Chemosphere 120 (2015) 1–7
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Reproductive responses of the earthworm (Eisenia fetida) to antiparasitic albendazole exposure Yuhong Gao a,b,⇑, Xuemei Li a, Jianjun Guo d, Xinsheng Sun c, Zhenjun Sun b a
College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China College of Resources and Environmental Sciences, China Agricultural University, Beijing 100094, PR China c College of Information Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China d Animal Husbandry Research Institute of Chengde, Chengde, Hebei Province 067000, PR China b
h i g h l i g h t s Earthworms were exposed to albendazole. Albendazole affected reproduction of earthworms. The cocoon number was sensitive to low concentration of albendazole. The ultrastructure of germ cells was sensitive to low concentration of albendazole.
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 13 May 2014 Accepted 13 May 2014
Handling Editor: A. Gies Keywords: Earthworm Veterinary drug Reproduction Ultrastructure Ecotoxicology
a b s t r a c t Albendazole (ABZ) is a veterinary drug with a high efficiency against helminths. Here reproductive responses of earthworms Eisenia fetida to ABZ exposure (0, 1, 3, 6, 9 and 12 mg kg 1 soil dry weight) were investigated for 56 d in chronic reproduction test, and deformed sperm were counted and morphological alterations in the seminal vesicles were qualitatively assessed by light and transmission electron microscopy. Results have showed that cocoon number of earthworms was more sensitive to low concentrations of ABZ than cocoon hatching success and hatching survival, showing a significant dose-related decrease in cocoon number at 3, 6, 9 and 12 mg kg 1. In short-time exposure of 14 d, the sperm deformity (%) of earthworms increased at 6, 9 and 12 mg kg 1, and the microstructural alteration in seminal vesicles was also observed at these concentrations, whereas ultrastructural alteration of germ cells, particularly morphology of mitochondria, was observed at 3 mg kg 1 and above, suggesting the high sensitivity of germ cell ultrastructure to low concentrations of ABZ in short-time exposure. The results can provide important information for prediction of ecologically significant toxic effects. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In recent years veterinary drug residues have been the subject of many environmental investigations and have been predicted to pose potential risk to the health of terrestrial organisms (Svendsen et al., 2002; Jjemba, 2006; Jensen et al., 2007; Römbke et al., 2010; Junge et al., 2011; Müller et al., 2013). One of their major routes of entry into the environment is likely through manure amended soil containing veterinary drug residues. Published data showed that these residues may indeed be present in manure
⇑ Corresponding author at: College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China. Tel.: +86 03127528470. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.chemosphere.2014.05.030 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
spread to land (Kay et al., 2005) and that this may subsequently lead to acute and sub-lethal effects on soil environment (Chee-Sanford et al., 2001). Albendazole (ABZ), a derivative of benzimidazole, exhibits a broad spectrum of gastrointestinal anthelmintic activity, and it has been used widely in livestock as antiparasitic drugs against nematodes, cestodes and trematodes (McKellar and Scott, 1990). When administered orally, ABZ is partially metabolized and absorbed in the gastrointestinal tract, with the drug being excreted unchanged in faeces and urine. The main bioactive metabolite, albendazole-sulfoxide, also an effective anthelmintic (Daniel-Mwambete et al., 2004), is excreted to the environment as well. Only a limited amount of information is available, however, on the environmental fate and transport of benzimidazole antiparasitics within the soil matrix (Kreuzig et al., 2007; Kim et al., 2010; Moenickes et al., 2011). Moreover,
2
Y. Gao et al. / Chemosphere 120 (2015) 1–7
few studies have been published on the potential impacts of ABZ on soil-dwelling organisms (Gao et al., 2007, 2012). Earthworms are sentinels for terrestrial systems on account of their decomposition activity, nutrient mineralization and promotion of microfloral activities. Consequently they have been widely used in ecotoxicological studies attempting to resolve the deleterious effects associated with various chemicals exposure (Lowe and Butt, 2007; Amorim et al., 2012; Wu et al., 2012). Reproductive response in earthworms to chemicals stress have been considered as a sensitive endpoint for ecological risk assessment on veterinary drugs, pesticides, metals and other xenobiotic contamination. Traditionally, single reproductive endpoints in earthworms exposed to veterinary drugs are measured through simple parameters such as cocoon production, hatching success and hatching survival in chronic toxicity test under laboratory conditions. In reality, however, the occurrence and fate of veterinary drugs in soil is very complex, which makes prediction of the resulting effects rather difficult and requires the study of multiple endpoints (Flammarion et al., 2002). In previous studies, the reproduction test on spermatogenesis in earthworms has been recognized for the potential to assess sublethal or chronic toxicity of pesticides and metals (Reinecke and Reinecke, 1997; Bustos-Obregon and Goicochea, 2002; Xiao et al., 2006). However, only little attention has been paid specifically to the effect of veterinary drug residues on spermatogenesis in earthworms. Our previous studies on the effect of ABZ on the earthworm, Eisenia fetida, demonstrated that the growth rate decreased at high concentrations of 90 and 270 mg kg 1 during 28-day exposure, and that gene expression (HSP90) was induced at 10 mg kg 1 and above during 14-day exposure (Gao et al., 2012). However, the potential effects of ABZ on reproduction of earthworms are unclear. To better understand the environmental and biological effects of ABZ in soil, in this study we employed four approaches (chronic reproduction test, sperm deformity, morphological alterations in the seminal vesicles by light and transmission electron microscopy) to determine the reproductive responses of earthworms to ABZ. The examined endpoints included survival and growth of adult earthworm, cocoon number, cocoon hatching success and hatching survival, biomass of juveniles and cocoons, sperm deformity percentage, microstructure and ultrastructure in seminal vesicles. 2. Materials and methods 2.1. Earthworms and chemical E. fetida were obtained from an earthworm farm at China Agricultural University and they were kept in the test soil in laboratory for a week prior to the start of the experiment. The individual earthworms were adults with well-developed clitellum (350 ± 20 mg). ABZ was purchased from Sigma with the purity above 98%. 2.2. Soil preparation The artificial soil was prepared according to OECD guideline 207 (1984). The soil comprised (by dry weight) 20% kaolin clay, 10% sphagnum peat and 70% quartz sand with particle size 0.05– 0.2 mm. Calcium carbonate was added to adjust the pH of the wetted substrate to 6.0 ± 0.5 (Wu et al., 2012).
The ABZ treated soil was then transferred to 1 L glass cylindrical containers (1000 g dry soil per container), which were placed in a well ventilated hood for acetone to evaporate overnight. Water was added to compensate for the lost weight due to acetone evaporation and then earthworms were added. Controls were prepared in a similar way except acetone added only. Eight replicate containers for each ABZ concentration were used, with ten earthworms per container (4 containers for chronic reproduction test and the rest for sperm deformity, light- and electron-microscopic observation. After covered with perforated lids, the containers were incubated in an environmental chamber under 23 ± 1 °C with 12-h exposure to light each day. Water was supplemented to ensure appropriate water content of the substrate by weighing the containers (Jensen et al., 2003). Chronic reproduction in earthworms was analyzed at day 28 and 56 of exposure, and sperm deformity (%), light and electron micrographs in seminal vesicles were investigated at day 14 of exposure. 2.4. Chronic reproduction test To ensure the growth and cocoon production of earthworms during the experiment, uncontaminated horse manure was used as a food source for the earthworms (Spurgeon et al., 2004). Within the initial 28-day exposure, 5 g dried and finely ground manure was moistened with 5 mL of distilled water and added to the upper layer of the soil per container weekly. At day 28 of the experiment, surviving earthworms were removed from the soil, washed in distilled water, dried on paper towels and then weighed. The cocoons and hatched juveniles were also hand-sorted, counted and weighed, then returned and incubated for another 28 d. At day 56 of the experiment, the number and weight of cocoons and hatched juveniles were recorded. 2.5. Sperm deformity At day 14 of exposure, earthworms for sperm deformity test were put on humid filter paper at room temperature to empty their gut content. The individual earthworms were anesthetized by placing them on moist cold filter paper in petri dishes for 10 min, and then in cold distilled water for 5 min, which helped to relax muscles for dissection. The dissection was carried out on the dorsal side, after which seminal vesicles were detached. The seminal vesicles were placed between two glass slides, and by exerting pressure the sperm were forced out of the vesicles. The sperm were then fixed in methanol and stained in Giemsa. The slides were observed under a light microscope (image at 400) to detect abnormalities in sperm morphology. One thousand sperms were counted for every slide and the sperm deformity (%) was calculated (Zang et al., 2000). 2.6. Light-microscopic observation At day 14 of exposure, eight earthworms at each concentration of ABZ were removed from the test soil and their seminal vesicles were detached. According to Chuang et al. (2006), the seminal vesicles were cut into 0.5 0.5 cm pieces and fixed in Bouin solution for no longer than 24 h. The fixed tissues were dehydrated and embedded in paraffin. Slices (6 lm section) were obtained and stained in Mayer’s Hematoxylin/Eosin, and then observed under a light microscope (image at 400). 2.7. Electron-microscopic observation
2.3. Treatments ABZ was dissolved in acetone, followed by mixing into the soil at concentrations of 1, 3, 6, 9 and 12 mg per kilogram dry weight.
At day 14 of exposure, eight earthworms at each concentration of ABZ were removed from the test soil and their seminal vesicles were detached for ultrastructural examination. The seminal
3
Y. Gao et al. / Chemosphere 120 (2015) 1–7
analysis of variance (ANOVA). Data sets were checked for normality and homogeneity of variance with the Shapiro–Wilk test and Levene’s test, respectively, prior to analysis by ANOVA. Post-hoc comparison (LSD test) was also carried out to check for differences between the exposed and control groups. All statistical analysis was performed using SPSS software (SPSS19.0).
400 300 200 100 0
0
1
0 day
3. Results
28 days
3.1. Survival and growth of adult earthworm
3
6
9
No mortality for adult earthworms was found after 28 d of exposure to any concentrations of ABZ, and there was no significant difference in the weight of adult earthworms between the control and exposed groups (Fig. 1).
12
Concentration (mg/kg) Fig. 1. Change in weight of Eisenia fetida after 28 d of exposure to ABZ. Error bars represent standard deviations.
vesicles were immersed in primary fixative (2.5% Glutaraldehyde in Phosphate buffer). After the post-fixation with 1% osmium tetroxide, the tissue blocks were dehydrated through gradient acetone series (50–100%), embedded in Araldite mixture and polymerized at 60 °C, and then the samples were sectioned with an ultramicrotome (Leica Ultracut UCT, Austria) and examined with a transmission electron microscope (Hitachi H-7500, Japan) after being counterstained with uranyl acetate and lead citrate. 2.8. Statistical analysis Differences in chronic reproduction test or assessment of sperm deformity were tested for treatment significance using one-way
8
8
*
6
6
4
4
** **
2 0
0
1
3
6
**
9
After 28 d of exposure, the number of cocoons per earthworm showed a clear concentration–response relationship and was significantly reduced over the range of concentrations from 3 to 12 mg kg 1 (Fig. 2A). The average weight per cocoon over the range of concentrations from 1 to 9 mg kg 1 was 8.91 mg and showed no significant difference compared to the control group (P > 0.05). However, when earthworms were exposed to the highest concentration of ABZ (12 mg kg 1), the average weight per cocoon was reduced to 6.85 mg and showed a significant difference compared to the control group (P < 0.05). The effect of ABZ on the hatching success of cocoons was also investigated during the entire exposure time of 56 d. After initial 28 d of exposure, the hatching success at the concentrations of 6,
10
A
**
2
weight of per coccon (mg)
No. of cocoons per worm
10
3.2. Chronic reproduction analysis
100
Hatching success (%)
Weight per worm (mg)
500
**
60 40 20 0
1
4
**
3
C **
2
* 1
** 0
0
1
3
6
9
Concentration (mg/kg)
12
Biomass of juveniles and cocoons per worm (mg)
No.of juveniles per cocoon
28 days 56 days
3
*
**
6
9
** 12
Concentration (mg/kg)
Concentration (mg/kg) 5
**
28 days 56 days
0
0
12
B
80
D
80
* 60 40
** 20
28 days 56 days
0 0
1
3
6
9
12
Concentration (mg/kg)
Fig. 2. Reproductive responses of Eisenia fetida to ABZ during 56-day exposure. (A) Number (solid line) and weight (broken line) of cocoons after 28 d; (B) hatching success of cocoons; (C) juveniles number per cocoon; and (D) biomass of juveniles and cocoons per earthworm. Statistical significance vs. control group: P < 0.05, P < 0.01. Error bars represent standard deviations.
4
Y. Gao et al. / Chemosphere 120 (2015) 1–7
subsequent increase was also observed at the two concentrations of ABZ (P < 0.01). Biomass of juveniles and cocoons was not significantly different over the range of ABZ concentrations from 1 to 9 mg kg 1 compared to the control group (P > 0.05) (Fig. 2D). However, at the highest concentration of 12 mg kg 1, the biomass was significantly reduced after 28 d (P < 0.01) and after 56 d of exposure (P < 0.05). 3.3. Sperm deformity
Fig. 3. The sperm deformity of Eisenia fetida after 14 d of exposure to ABZ. Statistical significance vs. control group: P < 0.05, P < 0.01. Error bars represent standard deviations.
The deformity (%) of earthworm sperm was not affected at ABZ concentrations of 1 and 3 mg kg 1 after 14 d of exposure, whereas the sperm deformity (%) increased significantly at the concentrations of 6 mg kg 1 and above, varied from 7.2% to 14.8% at the concentrations between 6 and 12 mg kg 1, while 1.6% of sperm deformity in the control group (P < 0.05) (Fig. 3). 3.4. Microstructural changes of seminal vesicles
9 and 12 mg kg 1 showed a significant reduction to 11.0%, 8.3% and 3.0%, respectively, while 14.7% of hatching success in the control group (P < 0.05) (Fig. 2B). After 56 d of exposure, the hatching success of cocoons in all groups increased compared to the hatching success after initial 28-day exposure, however, the hatching success at the higher concentrations of 9 and 12 mg kg 1 was significantly lower than the control group, with 58.9% and 47.9% of hatching success in the two treatment groups respectively, while 82.8% of hatching success in the control group (P < 0.01). The number of hatched juveniles per cocoon at the lower concentrations of 1, 3 and 6 mg kg 1 was not significantly different compared to the control group after 28 and 56 d of exposure (P > 0.05) (Fig. 2C), however, at the higher concentrations of 9 and 12 mg kg 1, a significant decrease in juvenile number was observed during initial 28-day exposure (P < 0.05), and the
A
Mature sperm
Under the light microscope, in comparison with normal seminal vesicles (from control earthworms), there was no difference in spermatogenesis in seminal vesicles at the concentrations of 1 and 3 mg kg 1. In seminal vesicles of control earthworms, a very marked differentiation of germ cells was observed, showing different stages of germ cells (Fig. 4A). Furthermore, a high percentage of mature sperm bundles were also observed. However, in the range of concentrations from 6 to 12 mg kg 1, some damage in the germ cells occurred and was aggravated with increasing concentrations of exposure (Fig. 4B–D). The mature sperm bundles at 6 and 9 mg kg 1 decreased and the disordered distribution of germ cells was also observed at 9 mg kg 1. When earthworms were exposed to the highest concentration of 12 mg kg 1, the germ cells were damaged seriously, exhibiting the decreased number of germ cells and disordered distribution in seminal vesicles (Fig. 4D).
B
Late spermatid Early spermatid
Mature sperm
30 µm
C
30 µm
30 µm
D
30 µm
Fig. 4. Microstructural changes in seminal vesicles of Eisenia fetida after 14 d of exposure to ABZ (image at 400). (A) Normal microstructure from control earthworms, showing germ cells in differentiation stages (arrow) and mature sperm bundles were very obvious (arrowhead); (B) seminal vesicles at 6 mg kg 1, showing the decreased number of mature sperm; (C) seminal vesicles at 9 mg kg 1, showing the decreased number of germ cells and their disordered distribution; and (D) seminal vesicles at 12 mg kg 1, showing the decreased number of germ cells and their disordered distribution.
5
Y. Gao et al. / Chemosphere 120 (2015) 1–7
B
A
500 nm
C
500 nm
E
1.3 µm
D
1.0 µm
F
1.4 µm
G
1.0 µm
Fig. 5. Ultrastructural changes in seminal vesicles of Eisenia fetida after 14 d of exposure to ABZ. (A) Normal mitochondria in spermatids, showing cristae that project into the matrix from control earthworms (arrow). The normal nucleus containing dense areas of chromatin were observed (arrowhead); (B) parallel cisternae of endoplasmic reticulum around the nucleus in the cytophore of control earthworms (arrow); (C) damaged mitochondria of spermatids at 3 mg kg 1, showing fewer cristae, diminished matrix content and edema (arrow); (D) damaged cytoplasmic organelles and spermatid nuclear at 6 mg kg 1, exhibiting irregular membrane (higher arrow) and diminished matrix content in mitochondria (lower arrow), mild cytoplasmic edema, and chromatin swelling (arrowhead); (E) dilated cisternae of endoplasmic reticulum at 9 mg kg 1 (arrow); (F) damaged cytoplasmic organelles at 9 mg kg 1, exhibiting no structured vacuole (arrow), cytoplasmic edema and diminished cytoplasmic organelles; and (G) seriously damaged spermatid at 12 mg kg 1, showing cytoplasmic edema, mitochondria collapse (arrow), partly dissolved nuclear membrane and chromatin edema (arrowhead).
3.5. Ultrastructural changes of seminal vesicles Under the electron microscope, some normal mitochondria and nucleus were observed in spermatids in the seminal vesicles of
control earthworms (Fig. 5A). In contrast, earthworms exposed to the concentration range from 3 to 12 mg kg 1 exhibited marked abnormalities in spermatids (Fig. 5C–G). The mitochondria were injured by ABZ and exhibited a progressive degeneration with
6
Y. Gao et al. / Chemosphere 120 (2015) 1–7
increasing ABZ concentrations. At the lower concentrations of 3 and 6 mg kg 1, some mitochondria showed diminished matrix content, irregular membranes and fewer cristae (Fig. 5C and D). When earthworms were exposed to the higher concentrations of 9 and 12 mg kg 1, the morphology of mitochondria was seriously damaged and exhibited the loss of inner structure and partly dissolved membrane (Fig. 5F), even collapsed at 12 mg kg 1 (Fig. 5G). The cytoplasmic edema was also observed in spermatids of earthworms exposed to 6 mg kg 1 and became more serious with increasing ABZ concentrations, accompanied with diminished the number of cytoplasmic organelles. Spermatid nuclear in exposed earthworms was damaged, exhibiting chromatin edema at 6 and 12 mg kg 1 and the nuclear membrane partly dissolved at 12 mg kg 1. In addition, the cisternae of endoplasmic reticulum in the cytophore were dilated significantly at 9 mg kg 1 compared to parallel endoplasmic reticulum in the control group (Fig. 5B and E).
4. Discussion The choice of sensitive parameters is crucial in assessing the potential toxicity of low concentrations exposure to chemicals in soil. The present study evaluated reproductive responses of the earthworm E. fetida to various concentrations of ABZ in multiple experiments. The results demonstrated that ABZ significantly affected the reproduction of adult earthworms, while survival and growth of adult earthworms were not affected for all ABZexposed groups. These effects could be attributed to a diversion of resources towards increasing maintenance and repair (Anderson et al., 2013). Lika and Kooijman (2003) stated that adult earthworms were likely to divert the allocation of resources away from reproduction, towards maintenance in cases where individuals were exposed to sub-optimal conditions. In this work, in response to low concentrations of ABZ, cocoon number was more sensitive than other reproductive parameters (cocoon weight, cocoon hatching success and hatching survival, biomass of juveniles and cocoons) during initial 28 d of exposure, showing a significant decrease at 3 mg kg 1 and above in a concentration-dependent manner. Previous studies demonstrated that chemicals affected the earthworm reproduction (cocoon production and hatching success) (Zhang et al. (2008); Alves et al., 2013) and previous work by Zhang et al. (2008) indicated that hatching success of cocoons was more sensitive to chemicals than cocoon production, which was in opposition to our result. The reason behind this difference is unclear. For this increasing toxicity on cocoon production, the possible explanation may be that the uptake of ABZ and its metabolite by earthworms depended on some combination of both ingestion of gastrointestinal contents and diffusion through external surface (Shalaby et al., 2009). For the toxicity on hatching success of cocoons, the possible explanation may be that earthworm cocoons absorbed this drug and its metabolite only by diffusion of eggshell. Therefore, during initial 28 d of experiment, the cocoon hatching success and hatched juvenile number were affected starting at concentrations of 6 and 9 mg kg 1, respectively, while the cocoon number was affected starting at 3 mg kg 1. With longer exposure time, the effect of ABZ on cocoon hatching success increased at higher concentrations. It may be attributed to the accumulation of ABZ or its degradation products in cocoons, which resulted in the increase of toxicity. Nevertheless, there is still lack of information on the fate of this drug in soil. Future studies should focus on the degradation, absorption and transformation of ABZ residues in soil, which is an extremely challenging task. It is worth noting that biomass of earthworms was not affected by ABZ concentrations below 12 mg kg 1 during entire exposure
time, despite other endpoints in chronic reproduction test were found to be sensitive to low concentrations of this drug. Published research suggested that biomass change of earthworms could be a good indicator of chemicals stress and link chemicals toxicity to energy dynamics and ultimately growth inhibition (Wu et al., 2012; Anderson et al., 2013). In this study, however, biomass change of earthworms was not optimal to assess the low concentrations of ABZ in soil. In the present study, a significant effect in spermatogenesis of earthworms was observed for short-term exposure of ABZ. The result suggests that the ultrastructural alterations of germ cells in seminal vesicles took place before the microstructural alterations, showing mitochondria in germ cells were damaged at 3 mg kg 1 of ABZ and above, while both the microstructural alterations and the increase of sperm deformity (%) were observed at 6 mg kg 1 and above. Therefore, the ultrastructural alterations of germ cells are speculated to be more sensitive to low concentrations of ABZ exposure than their microstructural alterations. Particularly, morphological alterations of mitochondria showed a progressive degeneration with increasing ABZ concentrations. It is well known that fundamental metabolic processes take place in the mitochondria, such as electron transport, oxidative phosphorylation, catabolism of fatty acids and amino acid metabolism (Reichert and Neupert, 2002). The damages of mitochondria might demonstrate a lower functional activity of the mitochondria followed by disturbing the pathways of energy metabolism. Our previous work concluded that the morphology of mitochondria in intestinal epithelium was damaged when earthworms were exposed to ABZ, which is in accordance with our present work (Gao et al., 2007). Though the sensitivity of microstructure in seminal vesicles and sperm morphology under a light microscope to ABZ was lower than that of ultrastructure of germ cells, these histological alterations can still provide crucial information for determining the short-time ecological implication of exposure (Reddy and Rao, 2008). Histopathological responses in earthworms have been reported as valuable indicators of terrestrial contamination in previous studies (Giovanetti et al., 2010; Kilic, 2011). The results of this study can shed light on early sub-lethal effects of ABZ on germ cell structure of earthworms.
5. Conclusions The reproductive responses of the earthworm E. fetida were observed during 56 d of exposure, while the growth and survival of adult earthworms were not affected. The result of this study suggests the cocoon number of earthworms was more sensitive to low concentrations of ABZ than other reproductive parameters (cocoon hatching success and hatching survival), with a significant decrease in cocoon number at 3 mg kg 1 and above. In short-term exposure of 14 d, the microstructural alterations in seminal vesicles and the increase of sperm deformity (%) were observed at 6 mg kg 1 and above, whereas the ultrastructure of germ cells, particularly morphology of mitochondria, were significantly affected at 3 mg kg 1 and above, suggesting the ultrastructure of germ cells is more sensitive than histological microstructure. The characterization and understanding of reproductive responses of earthworms to ABZ on multiple traits can provide important information for prediction of the pollution of ABZ residues in soil.
Acknowledgment This research was sponsored by Hebei Natural Science Foundation in China (No. C2012204074).
Y. Gao et al. / Chemosphere 120 (2015) 1–7
References
Alves, P.R., Cardoso, E.J., Martines, A.M., Sousa, J.P., Pasini, A., 2013. Earthworm ecotoxicological assessments of pesticides used to treat seeds. Chemosphere 90, 2674–2682. Amorim, M.J.B., Pereira, C., Menezes-Oliveira, V.B., Campos, B., Soares, A.M., Loureiro, S., 2012. Assessing single and joint effects of chemicals on the survival and reproduction of Folsomia candida (Collembola) in soil. Environ. Pollut. 160, 145–152. Anderson, C.J., Kille, P., Lawlor, A.J., Spurgeon, D.J., 2013. Life-history effects of arsenic toxicity in clades of the earthworm Lumbricus rubellus. Environ. Pollut. 172, 200–207. Bustos-Obregon, E., Goicochea, R.I., 2002. Pesticide soil contamination mainly affects earthworm male reproductive parameters. Asian J. Androl. 4, 195–199. Chee-Sanford, J.C., Aminov, R.I., Krapac, I.J., Garrigues-Jeanjean, N., Mackie, R.I., 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494–1502. Chuang, S.C., Lai, W.S., Chen, J.H., 2006. Influence of ultraviolet radiation on selected physiological responses of earthworms. J. Exp. Biol. 209, 4304–4312. Daniel-Mwambete, K., Torrado, S., Cuesta-Bandera, C., Ponce-Gordo, F., Torrado, J.J., 2004. The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs. Int. J. Pharm. 272, 29–36. Flammarion, P., Devaux, A., Nehls, S., Migeon, B., Noury, P., Garric, J., 2002. Multibiomarker responses in fish from the Moselle River (France). Ecotoxicol. Environ. Saf. 51, 145–153. Gao, Y., Sun, X., Gu, X., Sun, Z., 2012. Gene expression responses in different regions of Eisenia fetida with antiparasitic albendazole exposure. Ecotoxicol. Environ. Saf. 89, 239–244. Gao, Y., Sun, Z., Sun, X., Shi, W., 2007. Toxic effects of albendazole on Adenosine triphosphatase activity and ultrastructure in Eisenia fetida. Ecotoxicol. Environ. Saf. 67, 378–384. Giovanetti, A., Fesenko, S., Cozzella, M.L., Asencio, L.D., Sansone, U., 2010. Bioaccumulation and biological effects in the earthworm Eisenia fetida exposed to natural and depleted uranium. J. Environ. Radioact. 101, 509–516. Jensen, J., Diao, X., Scott-fordsmand, J.J., 2007. Sub-lethal toxicity of the antiparasitic abamectin on earthworms and the application of neutral red retention time as a biomarker. Chemosphere 68, 744–750. Jensen, J., Krogh, P.H., Sverdrup, L.E., 2003. Effects of the antibacterial agents tiamulin, olanquindox and metronidazole and the anthelmintic ivermectin on the soil invertebrate species Folsomia metaria (Collembola) and Enchytraeus crypticus (Enchytraeidae). Chemosphere 50, 437–443. Jjemba, P.K., 2006. Excretion and ecotoxicity of pharmaceutical and personal care products in the environment. Ecotoxicol. Environ. Saf. 63, 113–130. Junge, T., Meyer, K.C., Ciecielski, K., Adams, A., Schäffer, A., Schmidt, B., 2011. Characterization of non-extractable 14C- and 13C-sulfadiazine residues in soil including simultaneous amendment of pig manure. J. Environ. Sci. Health, Part B 46, 137–149. Kay, P., Blackwell1, P.A., Boxall, A.B.A., 2005. A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with field data. Environ. Pollut. 134, 333–341.
7
Kilic, G.A., 2011. Histopathological and biochemical alterations of the earthworm (Lumbricus terrestris) as biomarker of soil pollution along Porsuk River Basin (Turkey). Chemosphere 83, 1175–1180. Kim, H.J., Lee, D.S., Kwon, J.H., 2010. Sorption of benzimidazole anthelmintics to dissolved organic matter surrogates and sewage sludge. Chemosphere 80, 256– 262. Kreuzig, R., Blumlein, K., Holtge, S., 2007. Fate of the benzimidazole antiparasitics flubendazole and fenbendazole in manure and manured soils. Clean Soil Air Water 35, 488–494. Lika, K., Kooijman, S., 2003. Life history implications of allocation to growth versus reproduction in dynamic energy budgets. Bull. Math. Biol. 65, 809–834. Lowe, C.N., Butt, K.R., 2007. Earthworm culture, maintenance and species selection in chronic ecotoxicological studies: a critical review. Eur. J. Soil Biol. 43, S281– S288. McKellar, Q., Scott, E., 1990. The benzimidazole anthelmintic agents—a review. J. Vet. Pharmacol. Ther. 13, 223–247. Moenickes, S., Höltge, S., Kreuzig, R., Richter, O., 2011. Process dominance analysis for fate modeling of flubendazole and fenbendazole in liquid manure and manured soil. Sci. Total Environ. 410–411, 226–234. Müller, T., Rosendahl, I., Focks, A., Siemens, J., Klasmeier, J., Matthies, M., 2013. Short-term extractability of sulfadiazine after application to soils. Environ. Pollut. 172, 180–185. Reddy, N.C., Rao, J.V., 2008. Biological response of earthworm, Eisenia foetida (Savigny) to an organophosphorous pesticide, profenofos. Ecotoxicol. Environ. Saf. 71, 574–582. Reichert, A.S., Neupert, W., 2002. Contact sites between the outer and inner membrane of mitochondria-role in protein transport. Biochim. Biophys. Acta 1592, 41–49. Reinecke, S.A., Reinecke, A.J., 1997. The influence of lead and manganese on spermatozoa of Eisenia fetida (Oligochaeta). Soil Biol. Biochem. 29, 737–742. Römbke, J., Krogh, K.A., Moser, T., Scheffczyk, A., Liebig, M., 2010. Effects of the veterinary pharmaceutical ivermectin on soil invertebrates in laboratory tests. Arch. Environ. Contam. Toxicol. 58, 332–340. Shalaby, H.A., Abdel-Shafy, S., Abdel-Rahman, K.A., Derbala, A.A., 2009. Comparative in vitro effect of artemether and albendazole on adult Toxocara canis. Parasitol. Res. 105, 967–976. Spurgeon, D.J., Stürzenbaum, S.R., Svendsen, C., Hankard, P.K., Morgan, A.J., Weeks, J.M., Kille, P., 2004. Toxicological, cellular and gene expression responses in earthworms exposed to copper and cadmium. Comp. Biochem. Physiol. Part C 138, 11–21. Svendsen, T.S., Sommer, C., Holter, P., Grønvold, J., 2002. Survival and growth of Lumbricus terrestris (Lumbricidae) fed on dung from cattle given sustainedrelease boluses of ivermectin or fenbendazole. Eur. J. Soil Biol. 38, 319–322. Wu, S., Zhang, H., Zhao, S., Wang, J., Li, H., Chen, J., 2012. Biomarker responses of earthworms (Eisenia fetida) exposured to phenanthrene and pyrene both singly and combined in microcosms. Chemosphere 87, 285–293. Xiao, N., Jing, B., Ge, F., Liu, X., 2006. The fate of herbicide acetochlor and its toxicity to Eisenia fetida under laboratory conditions. Chemosphere 62, 1366–1373. Zang, Y., Zhong, Y., Luo, Y., Kong, Z.M., 2000. Genotoxicity of two novel pesticides for the earthworm, Eisenia fetida. Environ. Pollut. 108, 271–278. Zhang, B., Cox, S.B., McMurry, S.T., Jackson, W.A., Cobb, G.P., Anderson, T.A., 2008. Effect of two major N-nitroso hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites on earthworm reproductive success. Environ. Pollut. 153, 658–667.
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Reproductive responses of the earthworm (Eisenia fetida) to antiparasitic albendazole exposure Yuhong Gao a,b,⇑, Xuemei Li a, Jianjun Guo d, Xinsheng Sun c, Zhenjun Sun b a
College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China College of Resources and Environmental Sciences, China Agricultural University, Beijing 100094, PR China c College of Information Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China d Animal Husbandry Research Institute of Chengde, Chengde, Hebei Province 067000, PR China b
h i g h l i g h t s Earthworms were exposed to albendazole. Albendazole affected reproduction of earthworms. The cocoon number was sensitive to low concentration of albendazole. The ultrastructure of germ cells was sensitive to low concentration of albendazole.
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 13 May 2014 Accepted 13 May 2014
Handling Editor: A. Gies Keywords: Earthworm Veterinary drug Reproduction Ultrastructure Ecotoxicology
a b s t r a c t Albendazole (ABZ) is a veterinary drug with a high efficiency against helminths. Here reproductive responses of earthworms Eisenia fetida to ABZ exposure (0, 1, 3, 6, 9 and 12 mg kg 1 soil dry weight) were investigated for 56 d in chronic reproduction test, and deformed sperm were counted and morphological alterations in the seminal vesicles were qualitatively assessed by light and transmission electron microscopy. Results have showed that cocoon number of earthworms was more sensitive to low concentrations of ABZ than cocoon hatching success and hatching survival, showing a significant dose-related decrease in cocoon number at 3, 6, 9 and 12 mg kg 1. In short-time exposure of 14 d, the sperm deformity (%) of earthworms increased at 6, 9 and 12 mg kg 1, and the microstructural alteration in seminal vesicles was also observed at these concentrations, whereas ultrastructural alteration of germ cells, particularly morphology of mitochondria, was observed at 3 mg kg 1 and above, suggesting the high sensitivity of germ cell ultrastructure to low concentrations of ABZ in short-time exposure. The results can provide important information for prediction of ecologically significant toxic effects. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction In recent years veterinary drug residues have been the subject of many environmental investigations and have been predicted to pose potential risk to the health of terrestrial organisms (Svendsen et al., 2002; Jjemba, 2006; Jensen et al., 2007; Römbke et al., 2010; Junge et al., 2011; Müller et al., 2013). One of their major routes of entry into the environment is likely through manure amended soil containing veterinary drug residues. Published data showed that these residues may indeed be present in manure
⇑ Corresponding author at: College of Animal Science and Technology, Agricultural University of Hebei, Baoding, Hebei Province 071001, PR China. Tel.: +86 03127528470. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.chemosphere.2014.05.030 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
spread to land (Kay et al., 2005) and that this may subsequently lead to acute and sub-lethal effects on soil environment (Chee-Sanford et al., 2001). Albendazole (ABZ), a derivative of benzimidazole, exhibits a broad spectrum of gastrointestinal anthelmintic activity, and it has been used widely in livestock as antiparasitic drugs against nematodes, cestodes and trematodes (McKellar and Scott, 1990). When administered orally, ABZ is partially metabolized and absorbed in the gastrointestinal tract, with the drug being excreted unchanged in faeces and urine. The main bioactive metabolite, albendazole-sulfoxide, also an effective anthelmintic (Daniel-Mwambete et al., 2004), is excreted to the environment as well. Only a limited amount of information is available, however, on the environmental fate and transport of benzimidazole antiparasitics within the soil matrix (Kreuzig et al., 2007; Kim et al., 2010; Moenickes et al., 2011). Moreover,
2
Y. Gao et al. / Chemosphere 120 (2015) 1–7
few studies have been published on the potential impacts of ABZ on soil-dwelling organisms (Gao et al., 2007, 2012). Earthworms are sentinels for terrestrial systems on account of their decomposition activity, nutrient mineralization and promotion of microfloral activities. Consequently they have been widely used in ecotoxicological studies attempting to resolve the deleterious effects associated with various chemicals exposure (Lowe and Butt, 2007; Amorim et al., 2012; Wu et al., 2012). Reproductive response in earthworms to chemicals stress have been considered as a sensitive endpoint for ecological risk assessment on veterinary drugs, pesticides, metals and other xenobiotic contamination. Traditionally, single reproductive endpoints in earthworms exposed to veterinary drugs are measured through simple parameters such as cocoon production, hatching success and hatching survival in chronic toxicity test under laboratory conditions. In reality, however, the occurrence and fate of veterinary drugs in soil is very complex, which makes prediction of the resulting effects rather difficult and requires the study of multiple endpoints (Flammarion et al., 2002). In previous studies, the reproduction test on spermatogenesis in earthworms has been recognized for the potential to assess sublethal or chronic toxicity of pesticides and metals (Reinecke and Reinecke, 1997; Bustos-Obregon and Goicochea, 2002; Xiao et al., 2006). However, only little attention has been paid specifically to the effect of veterinary drug residues on spermatogenesis in earthworms. Our previous studies on the effect of ABZ on the earthworm, Eisenia fetida, demonstrated that the growth rate decreased at high concentrations of 90 and 270 mg kg 1 during 28-day exposure, and that gene expression (HSP90) was induced at 10 mg kg 1 and above during 14-day exposure (Gao et al., 2012). However, the potential effects of ABZ on reproduction of earthworms are unclear. To better understand the environmental and biological effects of ABZ in soil, in this study we employed four approaches (chronic reproduction test, sperm deformity, morphological alterations in the seminal vesicles by light and transmission electron microscopy) to determine the reproductive responses of earthworms to ABZ. The examined endpoints included survival and growth of adult earthworm, cocoon number, cocoon hatching success and hatching survival, biomass of juveniles and cocoons, sperm deformity percentage, microstructure and ultrastructure in seminal vesicles. 2. Materials and methods 2.1. Earthworms and chemical E. fetida were obtained from an earthworm farm at China Agricultural University and they were kept in the test soil in laboratory for a week prior to the start of the experiment. The individual earthworms were adults with well-developed clitellum (350 ± 20 mg). ABZ was purchased from Sigma with the purity above 98%. 2.2. Soil preparation The artificial soil was prepared according to OECD guideline 207 (1984). The soil comprised (by dry weight) 20% kaolin clay, 10% sphagnum peat and 70% quartz sand with particle size 0.05– 0.2 mm. Calcium carbonate was added to adjust the pH of the wetted substrate to 6.0 ± 0.5 (Wu et al., 2012).
The ABZ treated soil was then transferred to 1 L glass cylindrical containers (1000 g dry soil per container), which were placed in a well ventilated hood for acetone to evaporate overnight. Water was added to compensate for the lost weight due to acetone evaporation and then earthworms were added. Controls were prepared in a similar way except acetone added only. Eight replicate containers for each ABZ concentration were used, with ten earthworms per container (4 containers for chronic reproduction test and the rest for sperm deformity, light- and electron-microscopic observation. After covered with perforated lids, the containers were incubated in an environmental chamber under 23 ± 1 °C with 12-h exposure to light each day. Water was supplemented to ensure appropriate water content of the substrate by weighing the containers (Jensen et al., 2003). Chronic reproduction in earthworms was analyzed at day 28 and 56 of exposure, and sperm deformity (%), light and electron micrographs in seminal vesicles were investigated at day 14 of exposure. 2.4. Chronic reproduction test To ensure the growth and cocoon production of earthworms during the experiment, uncontaminated horse manure was used as a food source for the earthworms (Spurgeon et al., 2004). Within the initial 28-day exposure, 5 g dried and finely ground manure was moistened with 5 mL of distilled water and added to the upper layer of the soil per container weekly. At day 28 of the experiment, surviving earthworms were removed from the soil, washed in distilled water, dried on paper towels and then weighed. The cocoons and hatched juveniles were also hand-sorted, counted and weighed, then returned and incubated for another 28 d. At day 56 of the experiment, the number and weight of cocoons and hatched juveniles were recorded. 2.5. Sperm deformity At day 14 of exposure, earthworms for sperm deformity test were put on humid filter paper at room temperature to empty their gut content. The individual earthworms were anesthetized by placing them on moist cold filter paper in petri dishes for 10 min, and then in cold distilled water for 5 min, which helped to relax muscles for dissection. The dissection was carried out on the dorsal side, after which seminal vesicles were detached. The seminal vesicles were placed between two glass slides, and by exerting pressure the sperm were forced out of the vesicles. The sperm were then fixed in methanol and stained in Giemsa. The slides were observed under a light microscope (image at 400) to detect abnormalities in sperm morphology. One thousand sperms were counted for every slide and the sperm deformity (%) was calculated (Zang et al., 2000). 2.6. Light-microscopic observation At day 14 of exposure, eight earthworms at each concentration of ABZ were removed from the test soil and their seminal vesicles were detached. According to Chuang et al. (2006), the seminal vesicles were cut into 0.5 0.5 cm pieces and fixed in Bouin solution for no longer than 24 h. The fixed tissues were dehydrated and embedded in paraffin. Slices (6 lm section) were obtained and stained in Mayer’s Hematoxylin/Eosin, and then observed under a light microscope (image at 400). 2.7. Electron-microscopic observation
2.3. Treatments ABZ was dissolved in acetone, followed by mixing into the soil at concentrations of 1, 3, 6, 9 and 12 mg per kilogram dry weight.
At day 14 of exposure, eight earthworms at each concentration of ABZ were removed from the test soil and their seminal vesicles were detached for ultrastructural examination. The seminal
3
Y. Gao et al. / Chemosphere 120 (2015) 1–7
analysis of variance (ANOVA). Data sets were checked for normality and homogeneity of variance with the Shapiro–Wilk test and Levene’s test, respectively, prior to analysis by ANOVA. Post-hoc comparison (LSD test) was also carried out to check for differences between the exposed and control groups. All statistical analysis was performed using SPSS software (SPSS19.0).
400 300 200 100 0
0
1
0 day
3. Results
28 days
3.1. Survival and growth of adult earthworm
3
6
9
No mortality for adult earthworms was found after 28 d of exposure to any concentrations of ABZ, and there was no significant difference in the weight of adult earthworms between the control and exposed groups (Fig. 1).
12
Concentration (mg/kg) Fig. 1. Change in weight of Eisenia fetida after 28 d of exposure to ABZ. Error bars represent standard deviations.
vesicles were immersed in primary fixative (2.5% Glutaraldehyde in Phosphate buffer). After the post-fixation with 1% osmium tetroxide, the tissue blocks were dehydrated through gradient acetone series (50–100%), embedded in Araldite mixture and polymerized at 60 °C, and then the samples were sectioned with an ultramicrotome (Leica Ultracut UCT, Austria) and examined with a transmission electron microscope (Hitachi H-7500, Japan) after being counterstained with uranyl acetate and lead citrate. 2.8. Statistical analysis Differences in chronic reproduction test or assessment of sperm deformity were tested for treatment significance using one-way
8
8
*
6
6
4
4
** **
2 0
0
1
3
6
**
9
After 28 d of exposure, the number of cocoons per earthworm showed a clear concentration–response relationship and was significantly reduced over the range of concentrations from 3 to 12 mg kg 1 (Fig. 2A). The average weight per cocoon over the range of concentrations from 1 to 9 mg kg 1 was 8.91 mg and showed no significant difference compared to the control group (P > 0.05). However, when earthworms were exposed to the highest concentration of ABZ (12 mg kg 1), the average weight per cocoon was reduced to 6.85 mg and showed a significant difference compared to the control group (P < 0.05). The effect of ABZ on the hatching success of cocoons was also investigated during the entire exposure time of 56 d. After initial 28 d of exposure, the hatching success at the concentrations of 6,
10
A
**
2
weight of per coccon (mg)
No. of cocoons per worm
10
3.2. Chronic reproduction analysis
100
Hatching success (%)
Weight per worm (mg)
500
**
60 40 20 0
1
4
**
3
C **
2
* 1
** 0
0
1
3
6
9
Concentration (mg/kg)
12
Biomass of juveniles and cocoons per worm (mg)
No.of juveniles per cocoon
28 days 56 days
3
*
**
6
9
** 12
Concentration (mg/kg)
Concentration (mg/kg) 5
**
28 days 56 days
0
0
12
B
80
D
80
* 60 40
** 20
28 days 56 days
0 0
1
3
6
9
12
Concentration (mg/kg)
Fig. 2. Reproductive responses of Eisenia fetida to ABZ during 56-day exposure. (A) Number (solid line) and weight (broken line) of cocoons after 28 d; (B) hatching success of cocoons; (C) juveniles number per cocoon; and (D) biomass of juveniles and cocoons per earthworm. Statistical significance vs. control group: P < 0.05, P < 0.01. Error bars represent standard deviations.
4
Y. Gao et al. / Chemosphere 120 (2015) 1–7
subsequent increase was also observed at the two concentrations of ABZ (P < 0.01). Biomass of juveniles and cocoons was not significantly different over the range of ABZ concentrations from 1 to 9 mg kg 1 compared to the control group (P > 0.05) (Fig. 2D). However, at the highest concentration of 12 mg kg 1, the biomass was significantly reduced after 28 d (P < 0.01) and after 56 d of exposure (P < 0.05). 3.3. Sperm deformity
Fig. 3. The sperm deformity of Eisenia fetida after 14 d of exposure to ABZ. Statistical significance vs. control group: P < 0.05, P < 0.01. Error bars represent standard deviations.
The deformity (%) of earthworm sperm was not affected at ABZ concentrations of 1 and 3 mg kg 1 after 14 d of exposure, whereas the sperm deformity (%) increased significantly at the concentrations of 6 mg kg 1 and above, varied from 7.2% to 14.8% at the concentrations between 6 and 12 mg kg 1, while 1.6% of sperm deformity in the control group (P < 0.05) (Fig. 3). 3.4. Microstructural changes of seminal vesicles
9 and 12 mg kg 1 showed a significant reduction to 11.0%, 8.3% and 3.0%, respectively, while 14.7% of hatching success in the control group (P < 0.05) (Fig. 2B). After 56 d of exposure, the hatching success of cocoons in all groups increased compared to the hatching success after initial 28-day exposure, however, the hatching success at the higher concentrations of 9 and 12 mg kg 1 was significantly lower than the control group, with 58.9% and 47.9% of hatching success in the two treatment groups respectively, while 82.8% of hatching success in the control group (P < 0.01). The number of hatched juveniles per cocoon at the lower concentrations of 1, 3 and 6 mg kg 1 was not significantly different compared to the control group after 28 and 56 d of exposure (P > 0.05) (Fig. 2C), however, at the higher concentrations of 9 and 12 mg kg 1, a significant decrease in juvenile number was observed during initial 28-day exposure (P < 0.05), and the
A
Mature sperm
Under the light microscope, in comparison with normal seminal vesicles (from control earthworms), there was no difference in spermatogenesis in seminal vesicles at the concentrations of 1 and 3 mg kg 1. In seminal vesicles of control earthworms, a very marked differentiation of germ cells was observed, showing different stages of germ cells (Fig. 4A). Furthermore, a high percentage of mature sperm bundles were also observed. However, in the range of concentrations from 6 to 12 mg kg 1, some damage in the germ cells occurred and was aggravated with increasing concentrations of exposure (Fig. 4B–D). The mature sperm bundles at 6 and 9 mg kg 1 decreased and the disordered distribution of germ cells was also observed at 9 mg kg 1. When earthworms were exposed to the highest concentration of 12 mg kg 1, the germ cells were damaged seriously, exhibiting the decreased number of germ cells and disordered distribution in seminal vesicles (Fig. 4D).
B
Late spermatid Early spermatid
Mature sperm
30 µm
C
30 µm
30 µm
D
30 µm
Fig. 4. Microstructural changes in seminal vesicles of Eisenia fetida after 14 d of exposure to ABZ (image at 400). (A) Normal microstructure from control earthworms, showing germ cells in differentiation stages (arrow) and mature sperm bundles were very obvious (arrowhead); (B) seminal vesicles at 6 mg kg 1, showing the decreased number of mature sperm; (C) seminal vesicles at 9 mg kg 1, showing the decreased number of germ cells and their disordered distribution; and (D) seminal vesicles at 12 mg kg 1, showing the decreased number of germ cells and their disordered distribution.
5
Y. Gao et al. / Chemosphere 120 (2015) 1–7
B
A
500 nm
C
500 nm
E
1.3 µm
D
1.0 µm
F
1.4 µm
G
1.0 µm
Fig. 5. Ultrastructural changes in seminal vesicles of Eisenia fetida after 14 d of exposure to ABZ. (A) Normal mitochondria in spermatids, showing cristae that project into the matrix from control earthworms (arrow). The normal nucleus containing dense areas of chromatin were observed (arrowhead); (B) parallel cisternae of endoplasmic reticulum around the nucleus in the cytophore of control earthworms (arrow); (C) damaged mitochondria of spermatids at 3 mg kg 1, showing fewer cristae, diminished matrix content and edema (arrow); (D) damaged cytoplasmic organelles and spermatid nuclear at 6 mg kg 1, exhibiting irregular membrane (higher arrow) and diminished matrix content in mitochondria (lower arrow), mild cytoplasmic edema, and chromatin swelling (arrowhead); (E) dilated cisternae of endoplasmic reticulum at 9 mg kg 1 (arrow); (F) damaged cytoplasmic organelles at 9 mg kg 1, exhibiting no structured vacuole (arrow), cytoplasmic edema and diminished cytoplasmic organelles; and (G) seriously damaged spermatid at 12 mg kg 1, showing cytoplasmic edema, mitochondria collapse (arrow), partly dissolved nuclear membrane and chromatin edema (arrowhead).
3.5. Ultrastructural changes of seminal vesicles Under the electron microscope, some normal mitochondria and nucleus were observed in spermatids in the seminal vesicles of
control earthworms (Fig. 5A). In contrast, earthworms exposed to the concentration range from 3 to 12 mg kg 1 exhibited marked abnormalities in spermatids (Fig. 5C–G). The mitochondria were injured by ABZ and exhibited a progressive degeneration with
6
Y. Gao et al. / Chemosphere 120 (2015) 1–7
increasing ABZ concentrations. At the lower concentrations of 3 and 6 mg kg 1, some mitochondria showed diminished matrix content, irregular membranes and fewer cristae (Fig. 5C and D). When earthworms were exposed to the higher concentrations of 9 and 12 mg kg 1, the morphology of mitochondria was seriously damaged and exhibited the loss of inner structure and partly dissolved membrane (Fig. 5F), even collapsed at 12 mg kg 1 (Fig. 5G). The cytoplasmic edema was also observed in spermatids of earthworms exposed to 6 mg kg 1 and became more serious with increasing ABZ concentrations, accompanied with diminished the number of cytoplasmic organelles. Spermatid nuclear in exposed earthworms was damaged, exhibiting chromatin edema at 6 and 12 mg kg 1 and the nuclear membrane partly dissolved at 12 mg kg 1. In addition, the cisternae of endoplasmic reticulum in the cytophore were dilated significantly at 9 mg kg 1 compared to parallel endoplasmic reticulum in the control group (Fig. 5B and E).
4. Discussion The choice of sensitive parameters is crucial in assessing the potential toxicity of low concentrations exposure to chemicals in soil. The present study evaluated reproductive responses of the earthworm E. fetida to various concentrations of ABZ in multiple experiments. The results demonstrated that ABZ significantly affected the reproduction of adult earthworms, while survival and growth of adult earthworms were not affected for all ABZexposed groups. These effects could be attributed to a diversion of resources towards increasing maintenance and repair (Anderson et al., 2013). Lika and Kooijman (2003) stated that adult earthworms were likely to divert the allocation of resources away from reproduction, towards maintenance in cases where individuals were exposed to sub-optimal conditions. In this work, in response to low concentrations of ABZ, cocoon number was more sensitive than other reproductive parameters (cocoon weight, cocoon hatching success and hatching survival, biomass of juveniles and cocoons) during initial 28 d of exposure, showing a significant decrease at 3 mg kg 1 and above in a concentration-dependent manner. Previous studies demonstrated that chemicals affected the earthworm reproduction (cocoon production and hatching success) (Zhang et al. (2008); Alves et al., 2013) and previous work by Zhang et al. (2008) indicated that hatching success of cocoons was more sensitive to chemicals than cocoon production, which was in opposition to our result. The reason behind this difference is unclear. For this increasing toxicity on cocoon production, the possible explanation may be that the uptake of ABZ and its metabolite by earthworms depended on some combination of both ingestion of gastrointestinal contents and diffusion through external surface (Shalaby et al., 2009). For the toxicity on hatching success of cocoons, the possible explanation may be that earthworm cocoons absorbed this drug and its metabolite only by diffusion of eggshell. Therefore, during initial 28 d of experiment, the cocoon hatching success and hatched juvenile number were affected starting at concentrations of 6 and 9 mg kg 1, respectively, while the cocoon number was affected starting at 3 mg kg 1. With longer exposure time, the effect of ABZ on cocoon hatching success increased at higher concentrations. It may be attributed to the accumulation of ABZ or its degradation products in cocoons, which resulted in the increase of toxicity. Nevertheless, there is still lack of information on the fate of this drug in soil. Future studies should focus on the degradation, absorption and transformation of ABZ residues in soil, which is an extremely challenging task. It is worth noting that biomass of earthworms was not affected by ABZ concentrations below 12 mg kg 1 during entire exposure
time, despite other endpoints in chronic reproduction test were found to be sensitive to low concentrations of this drug. Published research suggested that biomass change of earthworms could be a good indicator of chemicals stress and link chemicals toxicity to energy dynamics and ultimately growth inhibition (Wu et al., 2012; Anderson et al., 2013). In this study, however, biomass change of earthworms was not optimal to assess the low concentrations of ABZ in soil. In the present study, a significant effect in spermatogenesis of earthworms was observed for short-term exposure of ABZ. The result suggests that the ultrastructural alterations of germ cells in seminal vesicles took place before the microstructural alterations, showing mitochondria in germ cells were damaged at 3 mg kg 1 of ABZ and above, while both the microstructural alterations and the increase of sperm deformity (%) were observed at 6 mg kg 1 and above. Therefore, the ultrastructural alterations of germ cells are speculated to be more sensitive to low concentrations of ABZ exposure than their microstructural alterations. Particularly, morphological alterations of mitochondria showed a progressive degeneration with increasing ABZ concentrations. It is well known that fundamental metabolic processes take place in the mitochondria, such as electron transport, oxidative phosphorylation, catabolism of fatty acids and amino acid metabolism (Reichert and Neupert, 2002). The damages of mitochondria might demonstrate a lower functional activity of the mitochondria followed by disturbing the pathways of energy metabolism. Our previous work concluded that the morphology of mitochondria in intestinal epithelium was damaged when earthworms were exposed to ABZ, which is in accordance with our present work (Gao et al., 2007). Though the sensitivity of microstructure in seminal vesicles and sperm morphology under a light microscope to ABZ was lower than that of ultrastructure of germ cells, these histological alterations can still provide crucial information for determining the short-time ecological implication of exposure (Reddy and Rao, 2008). Histopathological responses in earthworms have been reported as valuable indicators of terrestrial contamination in previous studies (Giovanetti et al., 2010; Kilic, 2011). The results of this study can shed light on early sub-lethal effects of ABZ on germ cell structure of earthworms.
5. Conclusions The reproductive responses of the earthworm E. fetida were observed during 56 d of exposure, while the growth and survival of adult earthworms were not affected. The result of this study suggests the cocoon number of earthworms was more sensitive to low concentrations of ABZ than other reproductive parameters (cocoon hatching success and hatching survival), with a significant decrease in cocoon number at 3 mg kg 1 and above. In short-term exposure of 14 d, the microstructural alterations in seminal vesicles and the increase of sperm deformity (%) were observed at 6 mg kg 1 and above, whereas the ultrastructure of germ cells, particularly morphology of mitochondria, were significantly affected at 3 mg kg 1 and above, suggesting the ultrastructure of germ cells is more sensitive than histological microstructure. The characterization and understanding of reproductive responses of earthworms to ABZ on multiple traits can provide important information for prediction of the pollution of ABZ residues in soil.
Acknowledgment This research was sponsored by Hebei Natural Science Foundation in China (No. C2012204074).
Y. Gao et al. / Chemosphere 120 (2015) 1–7
References
Alves, P.R., Cardoso, E.J., Martines, A.M., Sousa, J.P., Pasini, A., 2013. Earthworm ecotoxicological assessments of pesticides used to treat seeds. Chemosphere 90, 2674–2682. Amorim, M.J.B., Pereira, C., Menezes-Oliveira, V.B., Campos, B., Soares, A.M., Loureiro, S., 2012. Assessing single and joint effects of chemicals on the survival and reproduction of Folsomia candida (Collembola) in soil. Environ. Pollut. 160, 145–152. Anderson, C.J., Kille, P., Lawlor, A.J., Spurgeon, D.J., 2013. Life-history effects of arsenic toxicity in clades of the earthworm Lumbricus rubellus. Environ. Pollut. 172, 200–207. Bustos-Obregon, E., Goicochea, R.I., 2002. Pesticide soil contamination mainly affects earthworm male reproductive parameters. Asian J. Androl. 4, 195–199. Chee-Sanford, J.C., Aminov, R.I., Krapac, I.J., Garrigues-Jeanjean, N., Mackie, R.I., 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494–1502. Chuang, S.C., Lai, W.S., Chen, J.H., 2006. Influence of ultraviolet radiation on selected physiological responses of earthworms. J. Exp. Biol. 209, 4304–4312. Daniel-Mwambete, K., Torrado, S., Cuesta-Bandera, C., Ponce-Gordo, F., Torrado, J.J., 2004. The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs. Int. J. Pharm. 272, 29–36. Flammarion, P., Devaux, A., Nehls, S., Migeon, B., Noury, P., Garric, J., 2002. Multibiomarker responses in fish from the Moselle River (France). Ecotoxicol. Environ. Saf. 51, 145–153. Gao, Y., Sun, X., Gu, X., Sun, Z., 2012. Gene expression responses in different regions of Eisenia fetida with antiparasitic albendazole exposure. Ecotoxicol. Environ. Saf. 89, 239–244. Gao, Y., Sun, Z., Sun, X., Shi, W., 2007. Toxic effects of albendazole on Adenosine triphosphatase activity and ultrastructure in Eisenia fetida. Ecotoxicol. Environ. Saf. 67, 378–384. Giovanetti, A., Fesenko, S., Cozzella, M.L., Asencio, L.D., Sansone, U., 2010. Bioaccumulation and biological effects in the earthworm Eisenia fetida exposed to natural and depleted uranium. J. Environ. Radioact. 101, 509–516. Jensen, J., Diao, X., Scott-fordsmand, J.J., 2007. Sub-lethal toxicity of the antiparasitic abamectin on earthworms and the application of neutral red retention time as a biomarker. Chemosphere 68, 744–750. Jensen, J., Krogh, P.H., Sverdrup, L.E., 2003. Effects of the antibacterial agents tiamulin, olanquindox and metronidazole and the anthelmintic ivermectin on the soil invertebrate species Folsomia metaria (Collembola) and Enchytraeus crypticus (Enchytraeidae). Chemosphere 50, 437–443. Jjemba, P.K., 2006. Excretion and ecotoxicity of pharmaceutical and personal care products in the environment. Ecotoxicol. Environ. Saf. 63, 113–130. Junge, T., Meyer, K.C., Ciecielski, K., Adams, A., Schäffer, A., Schmidt, B., 2011. Characterization of non-extractable 14C- and 13C-sulfadiazine residues in soil including simultaneous amendment of pig manure. J. Environ. Sci. Health, Part B 46, 137–149. Kay, P., Blackwell1, P.A., Boxall, A.B.A., 2005. A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with field data. Environ. Pollut. 134, 333–341.
7
Kilic, G.A., 2011. Histopathological and biochemical alterations of the earthworm (Lumbricus terrestris) as biomarker of soil pollution along Porsuk River Basin (Turkey). Chemosphere 83, 1175–1180. Kim, H.J., Lee, D.S., Kwon, J.H., 2010. Sorption of benzimidazole anthelmintics to dissolved organic matter surrogates and sewage sludge. Chemosphere 80, 256– 262. Kreuzig, R., Blumlein, K., Holtge, S., 2007. Fate of the benzimidazole antiparasitics flubendazole and fenbendazole in manure and manured soils. Clean Soil Air Water 35, 488–494. Lika, K., Kooijman, S., 2003. Life history implications of allocation to growth versus reproduction in dynamic energy budgets. Bull. Math. Biol. 65, 809–834. Lowe, C.N., Butt, K.R., 2007. Earthworm culture, maintenance and species selection in chronic ecotoxicological studies: a critical review. Eur. J. Soil Biol. 43, S281– S288. McKellar, Q., Scott, E., 1990. The benzimidazole anthelmintic agents—a review. J. Vet. Pharmacol. Ther. 13, 223–247. Moenickes, S., Höltge, S., Kreuzig, R., Richter, O., 2011. Process dominance analysis for fate modeling of flubendazole and fenbendazole in liquid manure and manured soil. Sci. Total Environ. 410–411, 226–234. Müller, T., Rosendahl, I., Focks, A., Siemens, J., Klasmeier, J., Matthies, M., 2013. Short-term extractability of sulfadiazine after application to soils. Environ. Pollut. 172, 180–185. Reddy, N.C., Rao, J.V., 2008. Biological response of earthworm, Eisenia foetida (Savigny) to an organophosphorous pesticide, profenofos. Ecotoxicol. Environ. Saf. 71, 574–582. Reichert, A.S., Neupert, W., 2002. Contact sites between the outer and inner membrane of mitochondria-role in protein transport. Biochim. Biophys. Acta 1592, 41–49. Reinecke, S.A., Reinecke, A.J., 1997. The influence of lead and manganese on spermatozoa of Eisenia fetida (Oligochaeta). Soil Biol. Biochem. 29, 737–742. Römbke, J., Krogh, K.A., Moser, T., Scheffczyk, A., Liebig, M., 2010. Effects of the veterinary pharmaceutical ivermectin on soil invertebrates in laboratory tests. Arch. Environ. Contam. Toxicol. 58, 332–340. Shalaby, H.A., Abdel-Shafy, S., Abdel-Rahman, K.A., Derbala, A.A., 2009. Comparative in vitro effect of artemether and albendazole on adult Toxocara canis. Parasitol. Res. 105, 967–976. Spurgeon, D.J., Stürzenbaum, S.R., Svendsen, C., Hankard, P.K., Morgan, A.J., Weeks, J.M., Kille, P., 2004. Toxicological, cellular and gene expression responses in earthworms exposed to copper and cadmium. Comp. Biochem. Physiol. Part C 138, 11–21. Svendsen, T.S., Sommer, C., Holter, P., Grønvold, J., 2002. Survival and growth of Lumbricus terrestris (Lumbricidae) fed on dung from cattle given sustainedrelease boluses of ivermectin or fenbendazole. Eur. J. Soil Biol. 38, 319–322. Wu, S., Zhang, H., Zhao, S., Wang, J., Li, H., Chen, J., 2012. Biomarker responses of earthworms (Eisenia fetida) exposured to phenanthrene and pyrene both singly and combined in microcosms. Chemosphere 87, 285–293. Xiao, N., Jing, B., Ge, F., Liu, X., 2006. The fate of herbicide acetochlor and its toxicity to Eisenia fetida under laboratory conditions. Chemosphere 62, 1366–1373. Zang, Y., Zhong, Y., Luo, Y., Kong, Z.M., 2000. Genotoxicity of two novel pesticides for the earthworm, Eisenia fetida. Environ. Pollut. 108, 271–278. Zhang, B., Cox, S.B., McMurry, S.T., Jackson, W.A., Cobb, G.P., Anderson, T.A., 2008. Effect of two major N-nitroso hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites on earthworm reproductive success. Environ. Pollut. 153, 658–667.
