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

Graphical abstract


Method details
Many authors have been utilizing Chironomus sancticaroli species in ecotoxicological bioassays, assessing the effects of contaminants on the development of the organisms using, for example, larvae length and adult wing length, especially for the first population generation. The present methodology, of population generation tests, can aid the researchers to obtain more detailed results in laboratory experiments and also may support sustainable decision-making by environmental agencies. Despite some authors have already used the idea of generation test on Chironomidae family, there is a lack of methods in the literature for the development of longer assays in Brazilian species [1,2]. In order to evaluate this gap, the present study proposes a methodology for the creation and accomplishment of long-term toxicity tests using C. sancticaroli (C. xanthus or C. domizii) in the laboratory, with the objective of covering more than one generation of the species and contributing to refine lethal and sublethal effects of diverse substances to the aquatic biota. This method can be useful in environmental ecotoxicological effects (water and sediment sample) of chemical substances and to analyze sublethal effects of substances in low concentration (parts per billion or parts per million).

C. sancticaroli cultivation and maintenance
The cultivation and maintenance conditions of C. sancticaroli followed previous methodology proposed by [3][4][5][6]. The first individuals of C. sancticaroli cultivated in laboratory were obtained from samples in stabilization ponds of a chicken slaughterhouse (BR-Aves) in São Carlos (São Paulo, Brazil).
Cultures of C. sancticaroli were kept in plastic trays (38 cm long Â 33 cm wide Â 6 cm high), covered by a nylon mesh support (42 cm long Â 36 cm wide Â 38 cm height) to prevent winged adult organisms from escaping from the growing area, cultures were maintained under constant aeration. Inside the trays there were 0.6 cm of sand treated as substrate and deionized water under the following conditions: conductivity between 25-55 mS cm À1 , hardness between 12 and 16 mg L À1 for CaCO 3 , pH between 6.5 and 7.5, temperature of 25 AE 2 C and photoperiod of 12 h light/ 12 h dark [7,8]. The feeding comprised the addition of macerated solid fish (Tetramin1), offered every 7 days, (5.75 mg L -1 of final concentration).

Method validation
The organisms cultivated in laboratory were used to evaluate the best condition to proceed the C. sancticaroli generation test. This long-term ecotoxicological tests were carried out involving the entire life cycle of the C. sancticaroli species. The conditions tested are described in Table 1. All the parameters were described for each replicate.
Beakers with different material were evaluated considering the material available in the laboratory and distinct volume was dependent on: the solution volume to ensure a suitable depth of overlapping water and sediment and headspace to guarantee the swarming and mating development. The pots were firstly locked with tulle netting, but as the evaporation became an intense process, a plastic lid was used to prevent both evaporation and the scape of the midges. On the same line of reasoning, the replacement of test solution was proceeded order to compensate the evaporation.
The frequency of feeding was reduced in order to provide subsistence of the larvae and to avoid the appearance of fungi. Moreover, the number of larvae added from the second generation was decreased (from 40 to 60 to 20) to preventing intraspecies competition (food and mobilization) and avoiding mortality.

Chironomus sancticaroli generation test
The condition that prolonged the duration of the test and allowed the best sampling of the organism was chosen as the best condition to proceed the C. sancticaroli generation test and is described as Assay: 6 ( Table 1).
The tests are conducted with 4 replicates, using 1 L glass beakers or non-toxic 500-militer plastic pots, 340 mL or 500 mL of test solution (depending on the vessel chosen), 60 g of formulated sediment and feed with 2.5 mg of macerated fish food (Tetramin 1 ). The pots have to be closed to prevent evaporation. The temperature varying between 23 and 27 C and photoperiod of 12 h light / 12 h dark [3,9].
1) The Parental (P) generation ( Fig. 1): Procedure a, b and c: To start the test, an egg rope is used and placed in a Petri dish under slow and constant aeration until the egg mass larvae are fully developed (about 48 h). After larvae hatch the egg rope, 20 organisms are selected for each replicate in a Petri dish using a stereomicroscope and a glass Table 1 The conditions tested and used to evaluate the best condition to proceed the C. sancticaroli generation test in six assays.

Conditions tested
Assay: Pasteur (arranged in lab watch glass). As recommended by Organization for Economic Co-operation and Development (OECD) [10], the addition of one replicate (from 3 to 4) can avoid larvae stress and allows to sample 20 larvae each time, used to observe the larvae length and buccal deformities endpoints. These organisms are placed in 1 L glass beakers or 500-militer plastic pots containing formulated sediment, test solution, feed with constant aeration and closed with tulle netting for glass beakers or with plastic lid for plastic pots to prevent evaporation. During the first 10 days after the test is set up, is necessary to observe the level of the test solution and, when necessary, replace it. On the 10th day, food is added to all replicates. Procedure d: At the 11th day all alive larvae on the forth replicate is sampled and fixed on isopropyl alcohol to observe it development (larval length).
Procedure e: It is important that after 10 days of test the presence of adults is observed daily to ensure that the midges will be sample and to make any relocation when male and female were in different replicate. From the first emergence until all larvae become midge, the number of male and female have to be count (males are easily identified by their plumose antennae and thin body posture).
Emerging adults should remain in vessels until oviposition. If necessary, the female or male midge can be relocated in other replicate at the same test solution to provide swarming, mating and oviposition. After confirming the oviposition, the females are captured with an aspirator and placed in Eppendorf containing isopropyl alcohol for analysis of potential fecundity.
Procedure f: After the oviposition, the egg rope is carefully collected.
1) The filial (F1 and F2) generation (Fig. 2): Procedure a, b, c, d, e and f: The procedures from a to f should be followed as described for P generation.
Procedure g: After the oviposition from the previous generation, the egg rope of each replicate should be placed in Petri dishes containing culture water and gentle aeration for 48 h until the larvae of the I instar are visible and start swimming. After this period, 4 replicates were assembled by adding 20 larvae in each, using stereomicroscope and glass Pasteur, with the same condition of the P generation (test solution), starting the next generation test (F1 or F2). As a result of this study we could formulate a new methodology for C. sancticaroli generation test. It is an advanced because there is a necessity for a new ecotoxicological analysis that permits to conclude the lethal and sublethal effects of a substance and to verify environmental quality of water and sediment through generations. An important difference between our methodology and OECD n.233 [10] is that the hole life cycle of the organism occurs at the same vessel. So, it is possible have the control of number of larvae and adult's emergence. This change minimizes disturbing and damaging of midges and, also allows the targeting of midges among the replicates, facilitating the swarming, mating and oviposition.
Possible endpoints to be analyzed: This procedure allows the sequencing of the analysis of possible genetic mutations in organisms, such as alterations in larval development (absence or excess of teeth), alterations in adult wing morphometry, decrease in female wing length (fecundity) and on the body of the larvae (development). Following this conception of test, it is possible to analyze the following endpoints: Mortality: the number of organisms is counted at the number of the test, and the mortality ratio is calculated for each replicate; Larvae length: the larvae sampled are fix at isopropyl alcohol and placed on a sheet of glass above a graph paper. The length of the larvae is estimated in Image J software; Buccal deformities: the cephalic capsule is placed on a sheet of glass and analyzed with 10x magnification under an optical microscope. The presence of gap, addition or absence of teeth are considered buccal deformity; Adult emergence ratio: the number of adult midges was taken daily, and the ratio of male/female is calculated; Potential fecundity: the fecundity is estimated by the wing length following the methodology described by Trivinho-Strixino [6] (1980).

Financial support
This research was supported by CAPES (Coordination for higher Education Staff Development) and CNPq (National Council of Technological and Scientific Development).

Additional information
For the monitoring of the test it is suggested the daily annotation of the parameters: date; number of emerged females; number of emerged males; number of spawning; any change between vessel (if any adult needs to be changed in order to allow copulation, it should be noted as relocation (initial vessel -final vessel) ( Table 2). Also, any observations on dead pupae, larvae, appearance of fungus and any other difference should be noted.