Keywords

1 Introduction

Caenorhabditis elegans is a non-parasitic nematode which, due to its many convenient features has become an important model in biology research; for example, it was the first animal whose genome was completely sequenced. This nematode was proposed as a model organism by Sydney Brenner in 1965 (Garcia-Sancho 2012). Since then, it has been used in cell biological, genetic and neurobiological studies of higher eukaryotes. Between 1970 and 1980, the complete cell lineage of this worm, from the fertilized egg to adult, was characterized by laser ablation and microscopy (Sulston et al. 1983). Electron microscopy and serial sectioning allowed for the reconstruction of the entire nervous system (White et al. 1986), together with the genetic and genomic data generated in the 1990s (Coulson et al. 1991). This organism has become a powerful tool for the discovery and functional characterization of eukaryotic genes (Dimitriadi and Hart 2010). Many aspects of C. elegans as a toxicological model have been reviewed in an excellent paper by Leung et al. (2008). In this state-of-the-art review, the authors present an update on that report, focusing on toxicity end points and assessments for many types of environmental pollutants.

2 Biological Features of C. elegans

The body of an adult C. elegans is approximately 1 mm long. Its transparency allows viewing of cell types in all stages of development. It has a simple nervous system of 302 neurons as an adult, where each neuron has a unique position (Dimitriadi and Hart 2010; Giles and Rankin 2009). Most organisms are hermaphrodites, with two ovaries, oviducts, a cavity for storing sperm called the spermatheca, and uterus (L’Hernault 2009). Hermaphrodites produce sperm as L4 larvae and oocytes during early adulthood; they reproduce by self-fertilization and therefore cannot fertilize other hermaphrodites. The males, which appear spontaneously with a frequency of less than 0.3 %, are able to fertilize hermaphrodites. The reproductive cycle of C. elegans lasts 2.5–4 days at room temperature, and with a usual lifespan of 12–20 days (Giles and Rankin 2009).

Embryonic development culminates in the generation of an L1 larva of 550 cells, after 113 cells have died by apoptosis. After four larval stages, the hermaphrodite worm becomes an adult organism with 959 cell nuclei (some syncytial), 302 of which are neurons. Males have 1031 cell nuclei. The mature adult is fertile for 4 days, and it can live between 10 and 15 additional days. Each adult hermaphrodite lays between 200 and 300 eggs, at intervals of about 20 min. Furthermore, the cycle time depends upon the temperature of incubation (Garcia-Sancho 2012). When environmental conditions are adverse, for example, during food shortages, high temperatures, or high population densities, successful reproduction is unlikely. Under such conditions, C. elegans can halt its development passing into an alternative L3 stage called dauer, which can survive for months. During this stage, the nematode does not feed and its cuticle is tougher. Nematodes can re-enter the reproductive life cycle at L4 when conditions are more favorable (Wang et al. 2010b). Figure 1 shows the complete cycle of C. elegans .

Fig. 1
figure 1

Life cycle of C. elegans. Images were acquired using a dissection microscope Nikon smz 745T with 4× magnification

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3 Advantages of Using C. elegans as a Biological Model

C. elegans is used as a model in genetic research because of its convenient features. First, its transparency allows for transgenic proteins fused to fluorescent markers to be visible in living animals in in vivo experiments (Giles and Rankin 2009). Its generation time is short (4 days) and it occurs by self-fertilization, ensuring rapid reproduction in the laboratory (Zhuang et al. 2014) since each adult hermaphrodite produces 200–300 progeny (Megalou and Tavernarakis 2009).

Its excellent performance as a model in genetics has led to the development of many tools and resources, including thousands of characterized mutants and RNA interference libraries, useful for silencing gene expression (Giles and Rankin 2009; Megalou and Tavernarakis 2009). RNA interference (RNAi) with this organism is relatively simple, and therefore gene silencing is often used to dissect signaling pathways (Adam 2009).

C. elegans has been used in toxicological research, from the whole animal level to the level of individual cells (Zhuang et al. 2014). It is cultured in the laboratory in a nematode growth medium (NGM), which contains NaCl, agar, peptone, cholesterol, K3PO4, KH2PO4, K2HPO4 and MgSO4. Another suitable culture medium is K agar, which also contains KCl (Meyer et al. 2010). The worms are maintained in an incubator at 20 °C and the bacteria Escherichia coli OP50 is utilized as a food source (Giles and Rankin 2009). The K medium prepared with KCl and NaCl is the liquid used to transfer worms to fresh dishes and to carry out bioassays (Williams and Dusenbery 1990).

4 Applications in Medicine

C. elegans is a model o rganism that has been important in the studies carried out to identify and understand the functioning of the machinery in nuclear transportation (Adam 2009). It has helped to elucidate biochemical pathways involved in diseases, such as obesity (Finley et al. 2013; MacNeil et al. 2013), diabetes (Estevez et al. 2014; Shi et al. 2012), and Alzheimer’s disease (Diomede et al. 2014; Lublin and Link 2013). C. elegans is an excellent model to investigate aging because of its short lifespan, its susceptibility to oxidative stress and the similarities with the human aging process (Chatterjee et al. 2013; Pang and Curran 2014). This nematode has also been employed to identify biochemical pathways and mechanisms of action of new drugs, especially antihelmintics (Kumarasingha et al. 2014; Lublin and Link 2013; Wu et al. 2012b).

5 Toxicity Endpoints

Bioassays to assess the effects of a toxicant on C. elegans can be carried out through different endpoints. The normal procedure for acute exposure consists of the incubation of young adults in the K medium containing the toxicant at several concentrations, usually without food. In long term exposure assays, worms in the L1 stage are used; in this case E. coli OP50 is added as food (Zhuang et al. 2014). When worm reproduction is not required during an experiment, since brood size may affect the results, 5-fluorodesoxiuridine is used to inhibit DNA synthesis (Wu et al. 2012a). Endpoints can be grouped according to their effects on biological parameters, for instance, lethality, growth, locomotion, and reproduction. It is also possible to use molecular markers to determine oxidative stress, changes in gene or protein expression, DNA damage, or green fluorescence protein (GFP) expression. A classification of endpoints, commonly utilized in toxicity research using C. elegans as model is shown in Fig. 2.

Fig. 2
figure 2

End points toxicity on C. elegans. Toxicity studies with C. elegans could be carry out through two kinds of endpoints, those evaluate effects in nematode biology and those use molecular markers

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Some of the frequently used endpoints related to toxicity assessment using C. elegans are presented below. These assays are usually performed employing concentration-response curves.

5.1 Lethality

This assay is performed to determine the death rate derived from acute toxicity in a concentration-response curve basis. 10 ± 1 young adults are transferred in microplates which contain different concentrations of the toxicant and a negative control. The exposure is carried out at 20 °C during 24 h in the absence of food. Then, the number of live and dead worms is counted through visual inspection using a dissecting microscope (Williams and Dusenbery 1990; Ellegaard et al. 2012; Helmcke and Aschner 2010; Kim et al. 2012; Wu et al. 2012a; Zhuang et al. 2014). Death is assumed when there is no movement during an observation period of 30 s (Rui et al. 2013; Shen et al. 2009; Wang et al. 2009a; Wu et al. 2013).

5.2 Growth

The effect of a toxicant in the development of the nematode can be evaluated by measuring the body length of synchronous worms before and after exposure, then comparing them to a vehicle-control. The bodies of the worms are observed employing a light microscope with 10X magnification and with image analysis software, such as Image-Pro® Express, ImageJ, or Fiji (Boyd et al. 2010; Cha et al. 2012; Höss et al. 2009b; Meyer et al. 2010; Roh and Choi 2011; Shen et al. 2009; Wang et al. 2010a; Yu et al. 2013a, b). Some authors have reported the warming of the worms to 50 °C in order to make them straight and ease the process of measuring their length (Wang et al. 2009a). The immobilization of the worms can also be achieved by using sodium azide (Turner et al. 2013). Growth can also be evaluated by registering the length of a curve, drawn from the tip of the head to the tip of the tail along the dorsal-ventral half of the animal intestine, using the reference line. Width measurements are taken in the vulva, drawing a line on the ventral side of the animal between the front edge and the posterior periphery of the vulva (Rudel et al. 2013). Other authors have proposed measuring the surface area for the flat worm (Rui et al. 2013; Wu et al. 2013; Zhuang et al. 2014). Currently, some laboratories have high-tech equipment, such as COPAS Biosort, which measures the optical density of the worm as an endpoint of growth (Hunt et al. 2012, 2013). The advantage is that the COPAS Biosort can analyze hundreds of nematodes per minute, and it can also evaluate mortality and fluorescence statistics (Hunt et al. 2012; Sprando et al. 2009). For growth assays, some authors perform 24 h exposure periods with E. coli OP50 as food (Boyd et al. 2010; Cha et al. 2012; Roh et al. 2009), whereas in other studies, E. coli uvrA, previously killed by UVA radiation is used; in this case, the exposure is carried out for 72 h and feeding is re-dosed every 24 h (Turner et al. 2013).

5.3 Reproduction

Brood size is the end point used to evaluate whether a toxic environment affects reproduction of the nematodes, placing exposed adult or L4 worms onto fresh plates. The number of offspring at all stages is counted and compared with a control group (Cha et al. 2012; Gomez et al. 2009; Höss et al. 2009b; Höss et al. 2013; Kim et al. 2012; Leelaja and Rajini 2013; Menzel et al. 2009; Li et al. 2012b; Roh et al. 2009; Rui et al. 2013; Smith et al. 2013; Wang et al. 2009a, 2010a, b). Counting is facilitated by heating the worms to 50 °C and staining them with Bengal red (Höss et al. 2013). In several studies the fertility rate is calculated by counting the total number of larvae at the end of the test and dividing by the total progeny recovered to the total parents (Rudel et al. 2013). This assay may also be carried out using the COPAS Biosort by measuring optical density (Boyd et al. 2010). Moreover, the gonad size, obtained by image analysis under a microscope, has been utilized to evaluate the effects on reproductive organs (Wu et al. 2011). Toxic effects may also be seen as changes in the egg-laying pattern and the number of eggs or larvae at different time intervals (Gomez et al. 2009; Smith et al. 2013). Finally, the rate of egg laying can be estimated by placing adult worms exposed to fresh plates and counting the number of eggs laid in 1 h (Jadhav and Rajini 2009; Shashikumar and Rajini 2010).

5.4 Fertility

Reproductive toxicity can also be assessed by calculating the percentage of L4 larvae that develop fertilized eggs after exposure. Gravid hermaphrodites are considered to have at least one egg inside their bodies (Höss et al. 2009a, b; Roh and Choi 2011; Wang et al. 2009a). To count the number of eggs in the uterus, nematodes can be transferred to a bleach solution, which dissolves the body of the worm, directly exposing the eggs and allowing them to be counted under a light microscope (Wu et al. 2011).

5.5 Lifespan

Healthy worms at the L4 larval stage are exposed to a toxic agent, for example 24 h and then placing them on NGM plates with E. coli OP50. To prevent the production of offspring, 5-fluorodeoxyuridine is added. Worms are transferred to new plates every 3 days. The number of survivors is recorded daily until all animals die. The survival rate is calculated by dividing the number of live nematodes by the total number of nematodes, including both live and dead worms. The lifespan is defined as the time period between the L4 larval stage and death (Cha et al. 2012; Li et al. 2009, 2012b; Shen et al. 2009; Wang et al. 2010a; Zhuang et al. 2014).

5.6 Intestinal Autofluorescence

The intestinal l ysosomal lipofuscin deposits that accumulate over time in the nematodes generate autofluorescence, feature used as a marker of aging. Treated nematodes are placed on an agar pad on a glass slide, then the fluorescent signals are captured by a fluorescence microscope. A band filter of 525 nm is employed to detect the endogenous intestinal fluorescence, and images are analyzed using software such as Magnafire®. Lipofuscin levels can be measured using the software ImageJ, by determining the mean pixel intensity in the intestine of each animal. Adults need to be photographed on the same day to avoid the light variation related to the intensity of the fluorescence source (Boyd et al. 2010; Helmcke and Aschner 2010; Rui et al. 2013; Shen et al. 2009; Wang et al. 2010a; Wu et al. 2012a, c, 2013; Zhuang et al. 2014).

5.7 Locomotion

Effects on the locomotion of nematodes have been linked to a deterioration of the neural network which can be evaluated based on several criteria, such as head thrash, body bend frequency, and basic movements (Yu et al. 2013a). Each exposed nematode is transferred to a plate containing 60 μL of K medium on the top of the agar. After a recovery period of 1 min, the number of head trashes is counted for 1 min. A head trash is defined as a change in the direction of bending in the body. To test the body bend frequency, nematodes are collected in a second plate, and then the number of times that the body bends in a period of 20 s is recorded. The bend of the body is observed as a change in direction of the upper pharynx along the Y axis, assuming that the nematodes are moved along the X axis. To test the basic movements, the number of sinusoidal forward movements is counted at an interval of 20 s. Locomotion behavior of control and treated nematodes should be analyzed simultaneously to avoid possible influences of the light-darkness cycle (Giles and Rankin 2009; Li et al. 2009, 2012a, b; Matsuura et al. 2013; Roh and Choi 2011; Rui et al. 2013; Wu et al. 2012a, 2013; Xing et al. 2009a; Yu et al. 2013a, b; Zhuang et al. 2014). Alternatively, immobility is determined by counting the number of immobile worms, usually registering a response when touched by platinum wire (Jadhav and Rajini 2009; Leelaja and Rajini 2013; Roh and Choi 2011).

5.8 Metabolism

To assess the state of metabolism , the pharyngeal pumping speed and the average cycle length of defecation can be evaluated. For testing the pumping rate, the nematodes are placed on NGM agar plates with food. After a few minutes, the pumping movement of the pharynx is counted for a minute under a microscope (Jadhav and Rajini 2009). To test the average cycle length of defecation, every nematode is observed individually for a fixed number of cycles. A cycle is defined as the interval between initiation of two successive steps of muscle contraction (Liu et al. 2013; Wu et al. 2012a; Zhao et al. 2014a, b).

5.9 Development

The effects of toxicants on the development of nematodes can be investigated by counting the number of individuals in each stage of their life cycle: egg, L1, L2, L3, L4 and adults, at regular time intervals up to 96 h after treatment (Roh and Choi 2011). The development through the larval stages can be estimated using the following criteria: L1 if they have four or fewer gonadal cells; L2 if they possess over four gonadal cells which have begun to spread along the length of the animal; L3 if there is a further extension of the gonad, and vulval morphogenesis has started; L4 if there is a dorsal rotation of the gonad; and adults if they have observable eggs (Helmcke et al. 2009). Entrance into the dauer state can be used to analyze toxicity, since dauer formation is induced by causing starvation in nematodes. Usually, treated nematodes in state of gravidity are placed on agar plates until laying eggs at 20 °C. This progeny is changed to 27 °C, and 72 h later. The organisms in the dauer stage are counted (Wang et al. 2010b).

5.10 Feeding Behavior

Some toxics can affect the feeding and foraging behavior in C. elegans. Jones and Candido (1999) described a procedure to assess feeding behavior by monitoring the decline in the density of the bacterial food in liquid cultures of nematodes by measuring absorbance at 550 nm. Another method consists of the use of agar with round holes located equidistant from the center of the dish. Each hole is filled with bacterial suspension in K medium. Toxic solutions are placed in different holes, and nematodes are inoculated in the center of the plate. The number of nematodes in the interior of each hole is counted at various intervals of time. This test shows whether the test nematodes try to avoid contaminated food (Monteiro et al. 2014).

5.11 Oxidative Stress

Several markers of oxidative stress can be determined in worms after exposure to the examined agent, both within the organism and the supernatant (Helmcke and Aschner 2010; Leelaja and Rajini 2012; Shashikumar and Rajini 2010). The production of reactive oxygen species (ROS) and oxidative damage may be determined by fluorescence measurements, usually labeling the nematodes with 5-(y-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (Eom et al. 2013; Leelaja and Rajini 2013; Li et al. 2012a; Rui et al. 2013; Wu et al. 2012a, 2013; Zhuang et al. 2014), and reading with a laser scanning confocal microscope (Eom et al. 2013; Helmcke and Aschner 2010; Rudgalvyte et al. 2013; Liu et al. 2012; Wang et al. 2010a; Wu et al. 2012a). Oxidative damage on macromolecules has been analyzed by detecting carbonylated proteins (Wang et al. 2010a, b; Wu et al. 2011). Moreover, oxidative stress can be evaluated by quantifying changes in gene expression of oxidative stress-related genes by Real Time PCR or GFP reporters, such as sod-1, sod-2, sod-3, sod-4, sod-5, gst-4, gst-5, gst-8, gst-24 and gst-42 (Rui et al. 2013).

5.12 Patterns of Gene Expression

One method used to investigate the change in expression of genes in C. elegans exposed to environmental pollutants is the use of DNA microarrays or Real Time PCR (Eom et al. 2013; Menzel et al. 2009; Li et al. 2012a; Roh et al. 2009; Roh and Choi 2011; Rudgalvyte et al. 2013). As reference genes, act-1 (Zhuang et al. 2014; Wang et al. 2014a) and ubq-1 (Wu et al. 2011) are commonly used. This method has been applied to evaluate the effect on C. elegans gene expression for various environmental toxicants, such as river sediments (Menzel et al. 2009), veterinary drugs (Zhuang et al. 2014), sodium fluoride (Li et al. 2012b), nanoparticles (Eom et al. 2013), and metals (Roh et al. 2009; Wang et al. 2014b; Rudgalvyte et al. 2013) among others.

5.13 Protein Expression

Induction of proteins by exposure to pollutants can be evaluated by traditional techniques such as ELISA and Western Blot. The ELISA technique was used to determine HSP90 protein expression in wild type C. elegans exposure to zinc at different temperatures (Wang and Ezemaduka 2014). Western blots were used to evaluate the effect of lead on HSP90 expression (Wang et al. 2014b).

5.14 DNA Damage

C. elegans has been used to evaluate DNA damage through various techniques. One approach is the use of the qPCR technique to detect damage and DNA repair. This test works on the principle that DNA damage inhibits the progression of the polymerase used in qPCR (Roh et al. 2009; Li et al. 2012b). The amount of long PCR product provides a measure of the frequency of the injury (Leung et al. 2010). Another alternative is the comet assay, which was used to evaluate the genotoxic profile of river sediment (Menzel et al. 2009). More recently, the pathway of base excision repair has been proposed as a mechanism to assess the damage to DNA by specific qPCR (Hunter et al. 2012). Transgenic strains can also be used to assess DNA damage. The strain xpa-1 is deficient in the mechanism of nucleotide excision repair, and its growth is significantly affected when there is damage to DNA. Therefore, the growth assay on this strain is an indicator of genotoxicity (Leung et al. 2010). The transgenic strain hus-1::GFP is utilized to assess DNA damage. HUS-1::GFP foci represents DNA double-strand breaks, allowing quantification by counting the number of bright foci per 20 pachytene gonadal germ cells (Hofmann et al. 2002) which can be observed and counted under a fluorescence microscope (Wang et al. 2014a).

5.15 GFP Reporters

Transgenic nematodes carrying the GFP gene fused to various stress-inducible gene promoters have been developed for the study of various biochemical pathways. GFP strains are placed in wells containing the sample solutions and suitable controls. The plates are incubated at 20 °C, performing fluorescence readings within 4–6, 8–20, and 24–40 h for short, moderate and long exposures, respectively. GFP expression is quantified using a fluorometer with a wavelength of 485 nm excitation and 525 nm emission (Anbalagan et al. 2012; Anbalagan et al. 2013; De Pomerai et al. 2010; Roh et al. 2010; Roh and Choi 2011). Alternatively, the observation of fluorescence can be achieved under a light microscope, capturing images that are then analyzed by specialized software (Li et al. 2009; Shen et al. 2009; Wang et al. 2010a; Polak et al. 2014). The COPAS Biosort system has also been employed to measure fluorescence (Hunt et al. 2012; Turner et al. 2013). The transgenic strain F25B3.3::GFP with fluorescence expression in neurons has been utilized to study heavy metal (Du and Wang 2009; Helmcke et al. 2009) and pesticide toxicity (Negga et al. 2011).

5.16 RNA Interference (RNAi)

This technology has been widely used to study gene function. Bacterial RNAi is introduced for 48 h at room temperature for the expression of dsRNA. Double stranded (ds) RNA expression is induced in HT115 bacteria containing the gene- sequence of interest inserted in the L4440 vector, or else the empty vector as control (Kamath and Ahringer 2003). Approximately ten nematodes in stages L1–L3 are placed on the plate seeded with induced RNAi or vector-control bacteria and incubated at 20 °C. After 36–40 h, the worms are transferred to another plate seeded with the same bacteria and grown to adulthood, at which point cultures are synchronised by egg isolation, and the eggs transferred onto new plates with RNAi bacteria. To evaluate the efficiency of dsRNA feeding over 1000 worms are evaluated by using semiquantitative PCR (Cheng et al. 2014; Kumar et al. 2010; Roh and Choi 2011). RNAi can be used to evaluate genetic pathways involved in toxicant responses. For instance, RNAi has been involved in the transcription of the DAF-16 factor in an unpredicted upregulation of the cyp-34A9 reporter gene by exposure to high levels of cadmium (De Pomerai et al. 2008). In another case, gene knockdown by RNAi was used to determine the effects on reproduction due to PCB52 exposure; several genes were identified as having a crucial role, being the most remarkable the cytochrome P450s group (Menzel et al. 2007).

5.17 Cell Apoptosis

To assess apoptosis in the cells of the nematode, acridine orange is used. After exposure to the toxicant for 24 h, the nematodes are immersed in mixed medium with acridine orange at 20 °C for 2 h. Then they are placed on top of agar allowing them to recover for 10 min. Finally, they are examined under an inverted fluorescence microscope with an excitation wavelength of 515 and 488 nm absorption. Apoptotic cells appear yellow or yellow-orange showing increased DNA fragmentation, whereas intact cells are uniformly green (Li et al. 2012b; Wang et al. 2009b, 2014a, c). Another technique involves staining with SYTO 12 for 4 h at room temperature, followed by seeding with food for 30 min, washing with M9 buffer, and final observation under a fluorescence microscope with a red filter (Cha et al. 2012). Alternatively, the transgenic strain ced-1::GFP is used for visualization of apoptotic bodies in a fluorescence microscope (Cheng et al. 2014; Kumar et al. 2010).

5.18 Cell Cycle Arrest

To investigate whether the exposure to a toxicant causes cell cycle arrest in the germline, the number of cores of mitotic cells is determined by staining with 4′,6-diamidino-2-phenylindole. The number of mitotic nuclei present at the distal end of the germline is counted under a fluorescence microscope (Cheng et al. 2014; Kumar et al. 2010; Wang et al. 2014a).

5.19 Transgenerational Effects

Sublethal endpoints such as locomotion and growth can be evaluated in the offspring of exposed parents. Wild-type N2 nematodes at the L3 larval stage are exposed during the time when sperm, ova and eggs begin to form, providing a window of prenatal exposure (Yu et al. 2013b). Exposed worms are placed on several plates. Some of them are used for measuring the parents after 24 h of exposure, and the others, for obtaining the generations. This assay has been used to assess the effects of antibiotics (Yu et al. 2011) and heavy metals on the growth and locomotion of exposed parents and their first generation (Yu et al. 2013b).

6 Toxicity Assessments

Most currently known toxicants can be assessed using C. elegans as a model. The following are some research studies related to toxicity of environmental matrices, metals, pesticides, nanoparticles, and other chemicals.

6.1 Environmental Samples

C. elegans has been used as a model to assess the toxicity of environmental samples such as soils, sludges, and river sediment . The sediment of the Danube, the Rhine and the Elbe Rivers in Germany were studied by analyzing the changes in gene expression profiling using DNA microarrays of the entire genome. At the same time, the reproduction and DNA damage were evaluated using the comet assay technique (Menzel et al. 2009). In a study of the toxicity of contaminated soils from Germany, fertility, growth, and reproduction were evaluated using the wild type Bristol N2 strain (Höss et al. 2009b). Organic extracts of contaminated soil from Spain were evaluated using transgenic strains of C. elegans carrying GFP reporter genes driven by promoters sequences from five stress-related genes, hsp-16.2, gpx-6, hsp-6, gst-1, and cyp34A9; allowing the identification of different mechanisms of toxicity (Anbalagan et al. 2012). Aqueous extracts of the same soils were evaluated using 24 similar GFP transgenic reporter strains, correlating this data with the concentrations of metals present in the soil (Anbalagan et al. 2013). A summary of the results generated from these investigations is shown in Table 1.

Table 1 Evaluation of environmental samples using C. elegans as a model
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6.2 Pesticides

In the environment, C. elegans as a free-living nematode, is exposed to various pesticides used in agriculture as well as to persistent organic waste that can contaminate soil for long periods of time (Anbalagan et al. 2013). Some of the most recent studies relating to the toxicity of pesticides in C. elegans are summarized in Table 2. As many pesticides are neurotoxic, the well-defined nervous system of C. elegans is a suitable tool to assess the neurotoxicity induced by these chemicals (Gomez et al. 2009; Leelaja and Rajini 2012, 2013; Lewis et al. 2013; Negga et al. 2011; Roh and Choi 2008, 2011; Shashikumar and Rajini 2010; Meyer and Williams 2014). Fluorescence expression by GFP reporter genes has been employed to study the toxicity of pesticides such as Glyphosate, Paraquat, 2,4-days, Endosulfan, Cypermethrin, Carbendazim, Chlorpyrifos, Diuron, Rotenone, DDT, Deltamethrin, and Dichlorvos (Anbalagan et al. 2013). In another report, Chlorpyrifos was studied, and although it did not cause severe DNA damage, it inhibited growth of xpa-1 deficient strain, whose mechanism of nucleotide excision repair is deficient (Leung et al. 2010). The herbicide Glyphosate and the fungicide dithiocarbamate have been studied to assess mortality and neurological damage in C. elegans. Neuronal damage by exposure to these pesticides was verified by using the transgenic strain F25B3.3::GFP (Negga et al. 2011). The effect of Paraquat, Diquat, and Parathion on brood size was evaluated with COPAS Biosort, with Paraquat showing the highest toxicity (Boyd et al. 2010). Acetylcholinesterase activity of pesticides has also been assessed in nematodes exposed to Fenitrothion and Monocrotophos (Leelaja and Rajini 2013; Roh and Choi 2011). Studies with tributyltin reported that this biocide caused cell apoptosis in C. elegans via DNA double-strand breaks (DSBs) (Wang et al. 2014a). Furthermore, tributyltin chloride caused increased sterility and embryonic lethality by DSBs and checkpoint activation in the germline (Cheng et al. 2014). Insecticidal proteins such as Cry, used in transgenic corn, were studied and showed dose-dependent inhibitory effects on C. elegans reproduction (Höss et al. 2013).

Table 2 Evaluation of pesticide toxicity
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6.3 Metals

Metals , in particular those named as heavy metals, constitute one of the most important groups of environmental toxicants, and reach the ecosystems from sources such as oil refineries, mining, and industrial effluents, causing severe toxic effects on living systems. This group has been one of the most studied using C. elegans as a biological model. Several of the reports related to the effects of heavy metals on C. elegans are presented in Table 3. The effects of different metals such as Ag, As, Cr, Cd, Cu, Hg, Mn, Pb, Ni and Zn have been studied for several end points such as lethality (Williams and Dusenbery 1990), lifespan, fertility, growth, intestinal autofluorescence, GFP expression, morphology changes (Shen et al. 2009; Rudel et al. 2013; Hunt et al. 2012), neuronal damage, neurodegeneration, neuronal loss, and axonal degradation (Du and Wang 2009; Xing et al. 2009a, b). On the other hand, exposure to Zn, Cd, Hg, Cu, Fe, Cr, and As has also been monitored using GFP transgenic reporter strains (De Pomerai et al. 2010).

Table 3 Evaluation of metals and their derivatives
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6.4 Nanoparticles

The toxicological potential of nanoparticles (NPs) is receiving increased attention because of their massive release into the environment. Although a number of manufactured NPs are employed for medical and clinical purposes, the interaction between nanomaterials and biological systems remains unknown. For this reason, the NPs have joined the group of Emerging Contaminants and every year more studies on the subject are performed with C. elegans (Table 4). It has been considered that the main mechanism of nanotoxicity is oxidative stress (Zhao et al. 2014a). Toxicity of hydroxylated fullerene nanoparticles was studied using C. elegans, and it was demonstrated that water-soluble fullerol NPs have a potential for inducing apoptotic cell death (Cha et al. 2012). The study of TiO2 NPs was carried out by analyzing different toxicity endpoints such as lethality, reproduction, growth, locomotion, intestinal autofluorescence, and oxidative stress. TiO2 NPs caused severe deficits in gut development, defecation behavior, and changes in gene expression (Rui et al. 2013; Zhao et al. 2014a). The toxicity of TiO2, ZnO, and SiO2 NPs has been compared using endpoints including lethality, locomotion, growth, reproduction, and production of ROS. The order of toxicity was ZnO > TiO2 > SiO2 (Wu et al. 2013). In a study of the intake of silver NPs by image analysis, there was absorption of silver NPs in the body, transgenerational transfer, and inhibition of growth (Meyer et al. 2010). The lethal effects of AgNPs on C. elegans are increased if the exposure is through E. coli OP50 (Ellegaard et al. 2012). In other research, reduction was observed in survival and reproduction and there was interaction of Ag NPs with biological surfaces of C. elegans, causing severe edema (Kim et al. 2012).

Table 4 Assessment of nanotoxicity
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6.5 Drugs

Drugs and their metabolites have been classified within the group of emerging contaminants because of increased concentrations in the environment. The impact of several drugs on organisms has been evaluated using C. elegans as a model (Table 5). For instance, the effects of nicotine in plasticity and locomotion (Matsuura et al. 2013; Sobkowiak et al. 2011), changes in gene expression (Smith et al. 2013), and changes in microRNA expression (Taki et al. 2014); other studies show the effects of caffeine and methadone on reproduction (Boyd et al. 2010); and the effects of sulphamethoxazol on locomotion and growth of offspring of exposed parents (Yu et al. 2011), and on reproduction, growth, lifespan, pharynx pumping, lipid peroxidation, and gene expression (Liu et al. 2013); and the effect of 5-fluorouracil on reproduction and development (Kumar et al. 2010) among others.

Table 5 Evaluation of the toxicity of drugs
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6.6 Toxins

Natural toxins have also been studied using C. elegans as a biological model (Table 6). Microcystin , a toxin produced by toxic blooms of cyanobacteria on eutrophic waters, produced changes in the behavior of locomotion and GFP expression in C. elegans (Moore et al. 2014; Li et al. 2009; Saul et al. 2014). Aflatoxin β1, generated by Aspergillus fungi, inhibited growth and formed adducts with DNA by activation of the Cytochrome P system (Leung et al. 2010).

Table 6 Evaluation of toxicity of toxins
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6.7 Other Chemicals

In addition to the above groups, C. elegans has been reported as a biological model for assessing the toxicity induced by other chemicals (Table 7) such as sodium fluoride (Li et al. 2012b), vinyl chloride (Nam and An 2010), benzo pyrene [a] and β-naphthoflavone (Leung et al. 2010), ethyl methanesulfonate and DMSO (Boyd et al. 2010), NaAsO2, NaF, Na2B4O7, valproic acid, caffeine and DMSO (Sprando et al. 2009); and acrylamide which has been shown to induce a gst-4::GFP transgene (Leung et al. 2011).

Table 7 Evaluation of the toxicity of other chemicals
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7 Conclusion

C. elegans is a powerful, suitable and robust model for toxicological studies due to the transparency of its body, its short life cycle, easy fertilization, economical maintenance in the laboratory, large numbers of offspring and easy genetic manipulation. The use of this nematode has enabled the understanding of many biochemical pathways activated by environmental toxicants, allowing the study of multiple endpoints including lethality, growth, reproduction, fertility, and locomotion among others. Additionally, the ease of obtaining transgenic nematodes allows the possibility of studying direct changes in gene expression induced by toxicants or mixtures. Finally, this model may be used in the assessment of toxicity of several pollutants such as environmental samples, metals, pesticides, nanoparticles, drugs, and toxins, among others.

8 Summary

Caenorhabditis elegans is a nematode of microscopic size which, due to its biological characteristics, has been used since the 1970s as a model for research in molecular biology, medicine, pharmacology, and toxicology. It was the first animal whose genome was completely sequenced and has played a key role in the understanding of apoptosis and RNA interference. The transparency of its body, short lifespan, ability to self-fertilize and ease of culture are advantages that make it ideal as a model in toxicology. Due to the fact that some of its biochemical pathways are similar to those of humans, it has been employed in research in several fields.

C. elegans’ use as a biological model in environmental toxicological assessments allows the determination of multiple endpoints. Some of these utilize the effects on the biological functions of the nematode and others use molecular markers. Endpoints such as lethality, growth, reproduction, and locomotion are the most studied, and usually employ the wild type Bristol N2 strain. Other endpoints use reporter genes, such as green fluorescence protein, driven by regulatory sequences from other genes related to different mechanisms of toxicity, such as heat shock, oxidative stress, CYP system, and metallothioneins among others, allowing the study of gene expression in a manner both rapid and easy. These transgenic strains of C. elegans represent a powerful tool to assess toxicity pathways for mixtures and environmental samples, and their numbers are growing in diversity and selectivity. However, other molecular biology techniques, including DNA microarrays and MicroRNAs have been explored to assess the effects of different toxicants and samples.

C. elegans has allowed the assessment of neurotoxic effects for heavy metals and pesticides, among those more frequently studied, as the nematode has a very well defined nervous system. More recently, nanoparticles are emergent pollutants whose toxicity can be explored using this nematode. Overall, almost every type of known toxicant has been tested with this animal model. In the near future, the available knowledge on the life cycle of C. elegans should allow more studies on reproduction and transgenerational toxicity for newly developed chemicals and materials, facilitating their introduction in the market. The great diversity of endpoints and possibilities of this animal makes it an easy first-choice for rapid toxicity screening or to detail signaling pathways involved in mechanisms of toxicity.