organisms representing tropical and temperate environments indicates a warning of the potential harm of the microplastic presence in continental waters.

Toxicological Effects Caused by Sub-Lethal Concentrations of Ketoconazole in Chironomus Sancticaroli (Diptera, Chironomidae) Larvae Evaluated by Oxidative Stress Biomarkers

Toxicological Effects Caused by Sub-Lethal Concentrations of Ketoconazole in Chironomus Sancticaroli (Diptera, Chironomidae) Larvae Evaluated by Oxidative Stress Biomarkers

Accumulation of Nylon Microplastics and Polybrominated Diphenyl Ethers and Effects on Gut Microbial Community Of Chironomus Sancticaroli

Accumulation of Nylon Microplastics and Polybrominated Diphenyl Ethers and Effects on Gut Microbial Community Of Chironomus Sancticaroli

Multi-generational exposure to fipronil, 2,4-D, and their mixtures in Chironomus sancticaroli: Biochemical, individual, and population endpoints

Conventional farming delivers a range of pesticides to aquatic ecosystems leading to implications for the indigenous species. Due to the multiple applications and persistence of molecules, organisms may be exposed for a prolonged period over multiple generations. The present study outlines a full life-cycle design over three generations of Chironomus sancticaroli exposed to the insecticide fipronil, the herbicide 2,4-D, and their mixtures. The experiment started with newly hatched larvae from the parental generation and lasted with the emerged adults from the second generation. Five nominal concentrations of fipronil and 2,4-D were tested, as well as six combinations of both pesticides. As additional responses, the total carbohydrates and the lipid classes were evaluated in the parental generation. The first and second generations were more susceptible to the tested compounds compared with the parental ones. Survival of larvae and pupae was decreased by both pesticides and their mixtures along with the generations. Only fipronil impaired the survival of emerged adults. Both pesticides (isolated and in the mixture) altered the emergence and the fraction of males and females. Moreover, the number of eggs produced, and their hatchability decreased. Only one combination of the pesticides increased the content of carbohydrates. Fipronil, 2,4-D, and its mixture altered the profile of the lipid classes. All mixture treatments and the three highest concentrations of fipronil extinguished the population of C. sancticaroli at the end of the first generation. In the remaining treatments with the insecticide, the population did not survive the second generation. Only three concentrations of 2,4-D and the control persisted until the end of the experiment. The results indicate that a prolonged exposition to these pesticides may disrupt the natural populations of exposed organisms with consequences to ecosystems' functioning, considering the importance of chironomids to aquatic and terrestrial environments.

Toxicity of rhizomes of the invasive Hedychium coronarium (Zingiberaceae) on aquatic species

The production and release of chemical compounds by invasive plants can affect competitors and native species overall, destabilizing ecological interactions and harming ecosystem functioning. Hedychium coronarium is an invasive macrophyte common on Brazilian riparian areas that produces a wide variety of allelochemicals, but little is known about their effect on aquatic species. Here, we identified the major chemical compounds of the aqueous extract of H. coronarium rhizomes and assessed its toxicity, evaluating the growth inhibition of one alga (Raphidocelis subcapitata) and one macrophyte (Lemna minor), and the lethality of cladoceran (Ceriodaphnia silvestrii and Daphnia similis) and Chironomidae larvae (Chironomus sancticaroli). The majoritarian compounds of H. coronarium rhizomes were Coronarin D and Coronarin D Ethyl Ether. The aqueous extract was toxic for all tested species. We observed growth inhibition in R. subcapitata, as well as reduction in biomass in L. minor. Chironomus sancticaroli and cladoceran were the most sensible species. The aqueous extract of H. coronarium rhizomes was toxic on tested conditions, suggesting that the rhizome compounds may interfere on aquatic organisms and in the dynamic of trophic webs of aquatic ecosystems on invaded areas.

Ecotoxicity and Antimicrobial Inhibition Assessment of Effluent from an Anaerobic Bioreactor Applied to the Removal of Sulfamethoxazole and Ciprofloxacin Antibiotics from Domestic Sewage

This study evaluated the ecotoxicity effects of effluent from an anaerobic fixed bed biofilm reactor (AFBBR) removing the sulfamethoxazole (SMX - 403 ± 104 ng L−1) and ciprofloxacin (CIP - 294 ± 123 ng L−1) antibiotics from domestic sewage in some microorganisms (Brevundimonas sp., Escherichia coli, Ochrobactrum sp., Sphingomonas sp.) and benthic organisms (Allonais inaequalis and Chironomus sancticaroli). The AFBBR showed high removal efficiency of SMX (85 ± 10%) and CIP (81 ± 16%) and completely eliminated the acute ecotoxicological effect on the C. sancticaroli insect larvae. The bioreactor effluent did not cause any inhibition on the cell growth of the microorganisms. Nonetheless, the A. inaequalis was extremely sensitive to the reactor effluent (100% mortality), probably due to the death of bacteria and algae essential to the nutrition of the Oligochaeta. Regarding the adult insect emergency of C. sancticaroli, a reduction of 50% was observed after 17 days of exposure to the effluent with 40 μgSMX L−1, inhibiting the larval development, which did not occur for CIP. The antimicrobial inhibition by CIP present in the domestic sewage resulted in EC50 (median effective concentration) values of 2.5, 0.9, 3.1 and 0.1 mgCIP L−1 for Ochrobactrum sp., Brevundimonas sp., Sphingomonas sp. and E. coli, respectively. Assessing the effect of antibiotics in the effluent on the microorganisms’ growth by a disk diffusion test, only E. coli culture showed inhibition at the concentration of 5.0 mgCIP L−1. The study emphasized the potential of AFBBR to reduce ecotoxicity in the sewage on aquatic biota.

Chironomus sancticaroli generation test applied to chemical contaminants and freshwater sediment samples.

The use of ecotoxicological bioassays has been increasing due to the importance of understanding the effects of substances on biota and to help environmental agencies determine water and sediment qualities. The use of Chironomus sp. in laboratory bioassays is extensive, but there is still a lack of studies regarding the application of extended ecotoxicological tests, which evaluate different population generation responses and show a detailed impact on their development. The present study investigated the response of Chironomus sancticaroli, a Brazilian endemic insect, to 17α-ethinylestradiol hormone, caffeine anhydrous, LAS (linear alkylbenzene sulfonate), and environmental samples (sediments) in relation to wing length, fecundity, and larval length over three generations. Statistically, differences for all contaminants between P (parental), F1, and F2 generations (p ≤ 0.05) were observed, indicating that in the environment, the organism may be negatively affected when exposed to contaminants in successive generations. Different ecological responses were also noted in comparison to classic acute (96h) and chronic (10 days) ecotoxicological tests using Chironomus sancticaroli. Our results highlight that the generation test can present more detailed results regarding the effects of stressors on the organism's life cycle than do the classic bioassays.

Molecular and biochemical evaluation of effects of malathion, phenanthrene and cadmium on Chironomus sancticaroli (Diptera: Chironomidae) larvae

In-vitro effects of sub-lethal concentrations of malathion, phenanthrene (Phe) and cadmium (Cd) were tested on Chironomus sancticaroli larvae in acute bioassays by measuring biochemical and molecular parameters. Malathion was evaluated at 0.001, 0.0564 and 0.1006 mg L-1; Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 0.001, 3.2 and 7.4 mg L-1. The recovery test carried out at the highest concentration of each compound showed that survival of larvae exposed to Phe ranged from 4% to 5%, while the effects of malathion and Cd were irreversible, not allowing the emergence of adults. Results showed that malathion and Cd inhibited AChE, EST-α and ES-β activities at the two highest concentrations. Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 3.2 and 7.4 mg L-1 inhibited glutathione S-transferase activity. Oxidative stress was exclusively induced by the lowest concentration of malathion considering SOD activity once CAT was unaffected by the stressors. Lipid peroxidation was registered exclusively by malathion at the two highest concentrations, and total hemoglobin content was only reduced by Cd at the two highest concentrations. The relationship among biochemical results, examined using the PCA, evidenced that malathion and Cd concentrations were clustered into two groups, while Phe only formed one group. Four hemoglobin genes of C. sancticaroli were tested for the first time in this species, with Hemoglobin-C being upregulated by malathion. The toxicity ranking was malathion > Phe > Cd, while biochemical and molecular results showed the order malathion > Cd > Phe. Our results highlight the importance of combining different markers to understand the effects of the diverse compounds in aquatic organisms.

Genotoxic, metabolic, and biological responses of Chironomus sancticaroli Strixino & Strixino, 1981 (Diptera: Chironomidae) after exposure to BBP.

Benzyl butyl phthalate (BBP), which is widely used in industrial production, reaches the aquatic environment, mainly owing to improper disposal of plastic products. In the water, it remains adsorbed to sedimentary particles causing toxic effects in aquatic invertebrates such as Chironomidae, which are important in maintaining ecosystem dynamics and are an important link in the food chain. However, the effects of BBP on Chironomidae are still poorly known. Thus, the toxic effects of BBP on Chironomus sancticaroli at acute (48h), subchronic (8 d), and chronic (25 d) exposures of concentrations between 0.1 and 2000μg·L-1 were determined. Genotoxicity effects, changes in the oxidative stress pathway, and development and emergence of organisms were evaluated. Biochemical markers showed a reduction in cholinesterase (ChE) activity, indicating a neurotoxic effect on acute exposure (1-1000μg·L-1). The antioxidant pathway, glutathione S-transferase (GST) activity showed reduction on acute (0.1; 1-2000μg·L-1) and subchronic (1-2000μg·L-1) exposures and reduction in superoxide dismutase (SOD) activity at all evaluated concentrations, suggesting oxidative stress. In contrast, lipid peroxidation was not observed. DNA damage occurred on acute (10μg·L-1) and subchronic (10-2000μg·L-1) exposures, indicating genotoxic effects. At concentrations above 10μg·L-1, no emergence of adults occurred, while lower concentrations (0.1 and 1μg·L-1) showed a reduction in the number of adults, mainly males. The observed effects indicate that BBP is genotoxic and causes biochemical alterations presenting high toxicity at the population level.

Cell damage induced by polybrominated diphenyl ethers on Chironomus sancticaroli (Diptera: Chironomidae)

Abstract Histological effects of polybrominated diphenyl ethers (PBDEs) were observed in Chironomus sancticaroli larvae which underwent acute exposure. 2,2′,4-triBDE (BDE-17), 2,2′,4,4′-tetraBDE (BDE-47) and 2,2′,4,4′,5-pentaBDE (BDE-99) were evaluated at 0.5, 2.0 and 20 μg L-1. Cytoplasm vacuolisation of oenocytes was observed in the larvae exposed to BDE-17 and BDE-47. Cuenot cells were disrupted at the brush border as an effect of the three evaluated congeners highlighting BDE-47 at 2.0 μg L-1; 60% of larvae displayed this disruption. The midgut showed changes in the morphology of apex cells located next to the lumen of region I exposed to BDE-17 and BDE-47, while BDE-99 induced a narrowing of the lumen diameter. Significant cytoplasm vacuolisation of the larvae exposed to BDE-47 and BDE-99 was observed in region II of the midgut. Salivary glands showed acidophilic granules in the cytoplasm exposed to BDE-17 and BDE-47. The results showed that the tissues of C. sancticaroli were sensitive to flame retardants; these histopathologies can compromise the health and physiology of this organism, highlighting the concern with the presence of PBDEs in freshwater sediments.

Chironomus sancticaroli (Diptera, Chironomidae) as a Sensitive Test Species: Can We Rely on Its Use After Repeated Generations, Under Laboratory Conditions?

In ecotoxicological assays, previously selected and standardized organism tests are exposed to an environmental sample. Some species of the Chironomus genus have been extensively used in ecotoxicological assays. Among these, Chironomus tentans is usually utilized in the USA and Chironomus sancticaroli in Brazil. We conducted ecotoxicological bioassays to compare a population of C. sancticaroli, kept for 6years under laboratory conditions, with a sylvatic population of the same species, collected in the field. The aim was to test the hypothesis that populations of C. sancticaroli, maintained in the laboratory for long periods, could have a different response to stressors/substances. We analyzed the responses of C. sancticaroli for potassium chloride, zinc chloride, potassium dichromate, linear alkylbenzene sulphonate (LAS) and caffeine. The results showed no significant differences between the two populations in the analyses and seems to indicate the possible use of C. sancticaroli from populations kept in the laboratory for long periods.

Frequency of mentum deformity in Chironomus sancticaroli (Diptera: Chironomidae) in a laboratory culture

The midge Chironomus sancticaroli (Diptera: Chironomidae) has been used in ecotoxicological tests because it is sensitive to a variety of inorganic pollutants. Among the parameters used to evaluate the toxicity of a substance is the frequency of mentum deformity, which is part of the oral system of this organism. However, there is still no consensus on the baseline level (percentage) of acceptable deformities in laboratory cultures not exposed to pollutants. The determination of this variable is important to ensure the validity of bioassays and to compare cultures from different research and teaching institutions. Once this value is established, it will also be used to monitor the quality of organisms cultured, since factors such as inbreeding could increase the frequency of mentum deformity. Thus, the objective of this study was to quantify the percentage of mentum deformity in the fourth instar of C. sancticaroli larvae from the culture of the Laboratory of Aquatic Ecosystems, at Embrapa Meio Ambiente. The average frequency of mentum deformity obtained was 6,63%. It is believed that factors such as the renewal of the culture with the inclusion of spawns from the laboratories of other institutions, as well as the control of the quality of the dilution water and the sediment of the breeding may have contributed to a low frequency of mentum deformity of the culture observed in this study.

Chironomus sancticaroli generation test: A new methodology with a Brazilian endemic insect

This paper presents a new ecotoxicological test to investigate the response of a Brazilian endemic insect Chironomus sancticaroli through its life cycle and its future generations. This test can evaluate differences between the endpoints analyzed in diverse generations, describing the long-term impact of a substance or matrix effects along the time of exposure. Despite earlier papers already present the generation test with Chironomus riparius, there are still no studies with long-term test applied to C. sancticaroli. In this sense, this study evaluated different conditions for the development of a methodology that prolonged the duration of the test and allowed the best sampling of the organism on environmental toxicity tests. The distinct conditions tested were: volumes of test solution, frequency of feed, number of larvae, type of vessel and test solution replacement. The best condition for the C. sancticaroli generation test includes the exposition of 20 larvae to 340 or 500 mL of test solution, 60 g formulated sediment, feeding with Tetramin® each 10 days and, from a spawn, a new test will be prepared with the same characteristics of the previous one. This new methodology can reveal toxic effects along the exposure time and brings advances on toxicology area.•Prolonged testing makes it possible to analyze the long-term effects•The methodology "C. sancticaroli generation test" allows to evaluate the responses of organisms in different life cycles•The methodology allows to analyze the effects of substances in liquid medium and environmental quality through exposure to sediment