Various chemicals such as pesticides, plasticizers, and persistent pollutants are highly suspected to display endocrine-disrupting effects in animals and humans. Many studies have shown that environmental endocrine-disrupting chemicals could be the cause of reproductive disorders, endometriosis, and testicular cancer in animals and humans (Scippo et al., 2004).

The organochlorine pesticide chlordecone belongs to persistent organic pollutants, and demonstrates estrogenic activity by a series of assays such as MCF-7 cell proliferation and binding capacity to recombinant human steroid receptors (Hammond et al.,1979; Okubo et al., 2004; Scippo et al., 2004). Because it has been used in agriculture, a great quantity of chlordecone will eventually enter water bodies and affect aquatic organisms, including rotifers.

Rotifers are extremely important in freshwater ecosystems because their reproductive rates are the fastest among all the metazoan. They can convert primary (algal and bacterial) production into a form usable for secondary consumers (e.g., insect larvae, fish fry) (Nogrady et al., 1993). To date, some studies have been designed to investigate the effects of sublethal exposure to environmental endocrine-disrupting chemicals on the reproduction of rotifers (Ferrando et al., 1993; Ferrandez-Casalderrey et al., 1991, 1993; Gallardo et al., 1997, 2000; Janssen et al., 1994; Marcial et al., 2005; Preston et al., 2000; Preston and Snell, 2001; Radix et al., 2002; Rao and Sarma, 1986, 1990; Snell and Carmona, 1995; Xi and Feng, 2004), but none have dealt with chlordecone.

The main purpose of the current study was to assess the effects of sublethal exposure to chlordecone on development, survival, and reproduction of the freshwater rotifer Brachionus calyciflorus.

Materials and Methods

The rotifer B. calyciflorus used in this experiment was obtained by hatching resting eggs collected from sediments of Lake Jinghu and then clonally culturing them under controlled laboratory conditions. Stock rotifer cultures were kept at 25° ± 1°C on natural light and daily fed on 3 × 106 cells/mL of Scenedesmus obliquus. For mass cultures and for experiments, reconstituted hard water (Environment Protect Agency, EPA medium, prepared by dissolving 96 mg NaHCO3, 60 mg CaSO4, 60 mg MgSO4, and 4 mg KCl in 1 L of distilled water) (USEPA, 1985) was used as the medium. Algae were grown in a semicontinuous culture using HB-4 medium (Xi et al., 2001) renewed daily at 20%. Algae in exponential growth were centrifuged, then resuspended in EPA medium.

The pesticide chlordecone (standard grade, 99.8%; Sigma-Aldrich, Munich, Germany) was used as the toxicant. Stock solution of 1,000.0 mg/L was prepared by dissolving chlordecone in 100% acetone, then diluting it to the desired concentrations using EPA medium.

A preliminary range-finding test showed that 500 μg/L of chlordecone led to 100% mortality of the rotifers during the initial 24-h exposure, but that 50 μg/L of chlordecone did not cause any mortality. Therefore, we selected six toxicant concentrations (0.0005, 0.005, 0.05, 0.5, 5.0, and 50.0 μg/L), a blank control agent (EPA medium), and a solvent control agent (EPA medium and 0.5‰ [v/v] acetone), each comprising four replicates of 10 rotifers for this study.

The experiments were conducted in 24-well tissue culture plates and started by introduction of one neonate (<2 h old) into each well, which contained 0.5 mL of test solution with 3.0 × 106 cells/mL of S. obliquus. The rotifers were checked every 3 h during the initial 48 h, and the time of the first egg and neonate produced was recorded. Thereafter, the number of eggs and neonates produced and the number of original test individuals alive were recorded, and neonates were removed every 8 h. The original rotifers alive were transferred into freshly prepared test solution every 24 h. The life table experiments were conducted in darkness at 25° ± 1°C until each individual of every cohort had died.

On the basis of the data collected, the durations of principal development stages, mean life span, and lifetime egg production of the rotifers were calculated. Survivorship (lx) and fecundity (mx) were constructed for each cohort (replicate) using conventional life table techniques (Poole, 1974). Intrinsic rate of population increase (rm), net reproductive rate (R0), generation time (T), and life expectancy at hatching (e0) were calculated according to Krebs (1985) and Lotka (1913).

One-way analysis of variance (ANOVA), with concentration of chlordecone as the independent variable and each of the life history parameters as the dependent variable, followed by Dunnett’s test was conducted for pair-wise comparisons of each concentration of chlordecone and the solvent control agent relative to the blank control agent (Zar, 1999).

Results and Discussion

Chlordecone significantly influenced the durations of embryonic development, the juvenile period, the reproductive and postreproductive periods, the mean life span, and the lifetime egg production of the rotifers (p < 0.05, one-way ANOVA) (Table 1). As compared with the blank control agent, acetone at 0.5‰ had no marked effects on the durations of principal development stages, the mean life span, or the lifetime egg production of the rotifers (p > 0.05), but chlordecone at 5 and 50 μg/L shortened the duration of embryonic development (p < 0.01), exactly resembling the effects of glyphosate at 3 and 8 mg/L (Chu et al., 2005), but different from those of thiophanate-methyl and deltamethrin, which had no effect on the duration of embryonic development (Xi and Hu, 2003; Xu et al., 2005). Chlordecone at 5 and 50 μg/L prolonged the juvenile period of the rotifers (p < 0.01), similar to the effects of thiophanate-methyl at 1.2 and 1.8 mg/L, deltamethrin at 0.6 and 1.2 mg/L, and glyphosate at concentrations ranging from 3 to 10.5 mg/L (Chu et al., 2005; Xi and Hu, 2003; Xu et al., 2005), but contrary to those of deltamethrin at 2.4 and 3 mg/L (Xu et al., 2005).

Table 1. Mean values ± standard error of durations of embryonic development (ED), juvenile period (JP), reproductive period (RP), postreproductive period (PP), life span (ML) and lifetime egg production (NE) of Brachionus calycifloru s exposed to sublethal concentrations of chlordecone
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Chlordecone may directly or indirectly affect the duration of the juvenile period of the rotifers by altering the algal quality because the algal quality affects the duration of the juvenile period of the rotifers (Xi and Huang, 1999; Xi et al., 2001). However, chlordecone may directly affect only the embryonic development of the rotifers because the algal quality does not affect the duration of embryonic development of the rotifers (Xi and Huang, 1999). Further research should seek do discover how chlordecone directly affects the embryonic development time.

Chlordecone at 50 μg/L significantly prolonged the reproductive period of the rotifers (p < 0.05) (Table 1), exactly resembling the effects of deltamethrin at 0.6 and 1.2 mg/L, but contrary to those of deltamethrin at 2.4 and 3 mg/L (Xu et al., 2005), and different from those of glyphosate at 0.1 to 10.5 mg/L, which had no effect on the duration of the reproductive period of the rotifers (Chu et al., 2005). Chlordecone at 50 μg/L also prolonged the mean life span of the rotifers (p < 0.05) (Table 1), similar to the effects of glyphosate at 3 mg/L (Chu et al., 2005), but contrary to those of thiophanate-methyl at 0.6 to 4.2 mg/L and deltamethrin at 1.2 to 3.6 mg/L (Xi and Hu, 2003; Xu et al., 2005).

Chlordecone at 0.05 and 5 μg/L shortened the postreproductive period of the rotifers (p < 0.05) (Table 1), similar to the effect of deltamethrin at 1.2 to 3.6 mg/L (Xu et al., 2005), but different from that of glyphosate at 0.1 to 10.5 mg/L, which had no effect on the duration of the rotifers’ reproductive period (Chu et al., 2005).

Chlordecone at concentrations ranging from 0.5 to 50.0 μg/L increased the lifetime egg production of the rotifers (p < 0.05) (Table 1), in contrast to the effect of deltamethrin at 1.2 to 3.6 mg/L (Xu et al., 2005).

Based on the age-specific survival and fertility of the rotifers exposed to sublethal concentrations of chlordecone (Fig. 1), we calculated the intrinsic rate of population increase, the net reproductive rate, the generation time, and the life expectancy at hatching of the rotifers (Table 2). In contrast to the effects of all the other tested compounds such as pentachloro-phenol, 3,4-dichloroaniline, DDT, endosulfan, Cu, lindane, methylparathion, thiophanate-methyl, glyphosate, deltamethrin (Chu et al., 2005; Ferrandez-Casalderrey et al., 1991, 1993; Ferrando et al., 1993; Halbach, 1984; Janssen et al., 1993, 1994; Kooij- man and Metz, 1984; Rao and Sarma, 1986; Xi and Hu, 2003; Xu et al., 2005), chlordecone did not significantly influence the intrinsic rate of population increase or the life expectancy at hatching of the rotifers (p > 0.05, one-way ANOVA).

Fig. 1.
figure 1

The effect of sublethal exposure to chlordecone on the age-specific survivorship and fecundity of Brachionus calyciflorus. The arrow indicates the age at which the rotifers became reproductive

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Table 2. Intrinsic rate of population increase (rm), net reproductive rate (R0), generation time (T) and life expectancy at hatching (e0) of Brachionus calyciflorus exposed to sublethal concentrations of chlordecone (mean ± standard error)
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The reason for the disparity between our results and those of the aforementioned authors may be the difference in the concentration range of the tested compounds. In all of the aforementioned studies, the concentrations near to the LC50s were chosen to be the highest concentrations for all the compounds. In this study, however, considering the nominal concentration of chlordecone in natural water bodies, the concentration that did not result in mortality of rotifers during the initial 24-h exposure (50 μg/L) was chosen to be the highest concentration.

Chlordecone markedly influenced the net reproductive rate and the generation time of the rotifers (p < 0.05, one-way ANOVA). Compared with the blank control agent, acetone at 0.5‰ had no effect on the rotifers, but chlordecone at 0.5 to 50 μg/L increased the net reproduction rate (p < 0.05), which was identical to its effect on the lifetime egg production (Table 1), but contrary to the effects of pentachloro-phenol, 3,4- dichloroaniline, DDT, endosulfan, Cu, lindane, methylparathion, thiophanate-methyl, glyphosate, or deltamethrin on the net reproduction rates of B. calyciflorus and Brachionus patulus (Chu et al., 2005; Ferrandez-Casalderrey et al., 1991, 1993; Ferrando et al., 1993; Halbach, 1984; Janssen et al., 1993, 1994; Kooij-man and Metz, 1984; Rao and Sarma, 1986; Xi and Hu, 2003; Xu et al., 2005). In addition, chlordecone at 0.5 and 50 μg/L prolonged the generation time (p < 0.01), which contributed to the prolonged duration of the juvenile period (Table 1).

In conclusion, chlordecone at 5 and 50 μg/L shortened the duration of embryonic development, but prolonged the juvenile period and the generation time. Chlordecone at 50 μg/L prolonged the reproductive period and the mean life span. Chlordecone at 0.05 and 5 μg/L shortened the postreproductive period. Chlordecone at 0.5 to 50 μg/L increased the lifetime egg production and the net reproduction rate. However, chlordecone at 0.0005 to 50 μg/L did not significantly influence the intrinsic rate of population increase or the life expectancy at hatching of the rotifers, indicating that chlordecone at 0.0005 to 50 μg/L will not induce any significant effects on the growth of the natural B. calyciflorus population.