Zhang Hecai, Liu Tongyi, Liu Yanfang, Zhang Jie, Wang Zhige, Chen Guangwen

College of Life Sciences, Henan Normal University, Xinxiang 453007, China

Definition of Planarian Mortality in an Acute Toxicity Test: A Case Study onDugesiajaponicaExposed to 1-Octyl-3-methylimidazolium Bromide

Zhang Hecai, Liu Tongyi, Liu Yanfang, Zhang Jie, Wang Zhige, Chen Guangwen*

College of Life Sciences, Henan Normal University, Xinxiang 453007, China

Received 21 April 2016 accepted 12 June 2016

In order to define the mortality criteria of planarian objectively, a case study of Dugesia japonica exposed to 1-octyl-3-methylimidazolium bromide ([C8mim]Br) was performed followed by a recovery culture. The results showed that defining planarian mortality in light of its body disintegration was appropriate. If the disintegrated tissue of a planarian was more than 1/4 of its body length, it would completely degenerate. However, a planarian would regenerate the lost tissue and return to normal after a few days’ recovery culture if the disintegrated part was less than 1/4 of body length. Therefore, we propose to use body disintegration as the endpoint of planarian mortality, i.e., 1/4 body length degenerated is the critical threshold of mortality and survival of planarians when exposed to toxicants. This criterion could be adapted in the standardization of testing protocols and comparability of acute toxicity test or other toxicological research using planarian as the test animal.

1-octyl-3-methylimidazolium bromide; planarian; acute toxicity; mortality criterion; definition

Freshwater planarians, belonging to Phylum Platyhelminthes, Class Turbellaria, Order Tricladida, Suborder Paludicola, are distributed worldwide in unpolluted springs and streams. Traditionally, planarians have served as a favored animal model in regenerative biology and developmental biology due to their powerful regeneration capability[1-2]. Furthermore, planarians are very susceptible to environmental toxicants. Different agents such as heavy metals, herbicides and UV-irradiation given in sublethal doses can cause behavioral and morphological changes, histological and DNA molecular damages, as well as alterations of mitosis in planarians[3-6]. In addition, they are easily collected in large numbers and inexpensively kept in laboratory. All these characteristics make planarian one of the most suitable organisms for toxicological research.

Ionic liquids (ILs) are pure ionic compounds with melting points near room temperature (or by convention below 100 ℃)[7-8]. Due to their preponderant properties, such as almost no vapor pressure and nonvolatility, high thermal stability and nonflammability, and high solvent capacity and chemical stability, ILs have been applied in various areas of chemical industry[9-10]. With the application popularized and the research deepened, however, their greenness was questioned. Reports concerning the biotoxicity and evaluation of environmental hazards of ILs have being growing in literature[11-14].

In animal toxicology studies, the acute toxicity test with the aim of obtaining LD50/LC50of toxicants is not only the main method to determine the toxicity of toxicants, but also the basis and premise of a chronic toxicity test[15-16]. The determination of LD50/LC50is closely related to the mortality of test animals in acute exposure. Therefore, defining the death status of test animals objectively and further acquiring the number of deaths accurately are the keys of calculating the LD50/LC50appropriately. The poisoning symptoms of freshwater planarians in an acute toxic test differ from partial disintegration and other morphological changes, such as twisting, contraction, etc. to complete disintegration into debris[15].Whether a planarian with partial disintegration (a status before complete degenerating into debris) should be defined as death is an important issue in the acute toxicity test. However, it remains an open question at the moment because of the powerful regeneration capability of planarians. In the literature related to planarian toxicology, different researchers hold different views on planarian mortality criteria. In Camargo and Ward[17], death was defined as test animals not moving and not reacting to gentle prodding; in Li[18], the organisms without detectable movement were considered to be dead; whereas Alonso and Camargo[19-21]suggested that a planarian was considered to be dead when the body tissue started to degenerate. The definition of planarian mortality criteria will directly influence the determination of LC50of a toxicant and the accuracy of subsequent results. Therefore, establishment of a universal mortality criterion in planarian acute toxicity test is needed not only for the accuracy of toxicity evaluation but also for the standardization and comparability of planarian toxicology.

The aim of the present study was to define an objective criterion for planarian mortality via a case study of a common planarian species Dugesia japonica exposed to one type of normally used and investigated imidazolium-based ionic liquids 1-octyl-3-methylimidazolium bromide ([C8mim]Br), followed by a recovery experiment.

1 Materials and methods

1.1 Chemicals

The ionic liquid used in this study was 1-octyl-3-methylimidazolium bromide, which was purchased from Hubei Hengshuo Chemical CO., LTD. (Wuhan, China) with a chemical purity of 99%. The stock solution and all concentrations of testing chemicals were prepared in dechlorinated tap water.

1.2 Animals

Dugesia japonica used in this study was collected from Yu-quan stream (Qi County, China) in April, 2015. Animals have been maintained in dechlorinated tap water at laboratory and fed with raw fish spleen once a week. Planarians fasting for one week were used in the experiments.

為進(jìn)行PPO軌跡的參數(shù)優(yōu)化與最低能耗比較，引入電機(jī)輸入電能和機(jī)械能耗兩個(gè)量。討論電機(jī)輸入電能時(shí)，只計(jì)算機(jī)器人驅(qū)動(dòng)電機(jī)所消耗電能；在實(shí)驗(yàn)和仿真計(jì)算中，根據(jù)實(shí)際情況對(duì)再生能量進(jìn)行處理。

1.3 Toxic exposure and recovery

Fifty planarians with normal morphology and 10-15 mm in gliding length were selected and divided into five groups evenly. Every 10 animals were placed in a 100 mL Petri dish. One control group was cultured in dechlorinated tap water, while the other four were exposed to [C8mim]Br solution of 220 mg·L-1which was designed according to the LC50value determined by Shi et al.[15]. Animals were inspected every 24 hours with a stereomicroscope, and those presenting different poisoning symptoms were transferred into the Petri dishes containing dechlorinated tap water to recover by one planarian per Petri dish. The recovered animals were cultured till the final status (back to normal or complete disintegration to death) was observed. In order to keep the experimental conditions consistent, all the bioassays including the toxic exposure and the recovery culture were performed in a temperature incubator at (20±1) ℃. IL solution and dechlorinated tap water were renewed every day. The animals were not fed during the whole experiments.

2 Results and discussion

In our study, planarians in the control group retained normal morphology and moved normally in water, while those in the treatment groups presented different poisoning symptoms such as body twisting, contraction, depigmentation, head degeneration, tail degeneration and total degeneration. The earliest morpholocial change was body twisting or contraction. Planarians with these poisoning symptoms returned to normal in 1-2 days recovery culture (Table 1,Fig.1a-b’). Planarians with depigmentation would gradually become darker and return to normal body color after 3 days’ recovery (Table 1, Fig.1c-c’). Planarians with cavity repaired themselves by regeneration and returned to normal in 8-10 days recovery (Table 1, Fig.1d-d’). As for those with head disintegration or both head and tail disintegration, if the lost part was less than 1/4 of the body length, they regenerated the lost and returned to normal after 6 days’ recovery (Table 1, Fig.1e-f’); if the disintegrated tissue was more than 1/4 of the body length, regardless of the lost was head only or both head and tail, they disintegrated totally into debris in at most one week (Table 1, Fig.1g-h’). In addition, those with serious disintegration (lost part >3/5 body length) completely degenerated in one day after being transferred into dechlorinated tap water (photo not shown).

Fig. 1 Poisoning symptoms of planarians in toxic exposure and the corresponding final states after recovery cultureNote: a, twisting, a’, back to normal; b, contraction, b’, backto normal; c, depigmentation and contraction, c’, back to normal; d, cavity, d’,back to normal; e, head disintegration (lp<1/4 bl), e’, back to normal; f, both head and tail disintegration (lp<1/4 bl),f’, back to normal; g, head disintegration (lp>1/4 bl), g’, complete disintegration to death; h, both head and tail disintegration (lp>1/4 bl), h’, complete disintegration to death. lp, lost part, bl, body length; scale bar=3 mm.

Table 1 The toxic exposure followed by recovery culture of D. japonica

Note:lp, lost part; bl, body length.

The results of toxicity tests with internationally standardized species are important for evaluating the sensitivities of different organisms and the toxicity of diverse substances[22]. In comparison with insects, molluscs, and other invertebrate groups, the use of platyhelminths in toxicity tests is relatively rare. Paludicola planarians, however, have been utilized in several dispersed geographic regions as indicators of water quality and to evaluate the toxic effects of diverse substances[5-6,21]. Undoubtedly, the inconsistency in planarian mortality criterion will affect the evaluation of water quality and toxicants’ toxicity. To date, two indicators have been applied to define the mortality in planarian toxicology, one was movement and the other was body disintegration. Movement as an indicator is difficult to operate. For example, Camargo and Ward[17]defined death as test animals not moving and not reacting to gentle prodding, while Li[18]defined death as the organisms without detectable movement. By contrast, we suggest defining the mortality of planarian in light of its body disintegration in the toxic exposure. At present, there are two viewpoints to define the relationship between planarian mortality and disintegration. One claims that complete degeneration means mortality[23]and the other insists the beginning of degeneration means mortality[17,20-21]. In our opinion, the two definitions represent two extremes and were not very suitable for various circumstances. Based on our results, if the degenerated tissue of a planarian was more than 1/4 of its body length, it would finally degenerate totally into debris in at most one week; in contrast, if the degenerated tissue was less than 1/4 of its body length, it would regenerate the lost part and return to normal in approximately 6 days recovery. Therefore, that the degenerated part is more than 1/4 body length is the threshold of mortality and survival of a planarian exposed to the toxicant. Obviously, the complete degeneration criterion will lead to the higher LC50and lower toxicity of toxicants; whereas the inverse conclusion will be drawn if the criterion based on the beginning of degeneration is applied.

Acknowledgement：The authors would like to thank Dr. Jianyong Wu (U. S. Environmental Protection Agency) and Dr. Jiyong Wang (Department of Biological Science, Columbia University, US) for critical reading the manuscript.

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10.7524/AJE.1673-5897.20160421002

Foundation：This work was supported by the National Natural Science Foundation of China (31471965), the Natural Science Foundation of Henan Province (142300410457) and the National Student Innovation Training Project of Henan Normal University (201410476051).

Author introduction：Zhang Hecai (1977-), PhD, Associate Professor in Henan Normal University. Main research interests are planarian molecular systematics and toxicology. E-mail: zhcai9339@163.com

*Corresponding author， E-mail: chengw0183@sina.com

Zhang H, Liu T, Liu Y, et al. Definition of planarian mortality in an acute toxicity test: A case study on Dugesia japonica exposed to 1-octyl-3-methylimidazolium bromide [J]. Asian Journal of Ecotoxicology, 2016, 11(6): 291-295