Introduction

High levels of perchlorate (ClO4 −) have often been associated with environmental contamination, which attracted public concern regarding ClO4 − in drinking water, groundwater, soils, and foods (plants) in recent years (Smith et al. 2004; Ye et al. 2013; Nerenberg 2013; Smith et al. 2015). The concentration of ClO4 − was found in soil, ranging from 0.001 to 216 mg ClO4 −/kg in Northern China and from 0.001 to 25.8 mg ClO4 −/kg in Southern China (Gan et al. 2014). Furthermore, soil could also act as a potential exposure source of ClO4 − for crops, vegetables, and wildlife (Smith et al. 2004; Jackson et al. 2005; Shi et al. 2007).

In particular, ClO4 − is easily accumulated in nonedible aquatic and terrestrial plants, including parrot-feather (Myriophyllum aquaticum), smartweed (Polygonum punctatum), pickleweed (Allenrolfea occidentalis), sweet gum (Liquidambar styraciflua), water lily (Nymphaea odorata), and black willow (Salix nigra) (Nzengung et al. 1999; Urbansky et al. 2000) and is detectable in forage and edible vegetables such as soybean, alfalfa, and tomato (Jackson et al. 2005). These studies generally suggest that ClO4 − is taken up and accumulated mainly in the aboveground portion of plants, especially in the foliage (Urbansky et al. 2000; Jackson et al. 2005; Smith et al. 2004). Therefore, ClO4 − accumulation in plants is a major source of ClO4 − exposure for terrestrial organisms including human beings and herbivorous insect that primarily feed on the leaves of such plants (Yu et al. 2004; Lee et al. 2012; Grantz et al. 2014).

As the direct biological measure of chemicals bioavailability in organisms, bioaccumulations in organisms should be determined firstly in order to investigate the mechanism of toxic effect. ClO4 − accumulation has been detected in the tissues of rodents, fish, frogs, and aquatic insect at a contaminated site (Smith et al. 2004; Sorensen et al. 2006; Attanasio et al. 2011). ClO4 − accumulation in cattle was evaluated by monitoring heifer calves on a site with access to 25 μg/L contaminated water for 14 weeks (Kirk et al. 2003). The ClO4 − concentrations detected in the blood plasma (15 and 22 μg/L) was two times of that in the heifer calves drinking ClO4 − -contaminated water on consecutive sampling periods 4 and 6 weeks after the beginning of ClO4 − exposure (Cheng et al. 2004).

ClO4 − accumulation in organisms results in pleiotropic chronic effects on growth and reproduction, which has been reported in mammals, lampreys, amphibians, earthworms, and so on (Goleman et al. 2002a, 2002b; Landrum et al. 2006; Redick-Harris 2006; Sorensen et al. 2006), with a dose-mediated response. For example, the growth of mosquitofish (Gambusia holbrooki) was enhanced at 1 mg/L but inhibited at 10 mg/L when the fry were exposed to ClO4 − at 1, 10, and 100 mg/L for 4 weeks (Park et al. 2006).

Some studies also have shown that effects of chemicals exposure on organisms differ according to developmental stages, genders (Hirsch et al. 2003; Cid et al. 2010; Zhang et al. 2011; Sun et al. 2013). For example, Devkota (1992) found that cadmium (Cd) concentration in Aiolopus thalassinus steadily increased with the growth of muscle tissues and fat bodies during post-embryonic development (nymph-adult), when adults were feeding Cd-contaminated food. Furthermore, Cd accumulation in female grasshoppers (Oedipoda germanica and Calliptamus italicus) (accumulation factors 2.97–4.35) was always higher than that in males (accumulation factors 2.15–3.09) (Devkota and Schmidt 2000). Besides, as demonstrated by Goleman et al. (2002a), ammonium perchlorate (AP) at 59–14,140 μg/L caused a sex ratio change, significantly reducing the percentage of Xenopus laevis males at metamorphosis.

As a link between the base of the food web (e.g., vegetables) and higher order consumers (e.g., predators, birds, chicken), whether herbivorous insects possibly can accumulate ClO4 − in their bodies or not, which ultimately pose potential risks to their survival and growth with the generational-, sex-specific patterns, has been never studied. The phytophagous insect Spodoptera litura is one of the most important herbivorous pests distributed widely in terrestrial organism. In natural conditions, S. litura larvae feeds mainly on crops such as cotton, soybean, groundnut, tobacco, and then pupate in the soil of farmland (Punithavalli et al. 2014). For our experiment, considering that an acceptable artificial diet has been used in research on S. litura for several years (Chen et al. 2000), we chose ClO4 −-contaminated artificial diets. Based on the ClO4 − levels reported in contamination environment (5–400 mg ClO4 −/kg) (Smith et al. 2004; Sorensen et al. 2006), the ClO4 − concentrations were used in the present study ranged from 0 to 200 mg ClO4 −/kg. The primary objective of this study was to test the hypothesis that ClO4 − bioaccumulations in S. litura (insect at different development stages, pupae, and adults from female and male) directly impact on their life-history traits with the generational-, sex-specific pattern.

Materials and methods

Insect rearing and ClO4 − treatment

Eggs of S. litura were provided by a laboratory colony maintained at the Insectarium of Institute of Tropical and Subtropical Ecology, South China Agricultural University. Upon hatching, the 1st instar larvae were fed fresh artificial diet in a 500-mL plastic-box until the experiments were carried out. When the larvae of S. litura reached the 3rd instar stage, they were fed diets with different ClO4 − concentrations [0 (control), 20, 40, 60, 80, 100, and 200 mg ClO4 −/kg wet weight artificial diet (termed mg ClO4 −/kg)]. The artificial diet was designed and made according to Chen et al. (2000) and Shu et al. (2015). Briefly, the artificial diet was composed of 96 % of total dried weight (44.5 % soy power, 26.7 % wheat bran, 17.8 % yeast exact power, and 7 % agar) and the remaining 4 % of the mixture consisted of L-ascorbic acid (1.8 %), Nipagin (0.9 %), sorbic acid (0.9 %), and cholesterol agar (0.4 %). Each diet was identical, expect for differences in ClO4 −. After being sieved to 100 μm, the mixture of soy power, wheat bran, and yeast exact power was blended with deionized water with a proportion of 1:2.75 (m/V). Then, they were mixed with agar which was melted in heated deionized water with 1:25 (m/V) and sterilized with high-pressure stream for 30 min. When the temperature of sterilized mixture dropped below 50 °C, the remaining ingredients and different doses of NaClO4 · H2O (Sinopharm, Shanghai, China) were dissolved in sterilized deionized water and blended with mixture. All treatments were carried out at constant conditions of 26 ± 1 °C, 75 ± 5 % relative humidity and a 16-h light/8-h dark photoperiod in a climatic chamber. Pupae and adults were kept under the same conditions.

ClO4 − determination in S. litura

Insect preparation

A total of the 150 3rd instar larvae with similar size and weight (0.045–0.055 g) per concentration were divided into six groups. Each group of the 25 3rd instar larvae was kept in a 200 mL plastic-box with adequate fresh artificial diet (more than 12.5 g, 0.25 g artificial diet/day per larvae). Each group represented a replication, and six replications were used for each treatment.

Body samples and feces (the larvae insect produced before molting) were collected from the 4th, 5th, 6th instar larvae. Two 4th, 5th, and 6th instar larvae were randomly selected per replicate, respectively. In addition, the two second-day male or female pupae, 24-h-old male or female adults, and pupae and adults with no distinguishable gender were randomly collected per replicate. Random three among the six replicates were applied for ClO4 − measurement and analysis. Seven ClO4 − concentrations of fresh artificial diet and insect samples were collected and oven-dried at 50 °C until used for ClO4 − extraction.

ClO4 − extraction of insect

The ClO4 − extraction was slightly modified according to He et al. (2013). Briefly, the dry insect samples described above and dry artificial diet were firstly cut into small pieces and mixed thoroughly. Then, 0.5 g samples were homogenized in 10 mL ultrapure water. The mixture was carefully transferred into a 50 mL conical flask and extracted in a mechanical shaker at 200 r/min for 4 h at room temperature. After the mixture was centrifuged for 25 min at 8000 r/min, the supernatant solution was sterile filtered with a 0.45 μm filter. And organic matter in the solution was removed by filtering through a C18 SPE column (ENVI-18). The filtrate was stored with headspace to reduce the potential for degradation by any remaining anaerobic organisms and hold for less than 15 days at 4 °C until ClO4 − determination (the storage method according to U.S. EPA 2005).

ClO4 − analysis

Analysis of ClO4 − was carried out using a Dionex ICS-900 Ion Chromatography System equipped as described by Jackson et al. (2005). The chromatographic column was an IonPac AG20-HC (4 × 50 mm) guard column and an IonPac AS20-HC (4 × 250 mm) analytical column, and the temperature was maintained at 30 °C. The mobile phase was 30 mM KOH, and the flow rate was 1.0 mL/min. After filtering through a 0.22 μm filter, 250 μL sample solution was injected into the system. The retention time of ClO4 − was 11.993 min. The concentration of ClO4 − in the samples was quantified by external calibration. Standard curves were calculated from the injection of 1 to 100 mg/L calibration standards.

Effects of ClO4 − exposure on survival and growth of S. litura

Fifty 3rd instar larvae with similar size and weight (0.045–0.055 g) from every concentration were chosen and transferred to plastic boxes (10 cm diameter) to be reared individually with adequate diet. Each set of bioassays was performed in triplicate. The number of surviving larvae was recorded each day. The number of larvae that successfully underwent pupation and emergence was also recorded. The survival rates of S. litura at different developmental stages were calculated as follows: survival rate of larvae = (the number of the 6th larvae/50) × 100; pupation rate = (the number of pupae/number of the 6th larvae) × 100; emergence rate = (the number of adults/number of pupae) × 100.

The life period of S. litura at different development stages was recorded, including the period of larvae (defined as the days from the starting of the experiment, i.e., the pupation of the 3rd instar larvae to pupae) and the period of pupae (defined as the days from the emergence of pupae to adults). In addition, the duration of the 1st and 2nd molting was also observed.

The body weights of the second-day male or female pupae, 24-h-old female and male adults were recorded. Additionally, the male-female ratio was recorded.

Statistical analysis

Significant difference analyses among treatment experiments were carried out by using SPSS (version 17.0). The data were conducted by one-way analysis of variance (ANOVA) and the means compared using significant difference (Tukey) method at 5 % level. A multi-factor ANOVA was used to distinguish the interaction effects of sex, developmental stage, and ClO4 − exposure concentration. The statistical significance of ClO4 − bioaccumulation in male and female was determined by an independent sample t test at the 0.05 level. Likewise, the significance of weights of male and female of pupae or adults was determined by an independent sample t test at the 0.05 level. The relationships between ClO4 − accumulations in the feces of the 4th, 5th, 6th instar larvae and pupae or adult S. litura and ClO4 − concentrations in the dry artificial diet were analyzed by linear regressions. The percentages of survival were arcsine square-root transformed before the analysis. Other data were log transformed when necessary to verify variance homogeneity.

Results

ClO4 − accumulation in feces of S. litura larvae

With the increase of ClO4 − concentrations in the fresh artificial diet, a significant concentration-dependent increase was shown in ClO4 − concentrations in the dry artificial diet. The regression equation of linear relationship between ClO4 − concentration in fresh diets (X-axis) and its corresponding concentration in dry diets (Y-axis) was Y = 4.115x + 120.9 (R 2 = 0.944, P = 0.001). ClO4 − accumulations in the feces of the 4th, 5th, and 6th instar larvae were shown in Fig. 1a. ClO4 − accumulations in the feces of 6th instar larvae fed on diets with 100 and 200 mg ClO4 −/kg were 1.73 and 2.48 times of that in the 4th instar larvae, respectively. Additionally, 100 and 200 mg ClO4 −/kg diet administration for the 6th instar larvae resulted in fecal concentrations 1.62 and 1.80 times that of the 5th instar larvae, respectively. ClO4 − accumulations in the feces of the 4th, 5th, and 6th instar larvae increased with the increase of ClO4 − concentrations in the dry artificial diets, following a dose-linear relationship (Table 1). A multi-factor ANOVA results showed that developmental stage individually and mutually affected the ClO4 − concentration in the feces (Table 2).

Fig. 1
figure 1

Perchlorate (ClO4 −) accumulation in the feces (a) and its bioaccumulation in the larvae of S. litura (B-larvae, C-pupa, adult) at different stages. ClO4 − was not detected in the control groups. All values are presented as mean ± standard error (n = 3)

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Table 1 Linear relationship between perchlorate (ClO4−) concentration in dry diet (X) and its residues in feces or bioaccumulation in the body (Y) at different larval stages
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Table 2 A multi-factor ANOVA of ClO4− accumulation in S. litura exposed to ClO4−
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ClO4 − accumulations in S. litura at different developmental stages

The ClO4 − accumulations in the 4th, 5th, 6th instar larvae, pupae and adults were shown in Fig. 1b, c, respectively. ClO4 − accumulations in pupae and adults were higher than those of the 4th, 5th, and 6th instar larvae. Furthermore, when insects were exposed to 80 mg ClO4 −/kg, the lowest ClO4 − concentration, 12.01 ± 0.21 mg ClO4 −/kg, was detected in the 6th instar larvae. The ClO4 − accumulations in adults reached 1302.87 ± 3.87 mg ClO4 −/kg. In addition, the ClO4 − accumulations in adults when larvae were fed on diets with 200 mg ClO4 −/kg were 1.85, 4.44, 23.62, and 60.96 times that in pupae, the 4th, 5th, and 6th instar larvae, respectively. ClO4 − accumulations in the adults, pupae, 4th, 5th, and 6th instar larvae increased with the increase of ClO4 − concentrations in the dry artificial diets and also exhibited a dose-response relationship. The regression equations of ClO4 − accumulations in adults, pupae, the 4th, 5th, and 6th instar larvae were shown in Table 1. In addition, a multi-factor ANOVA results showed that ClO4 − exposure concentration and developmental stage individually and mutually affected the ClO4 − accumulations in S. litura (Table 2).

ClO4 − accumulation in male or female pupae and adults

ClO4 − accumulations in male or female pupae and adults given diets with different ClO4 − concentrations were shown in Fig. 2. A multi-factor ANOVA results also showed that ClO4 − exposure concentrations had a significant differences in males and females of pupae and adults (Table 2). For insect given diets containing 200 mg ClO4 −/kg, the ClO4 − concentration in female pupae was 790.1 ± 2.59 mg ClO4 −/kg, significantly higher than that of male pupae (341.74 ± 1.64 mg ClO4 −/kg). Similarly, the ClO4 − accumulation in female adults (1268.48 ± 6.51 mg ClO4 −/kg) was significantly higher than that in male adults (848.36 ± 6.38 mg ClO4 −/kg) in the 200 mg ClO4 −/kg treatment. With increased ClO4 − concentration in the fresh artificial diet, a significant concentration-dependent increase was shown in ClO4 − accumulation in male and female pupae (Fig. 2A). Similarly, exposure to different levels of ClO4 − significantly affected the ClO4 − accumulations in male and female adults among treatments (Fig. 2B).

Fig. 2
figure 2

Sex-dependent perchlorate (ClO4 −) bioaccumulation in the pupae (A) and adults (B), respectively, after various ClO4 − exposures. All values are presented as mean ± standard error (n = 3) and bars with different letters represent significantly different means in the male or female groups (P < 0.05, Tukey’s test, one-way ANOVA). Independent sample t test was used to detect sex different in ClO4 − bioaccumulation at the 0.05 level by an asterisk

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Effect of ClO4 − exposure on survival and growth of S. litura

In all treatments, the differences in larvae survival, pupation, and emergence rates among the seven ClO4 − treatments were not significant (Fig. 3A). The lowest larvae survival, pupation, and emergence rates of the 200 mg ClO4 −/kg treatment were 85, 93, and 94 %, respectively. During the concentration of 200 mg ClO4 −/kg exposure, the duration of the first molting, larvae duration, and pupae duration were 1.70 ± 0.3, 11.42 ± 0.19, and 29.67 ± 0.58 days, respectively, which were significantly longer than those of the control (Fig. 3B).

Fig. 3
figure 3

Toxic effects of perchlorate (ClO4 −) on the survival, pupation, emergence rate (A) and different developmental stages, e.g., the first or second molting, larval and pupal development (B) in S. litura. All values are presented as mean ± standard error (n = 3) and bars with different letters represent significantly different means in the treatment groups (P < 0.05, Tukey’s test, one-way ANOVA)

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Effect of ClO4 − exposure on sex ratio and weights of female or male pupae and adults

When the 3rd instar larvae fed the artificial diet with different ClO4 − concentrations, the sex differentiation of S. litura was biased (Fig. 4). The sex ratios (number of male/number female pupae) were ranked in the following order: 200 mg ClO4 −/kg (0.40) < 100 (0.59) < 80 (0.81) < 60 (0.97) = 40 < 20 mg ClO4 −/kg (1.00) = control. Statistical analysis indicated that the weights of pupae and adults form ClO4 − treatments were significantly higher than those of control (Fig. 5). In addition, the weights of female pupae and adults exposed to 80–200 mg ClO4 −/kg were significantly higher than that of male pupae. The weights of females were significantly positively correlated with ClO4 − concentrations in the dry artificial diet (R = 0.818, P = 0.025 for female pupae weights; R = 0.843, P = 0.017 for female adult weights).

Fig. 4
figure 4

Effect of perchlorate (ClO4−) on sex differentiation of S. litura. All values are presented as mean ± standard error (n = 3) and bars with different letters represent significantly different means in the treatment groups (P < 0.05, Tukey’s test, one-way ANOVA)

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Fig. 5
figure 5

Toxic effects of perchlorate (ClO4−) on sex dependent in the pupae (A) and adults (B) weight, respectively. All values are presented as mean ± standard error (n = 3) and bars with different letters represent significantly different means in the male or female groups (P < 0.05, Tukey’s test, ANOVA). An asterisk represented for significances of sex-dependent in pupae or adults weights were analyzed by independent sample t test at 0.05 level

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Discussion

Our study showed that low concentrations (20–80 mg ClO4 −/kg) had no effect on insect survival, pupation and emergence rate (Fig. 3A). Insect receiving the highest ClO4 − exposure (100–200 mg ClO4 −/kg) had significantly longer larval development time (Fig. 3B). ClO4 − exposure appeared to have slight effects on earthworm (Eisenia fetida) reproduction at environmentally relevant concentrations. And the chronic ClO4 − exposure may also result in a reduction in earthworm weight, which could influence survival over the long term (Smith 2006; Landrum et al. 2006). Park et al. (2006) results also suggest that ClO4 − did not induce acutely toxic effects but may have had mild stimulatory or chronic effects on fitness parameters in mosquitofish (Gambusia holbrooki) at environmentally relevant concentrations (1–100 mg/L).

In the present study, effects of ClO4 − exposure on S. litura presented sex-specific pattern, where the weights of female were higher than that of males (Fig. 5). Furthermore, high ClO4 − concentrations (80–200 mg ClO4 −/kg) significantly decreased the male/female (Fig. 4), which was consistent with some studies that effects of ClO4 − exposure resulted in skewed sex ratios of organisms (Goleman et al. 2002a; Smith 2006; Mukhi et al. 2007). Goleman et al. (2002a) showed that environmentally relevant concentrations of AP inhibit thyroid function and alter sex ratios in developing Xenopus laevis. In addition, Mukhi et al. (2007) also found that exposure of larval-juvenile zebrafish to ClO4 − induced hypothyroid-like conditions and significantly skewed the phenotypic sex ratio towards females relative to the 1 male: 1 female ratio observed in untreated fish. The detail results shown that compared to the control group (48 % female), the sex ratio in both ClO4 − treatments showed a concentration-dependent trend to change in favor of females (100 ppm ClO4 −, 58 % female and 40 % male; 250 ppm ClO4 −, 65 % female and 34 % male). These results suggested that female S. litura larvae presented higher tolerance to higher ClO4 − exposure, and the different effects on S. litura were possibly found between two genders. Stone et al. (2002) pointed out that the female of ground beetles (Pterosthicus oblongopunctatus) had a stronger induction of detoxifying enzyme activity than male. Smith (2006) examined that long-term exposure to ClO4 − in mammal results in decreased plasma thyroid hormone concentrations, and alters gonadal differentiation. These effects were observed at AP inhibits thyroid activity, hormones and alters gonadal differentiation in developing organism. It was predicted that ClO4 − exposure may affect sex ratios of S. litura through alterations in hormone secretion and gonadal differentiation. However, the mechanism of ClO4 − exposure affecting on sex ratios need further investigation.

Our results clearly indicated that S. litura larvae could ingest ClO4 − from diets, and the larger amounts of ClO4 − could be excreted via feces. Furthermore, the different excretion was found in larvae at the different development stage, where ClO4 − accumulations in the feces was ranked in the order: the 6th > 5th > 4th instar larvae (Fig. 1a). This suggested that S. litura 6th instar larvae had increased the ability of excretion to reduce the ClO4 − accumulations in insect body. Our results of ClO4 − accumulations in body well reflected this case, where ClO4 − accumulation in the 6th instar larvae was lower than those of the 4th and 5th instar larvae. Groenendijk et al. (1999) found that the imagoes Chironomus riparius of Cd exposure showed a 100-fold increase in Cd body burdens in comparison with larval body burdens. This strongly suggests that a larger larvae is most likely attained by both a higher metal excretion capacity and a highly efficient capacity to shed accumulated ClO4 − during metamorphosis.

In our study, ClO4 − accumulations in insect body and ClO4 − exposure concentrations were concentration-dependent response. Patterns of ClO4 − accumulation in S. litura body differ according to different developmental stage and sex, where ClO4 − accumulation in larvae decreased gradually with the progression of development; in addition, the ClO4 − accumulation in adults and pupae were more than that of insect larvae (Fig. 1b, c ). A multi-factor ANOVA results also showed that sex and developmental stage individually and mutually affected the ClO4 − accumulations in the insects (Table 2). Besides, the ClO4 − accumulations in female pupae or adults were significantly higher than that of males after exposure (Fig. 2).

This developmental stage-, sex-specific accumulation pattern has been also found in the insects species exposed to other chemicals (Heliöväara and Väisänen 1990; Cid et al. 2010; Zhang et al. 2011; Kafel et al. 2012). For example, Osman et al. (2015) found that a significant increase in soil Cd concentration leading to sex-specific difference in Cd accumulation in ground beetle (Blaps polycresta). Sexual differences in Cd, Cu, and Mn bioaccumulation in males and females of aquatic insects (Ephoron virgo) also have been described by Cid et al. (2010).

It has been reported that the differences in pollutants bioaccumulation between male and female may have resulted in differences in body size (Teder 2014) and food digestion (Sun et al. 2013). Zhang et al. (2011) also showed that Cd accumulation in Chinese rice grasshopper (Oxya chinensis) lead to a significant differences in insect’s developmental stage and genders of antioxidant enzyme activities. In the present study, skewed sex ratios of female appeared to increase at higher ClO4 − concentrations. Furthermore, the ClO4 − accumulations in female insect were significantly higher than that of males. This suggested that the sex-specific difference in ClO4 − bioaccumulation was significant which might have led to the alteration in the sex ratio.

In summary, sex and developmental stage individually and mutually affected the ClO4 − accumulations in the S. litura. Besides, the ClO4 − accumulations in female pupae or adults were significantly higher than that of males after exposure. Furthermore, high ClO4 − concentrations significantly skewed the phenotypic sex ratio towards females and increased with the weights of female adults. McLain et al. (2015) found that the largest female Chauliognathus pennsylvanicus mated to the smallest male have an expected fecundity of about 141 eggs. However, the smallest female, mated to the smallest male, would be expected to produce 58 eggs. The research suggests that, on average, the increases in female size result in about half the gain in fecundity as mated to the smallest male. The previous results show that 80–200 mg ClO4 −/kg exposure leads to an increase in female adult size which may translate to higher fecundity of S. litura. Therefore, the further study is necessary to determine the effect of long-term exposure to ClO4 − on the fecundity and fertility of offspring insects, hormone secretion, and gonadal differentiation in developing insect.