Ecotoxicological assessment of lanthanum with Caenorhabditis elegans in liquid medium

Abstract

With their widespread applications in industry, agriculture and many other fields, more and more rare earth elements (REEs) are getting into the environment, especially the aquatic systems. Therefore, understanding the aquatic ecotoxicity of REEs has become more and more important. In the present work, Caenorhabditis elegans (C. elegans) was used as a test organism and life-cycle endpoints were chosen along with elemental assay to evaluate the aquatic toxicity of lanthanum (La), a representative of REEs. The results show La³⁺ had significant adverse effects on the growth and reproduction of worms above a concentration of 10 μmol L⁻¹. The elemental mapping by microbeam synchrotron radiation X-ray fluorescence (μ-SRXRF) illustrated how La treatment disturbed the metals distribution in the whole body of a single tiny nematode at lower levels. Our results suggested that the high-level REEs in some polluted water bodies would lead to an aquatic ecological crisis. The assessment we performed in the present work could be developed as a standardized test design for aquatic toxicological research.

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Introduction

The aquatic system is the most active element in the natural environment, which is closely related to human beings and other creatures. It also plays the most important role in the ecosystem cycling of materials, including the translocation, transformation and fate of heavy metals. Rare earth elements (REEs), are an interesting group of 15 chemically active, mainly trivalent metals. Although REEs are not abundant in the earth's crust, Ce, the most plentiful element of REEs, is about 100 times more abundant than cadmium (Cd), one of the most well-known heavy metals in toxicology. Because of their particular magnetic, catalytic and optical properties, diversified medical and biological effects,¹ REE compounds show a broad spectrum of applications in industry, agriculture and in the clinic.^2–4 In China, REE-containing fertilizers are applied to over 6 million hectares of farm lands,⁵ which would increase the amount of REEs in soil. Though most of the REEs would be gradually fixed on the surface of soil particles and lose their bioavailability, 10% of the total amount would remain soluble when they are mixed with the soil.⁶ These soluble REEs can enter into the surface water and groundwater through surface runoff and seepage. The consequences of REEs in the aquatic system demand ecotoxicological assessment.

The C. elegans (nematode) has been widely used in developmental biology studies and ecotoxicological assessment because of its ease of use, short lifespan, cellular simplicity and sensitivity to environment variables.^7–10 In the present work, C. elegans was used as a test organism for ecotoxicological assessment of lanthanum, a representative of REEs. Life-cycle endpoints were chosen along with elemental assay to evaluate the aquatic toxicity of La.

Experimental

Test media

Escherichia coli strain OP50 as a food source for C. elegans was prepared using standard procedure.¹¹ The culture was centrifuged at 3600 rpm for 10 min and then resuspended in an equal volume of K-medium (2.4 g KCl, 3.1 g NaCl, 1 L ultrapure water). This treatment was repeated three times and then the concentration of bacteria was diluted to approx 10⁹ cells ml⁻¹. Lanthanum chloride was prepared from La₂O₃ (99.99%, China Medicine Group Shanghai Chemical Reagent Corporation, Shanghai) and the concentration of stock solution was 10 mmol L⁻¹. Small volumes of LaCl₃ stock solution were added to the bacterial suspension in K-medium and the final concentrations were 0, 0.1, 1, 5, 10, 20, 50, and 100 μmol L⁻¹ (μM) of La. MTT colorimetric assay was employed to determine the survival of E. coli after La exposure for 24 h.¹²

Lanthanum determination

After E. coli OP50 was suspended in K-medium with or without lanthanum for 24 h, the test media were centrifuged at 3600 rpm for 10 min to obtain supernatants and pellets of OP50. The content of La in bacteria was determined by inductively coupled plasma-mass spectrometer (ICP-MS). Briefly, the E. coli samples were dried at −50 °C for approximately 48 h in a freeze-drier and then 10–20 mg samples were microwave-digested (Mars Xpress, CEM, Germany) with a 2 mL mixture of 3 : 1 HNO₃/H₂O₂ (MOS grade). The La contents in supernatants were also determined by ICP-MS after filtration (0.45 μm, Millipore).

Test organism and culture

Caenorhabditis elegans var. Bristol, strain N2 (C. elegans), was maintained in stocks of dauer larvae (an alternative juvenile stadium, that occurs at lack of food) at 20 °C in Petri dishes on nematode growth medium (NGM). To obtain synchronized cultures, gravid hermaphrodites were lysed in an alkaline hypochlorite solution as described previously.¹¹

Test procedure

The nematode bioassay with C. elegans was carried out following a well-developed test design¹⁰ with a few modifications. Briefly, 1 ml aliquot of bacterial suspension in K-medium (Escherichia coli OP50, ∼10⁹ cells ml⁻¹) with or without lanthanum was added to polystyrene multiwells (24 wells, Corning, New York, USA). At the beginning of the test (0 h postexposure), synchronized first-stage juvenile hermaphrodites were picked from NGM and transferred individually to each well. Eight replicates were set up for each treatment. The cultures were maintained at 20 °C in an incubator and transferred daily to new wells and growth was assessed by measuring daily change in body length with image analysis drawing tool (Scion Image software, Scion Corp., Frederick, MD). At the end of the exposure period, the plates were heated in water baths (50 °C) for 5 min to kill the worms and the samples were then fixed in a 5% formalin solution with rose bengal (0.5 g L⁻¹) added to stain the worms for easier recovery. The number of eggs inside gravid worms and the number of progeny were counted under a light microscopy (Olympus IX 71, Japan). The test was repeated 6 times (48 individuals for each treatment).

Elemental mapping by μ-SRXRF

At 48 h postexposure, C. elegans exposed to 0, 1 and 20 μM of La³⁺ were washed 5 times with ultrapure water and were transferred on a polycarbonate film previously fixed on a plastic frame. Elemental mapping in C. elegans was carried out by μ-SRXRF at BL15U, Shanghai Synchrotron Radiation Facility, China. The storage ring ran at an energy of 3.5 GeV with an average current intensity of 300 mA. The beamline was equipped with a Si/Rh-coated K–B mirror-pair to focus the beam and the beam size at sample location was 2.3 × 3.5 μm². C. elegans were mounted on X–Z translation stages and the sample platform was moved along the X and Z directions at an interval of 5 μm for each step. The fluorescence intensities of La, Ca, Cu, Fe, K, Zn, and Compton scattering were collected up to 2 s for each point with a Si (Li) detector. In order to correct the effect of the synchrotron radiation beam flux variation on the signal intensity, the fluorescence intensity was normalized to the incident X-ray intensity, and Compton scattering was used as an internal standard to compensate the differences in thickness and density of the samples.¹³

Statistical analyses

For life-cycle endpoints, one-way ANOVA followed by post hoc Bonferroni multiple comparisons or Kruskal-Wallis H ANOVA followed by Mann-Whitney U test was run to test for significant differences between treatments where appropriate. For lanthanum contents, one-way ANOVA was employed and least significant difference (LSD) multiple comparison was applied to examine significance. Significant difference was defined as that with a p value <0.05.

Results and discussion

Effects of La³⁺ on E. Coli

When the E. coli were suspended in K-medium with or without lanthanum, all of the test media were homogeneous with slight blue opalescence at 0 h. But soon, the E. coli exposed to a high level of La³⁺ agglomerated and precipitated. The rapid flocculation may be due to the absorption of La³⁺ on the cell envelope:¹⁴ the accumulation of cations on the surface would attenuate the negative surface charge of E. coli, which is critical for the dispersion of bacteria in liquid medium. After exposure for 24 h, no obvious precipitation was found in the test media containing 0, 0.1, 1, 5, 10, and 20 μmol L⁻¹ La³⁺. The results of the MTT assay (Fig. 1) also showed that the survivals of E. coli introduced to 0, 0.1, 1, 10, and 20 μmol L⁻¹ La³⁺ were above 80% after exposure for 24 h and the survivals in 50 and 100 μmol L⁻¹ La³⁺ groups were below 80%. To minimize the impact of food abundance on the growth of C. elegans, the latter two doses were not chosen for the further assessment.

Fig. 1 The survival of E. coli after La³⁺ exposure for 24 h. Mean ± SEM; N = 8; **: p < 0.01.

After E. coli was suspended in K-medium with or without lanthanum for 24 h, the lanthanum contents in E. coli and medium were determined by ICP-MS. The previous research suggested a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner membrane.^14,15 The present work showed that the La contents in bacteria significantly increased after La³⁺ treatment, indicating a good adhesion of La³⁺ to E. coli (Table 1).

Table 1 Lanthanum contents in E. coli (μg g⁻¹ dry weight) and medium (μg L⁻¹)

	Treatment
	0 (CT)	0.1 μM	1 μM	5 μM	10 μM	20 μM
Mean ± SD; N = 8; *: 0.01 ≤ p < 0.05, **: p < 0.01.
E. coli	0.22 ± 0.04	55.2 ± 0.9**	544 ± 13**	3027 ± 143**	5783 ± 399**	10 832 ± 488**
Medium	<0.02	0.03 ± 0.03**	0.33 ± 0.14**	3.65 ± 0.40**	18.9 ± 3.4**	242 ± 45**

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Life-cycle endpoints

Nonparametric Kroskal-Wallis H test showed that lanthanum exposure had a significant effect on the growth of C. elegans (χ²[5, 259] = 39.02, p < 0.01; χ² [5, 268] = 64.38, p < 0.01; χ² [5, 260] = 131.25, p < 0.01; Fig. 2); post hoc comparisons revealed that the body length of C. elegans exposed to 10 μM and 20 μM La³⁺ were significantly shorter than those of the controls at 24 h, 48 h and 72 h postexposure (p < 0.01); C. elegans treated with 0.1 μM and 1 μM La³⁺ were significantly larger that those of the control at 72 h postexposure (p < 0.01).

Fig. 2 The effect of lanthanum exposure on the growth of C. elegans. Mean ± SEM; N = 42 ∼ 48; *: 0.01 ≤ p < 0.05, **: p < 0.01.

All of the hermaphrodites were gravid at 72 h postexposure, but ANOVA analyses indicated that La³⁺ treatment had significant effects on the amount of eggs in body (F[5, 261] = 38.23, p < 0.01, Fig. 3) and the brood size (χ² [5, 242] = 122.19, p < 0.01, Fig. 3) per adult. Post hoc Bonferroni multiple comparisons showed that the mean amount of eggs in gravid worms exposed to 1 μM La³⁺ was markedly higher than that of control (0.01 ≤ p < 0.05) while the one of those exposed to 20 μM La³⁺ was significantly lower (p < 0.01); Mann-Whitney U test showed the brood sizes of hermaphrodites exposed to 5 μM, 10 μM and 20 μM were significantly smaller than that of control.

Fig. 3 Effects of lanthanum exposure on number of eggs in body and brood size of C. elegans at 72 h postexposure. –■–: eggs in body per adult; –⋄–: brood size per adult. Mean ± SEM; N = 40 ∼ 48; *: 0.01 ≤ p < 0.05, **: p < 0.01.

Because of the short life cycle of C. elegans, ecologically relevant sublethal endpoints like growth and reproduction can be readily assessed within 72 h. Data of the three endpoints indicated that exposure to high level La had adverse effects on C. elegans. The brood size was the most sensitive index to La exposure and EC50 was 14.3 μM (1.97 mg L⁻¹, calculated with Sigmaplot, Systat Software). The EC50s for body length and number of eggs in body were higher than 20 μM. In the previous work, the aquatic toxicities of Cd, Cu, and Pb to C. elegans were determined, and the 24-h EC50 for movement, feeding, and growth and a 72-h EC50 for reproduction were ranged in (14.4–19.4 mg L⁻¹ Cd), (2.84–4.07 mg L⁻¹ Cu), (7.54–9.76 mg L⁻¹ Pb).¹⁶ Though the parameters chosen in the present work were a little different, the results as a whole implied that the toxicological behavior of La³⁺ in liquid medium was similar to those of Cd²⁺, Cu²⁺ and Pb²⁺.^16,17 Some positive effects of lanthanum were found in C. elegans exposed to 1 μM La or lower. Similar stimulus effects of low-level La treatment were also observed in many other species.^5,18

The low level of EC50 value for brood size highlighted the aquatic toxicity of REEs, which might be a reflex of the common toxicological features of heavy metals. The previous environmental survey reported that the contents of total REEs in the major river systems of China were quite low (10⁻³ ∼ 10⁻¹ μg L⁻¹);¹⁹ the content of total REEs in well water in the Gannan rare earth mining area was 9.18 μg L⁻¹.²⁰ According to our study, La exposure at natural background level had negligible adverse effects. However, the REEs concentration in some polluted river would raise to 200∼300 mg L⁻¹,²¹ which might pose a risk to aquatic ecosystems. Thus the direct release of water waste containing high-level REEs to environment should be cautioned.

Elemental mapping

Elemental mapping by μ-SRXRF indicated that lanthanum was accumulated in the body of C. elegans in a dose-dependent manner (Fig. 4). There are two possible routes for La to enter into the body of C. elegans: absorption of La³⁺ from medium on the body surface and La uptake into the alimentary system. The elemental mapping results showed that the La³⁺ in the body was just located above the excretory pore of C. elegans and the concentration of La on the tail region was extremely low. It suggested that the amount of La entering into worms through the body surface was negligible. The accumulation of La in C. elegans is mostly due to the intake of E. coli containing high level of La.

Fig. 4 The elemental mapping in C. elegans by μ-SRXRF.

Moreover, lanthanum treatment could disturb the distribution pattern of other elements. The concentrations of Ca, K and Zn in worms declined markedly while the concentration of Fe increased after a 48 h-exposure to 20 μM La. There was no obvious difference in Cu concentrations between worms in the control group and the 20 μM group when individual variations were taken into account. The elemental concentrations in many cases could serve as good indicators for the physiological and pathological conditions of animals. For example, the homeostasis of Ca in the body is critical for many cellular functions,^22,23 so the lanthanum-induced calcium disorder might lead to cell damage or dysfunction.²⁴ Of course, further research is needed to elucidate the interaction of these elements and the mechanism of these changes.

The great variability of metal concentrations in C. elegans suggested that the elemental mapping assay by μ-SRXRF could be developed as a sensitive technique to illustrate the response of C. elegans to toxicant, especially to the heavy metals. Sediment could also be introduced into the testing media as described in the previous work,^9,10 to test the whole-sediment toxicity. Due to the tiny size of C. elegans (the adults are about 1.3 mm in length with about 1000 somatic cells), quantitative techniques with higher resolution and sensitivity are needed to study the tissue-specific accumulations of trace elements.

However, the high-resolution quantitative imaging of elements in tiny organisms or tissue slices is still a challenge in analytical chemistry as well as biomedical research. Among those methods for elemental mapping, the laser ablation inductively coupled plasma mass spectrometry (LA-ICP/MS) and X-ray fluorescence analysis offer a fast and precise spatially resolved measurement of elements in situ at the trace or ultratrace level.^25,26 Recently, our laboratory developed a method for quantitative imaging of trace elements in sections of bio-tissues using μ-SRXRF, and the spatial distributions of trace elements in the coronal section of brain successfully obtained by using matrix-matched standards for the calculation of the concentrations of metals.²⁷ In the near future, the beamline at BL15U, Shanghai Synchrotron Radiation Facility could provide beams with spot sizes around 100 nm using zone plates. The smaller beam sizes would introduce an excellent opportunity to perform elemental mapping with resolution superior to LA-ICP/MS. It could provide more information about the elemental distribution in C. elegans, e.g. to distinguish the different accumulation patterns of trace elements between muscles and the alimentary system, which could hardly be obtained by other techniques.

Recently, the rapid development of nanotechnology and the broad application of nanomaterials have attracted a lot of concern about their toxic effects on both human health and the environment.^28,29C. elegans was also an ideal model to investigate the toxicological effects of engineered nanomaterials. Such investigations have double meanings: the experimental results contribute not only to the understanding of the underlying mechanisms for nanotoxicity, but also to the assessment of ecotoxicity of the special nanomaterials. The potential ecotoxicity of several metal nanoparticles was assessed using C. elegans with the toxic endpoints, such as body length, reproduction, and behaviors.^30–33 However, no information is available about the intake dose, body distribution and metabolism of nanomaterials in worms in these reports. So, data obtained by elemental mapping using μ-SRXRF would be an important complement to the previous work.

Conclusion

Abnormal growth, reproduction and elemental distribution were found in C. elegans exposed to 10 and 20 μM La; no obvious effects of lanthanum exposure on C. elegans were found when the exposure level was 1 μM or lower. Our results suggested that the REEs exposure at natural background level had negligible adverse effects on C. elegans, but the high-level REEs in some polluted water bodies would lead to an aquatic ecological crisis. Thus the direct release of water waste containing high-level REEs into environment should be cautioned. The assessment of life-cycle endpoints in C. elegans along with quantitative imaging of trace elements using μ-SRXRF could be developed as a standardized test design for aquatic toxicological research.

Acknowledgements

This work is financially supported by the Ministry of Science and Technology of China (grant No. 2011CB933400), the National Natural Science Foundation of China (Grant No. 10905062, 10875136, 11005118), and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant No KJCX3.SYW.N3).

References

1. K. Wang, R. Li, Y. Cheng and B. Zhu, Coord. Chem. Rev., 1999, 190, 297–308.
2. N. Dobrynina, M. Feofanova and I. Gorelov, J. Inorg. Biochem., 1997, 67, 168–168.
3. Q. Tu, X. Wang, L. Tian and L. Dai, Environ. Pollut., 1994, 85, 345–350.
4. F. Albaaj and A. J. Hutchison, Int. J. Clin. Pract., 2005, 59, 1091–1096.
5. B. Guo, W. Zhu, B. Xiong, Y. Ji, Z. Liu and Z. Wu, in Rare Earths in Agriculture, China Agricultural Science and Technology Press, Beijing, 1990.
6. X. Pang, X. Xing, D. Wang and A. Peng, Agro-environ. prot., 2001, 20, 319–321.
7. P. Williams and D. Dusenbery, Environ. Toxicol. Chem., 1990, 9, 1285–1290.
8. K. Guven, J. Duce and D. de Pomerai, Aquat. Toxicol., 1994, 29, 119–137.
9. S. Höss, T. Henschel, M. Haitzer, W. Traunspurger and C. Steinberg, Environ. Toxicol. Chem., 2001, 20, 2794–2801.
10. W. Traunspurger, M. Haitzer, S. Höss, S. Beier, W. Ahlf and C. Steinberg, Environ. Toxicol. Chem., 1997, 16, 245–250.
11. I. A. Hope, in C. elegans: A practical approach, Oxford University Press, New York, 1999.
12. M. Stevens and S. Olsen, J. Immunol. Methods, 1993, 157, 225–231.
13. Y. Gao, N. Liu, C. Chen, Y. Luo, Y. Li, Z. Zhang, Y. Zhao, B. Zhao, A. Iida and Z. Chai, J. Anal. At. Spectrom., 2008, 23, 1121–1124.
14. P. Liu, Y. Liu, Z. Lu, J. Zhu, J. Dong, D. Pang, P. Shen and S. Qu, J. Inorg. Biochem., 2004, 98, 68–72.
15. M. Bayer and M. Bayer, J. Bacteriol., 1991, 173, 141.
16. G. L. Anderson, W. A. Boyd and P. L. Williams, Environ. Toxicol. Chem., 2001, 20, 833–838.
17. S. Höss, T. Henschel, M. Haitzer, W. Traunspurger and C. E. W. Steinberg, Environ. Toxicol. Chem., 2001, 20, 2794–2801.
18. R. Zhao, Y. Liu, Z. Xie, P. Shen and S. Qu, J. Biochem. Biophys. Methods, 2000, 46, 1–9.
19. Z. Chai, Y. Wang, X. Qian, S. Ma, X. Mao, J. Sun, Y. Yang, R. Yang, X. Li, B. Chen, Q. Qian and S. Han, in Applications of Neutron Activation Analysis in Environmental Science, Biology and Geoscience, Atomic Energy Press, Beijing, 1992.
20. J. Zhu, Z. Yuan, X. Wang and S. Yan, J Environ Health, 2002, 19, 443–444.
21. Q. Hu and Z. Ye, Environ. Pollut. Control, 1995, 17, 28–30.
22. T. R. Cheek, Curr. Opin. Cell Biol., 1991, 3, 199–205.
23. T. Fujita and G. M. A. Palmieri, J. Bone Miner. Metab., 2000, 18, 109–125.
24. X. He, Z. Zhang, H. Zhang, Y. Zhao and Z. Chai, Toxicol. Sci., 2008, 103, 354–361.
25. J. Becker, A. Matusch, C. Depboylu, J. Dobrowolska and M. Zoriy, Anal. Chem., 2007, 79, 6074–6080.
26. M. West, A. Ellis, P. Kregsamer, P. Potts, C. Streli, C. Vanhoofe and P. Wobrauschekc, J. Anal. At. Spectrom., 2007, 22, 1304–1332.
27. H. Wang, M. Wang, B. Wang, X. Meng, Y. Wang, M. Li, W. Feng, Y. Zhaoa and Z. Chaia, J. Anal. At. Spectrom., 2010, 25, 328–333.
28. A. Nel, T. Xia, L. Madler and N. Li, Science, 2006, 311, 622–627.
29. Y. Zhao, G. Xing and Z. Chai, Nat. Nanotechnol., 2008, 3, 191–192.
30. J. Roh, S. Sim, J. Yi, K. Park, K. Chung, D. Ryu and J. Choi, Environ. Sci. Technol., 2009, 43, 3933–3940.
31. H. Ma, P. Bertsch, T. Glenn, N. Kabengi and P. Williams, Environ. Toxicol. Chem., 2009, 28, 1324–1330.
32. H. Wang, R. Wick and B. Xing, Environ. Pollut., 2009, 157, 1171–1177.
33. J. Roh, Y. Park, K. Park and J. Choi, Environ. Toxicol. Pharmacol., 2010, 29, 167–172.

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Footnote

† The first two authors contributed equally.

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