The induction of metallothioneins during pulsed cadmium exposure to Daphnia magna: Recovery and trans-generational effect

Ecotoxicology and Environmental Safety 126 (2016) 71–77

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

The induction of metallothioneins during pulsed cadmium exposure to Daphnia magna: Recovery and trans-generational effect Shuang Li a,n, Lianxi Sheng b, Jingbo Xu b, Haibin Tong c, Haibo Jiang b a

School of Forestry, Beihua University, Jilin 132013, PR China State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of Environment, Northeast Normal University, Changchun 130117, PR China c Life Science Research Center, Beihua University, Jilin 132013, PR China b

art ic l e i nf o

a b s t r a c t

Article history: Received 14 August 2015 Received in revised form 9 October 2015 Accepted 15 October 2015

Although the importance of pulse exposure has gained ground in recent years, there were few studies on recovery and trans-generational effect of it. Two successive generations Daphnia magna were exposed to cadmium (Cd) pulses for 6 h at the concentrations from 40 to 100 mg/l. The changes of tolerance and induction of MTs in exposed D. magna and their offspring were measured. The reduced tolerance of exposed D. magna was returned to levels similar to control after about 9 days in a generation. The level of MT still increased up to 3 days after exposure. In the experimental range, exposure duration played a decisive role in MT induction. The tolerance of F1 was lower than F0 and decreased with increasing pulsed concentrations of F0. Exposed to the same pulse, the MT levels of F1 were higher than the MT levels of F0, but the more obvious detoxification of MT in F1 had not been found. Our results suggest that pulsed cadmium exposure had impact on offspring of exposed organism and the risk assessment should take trans-generational effect into account. & 2015 Elsevier Inc. All rights reserved.

Keywords: Pulsed exposure Recovery Trans-generational effect Metallothioneins Cd

1. Introduction Pulsed toxicant exposures have been studied for decades of years, which are generated by surface water runoff, precipitationassociated hydrologic dilution, dispersion, as well as degradation activity. Most studies focused on lethal effect on exposed individual (Diamond et al., 2006; Ashauer et al.,2007; Hoang et al., 2007; Naddy et al., 2000; Angle et al., 2010). Only a few sub-lethal responses have been published, including reproductive effects during nitrite exposure (Alonso and Camargo, 2009), altered growth rates during pesticide exposures (Jarvinen et al., 1988), changes in osmoregulation (Davenport, 1977), thiobarbituric acid reactive substances and total glutathione (Amachree et al., 2013) during Cu exposures. Better understanding of pulsed toxicant exposure will help us to predict accurately the risk to population in the field. As an important characteristic of pulsed exposure, recovery even influenced the result of it (Wang and Hanson, 1985). Some researchers have quantified the effect of recovery time (Naddy et al., 2000; Naddy and Klaine, 2001; Reynaldi and Liess, 2004; Diamond et al., 2006; Zhao and Newman, 2006; Hoang et al., n

Corresponding author. E-mail address: [email protected] (S. Li).

http://dx.doi.org/10.1016/j.ecoenv.2015.10.015 0147-6513/& 2015 Elsevier Inc. All rights reserved.

2007) on pulsed exposure. Only Naddy and Klaine (2001) reported recovery occurring when the amount of accumulating toxic substance in organism did not exceed critical toxic threshold or when clearance period was long enough between consecutive pulses. There is few report on how does recovery happen and how do biomarkers change during pulsed exposure. More importantly, without the information on offspring of exposed organism, the “recovery” of pulsed exposure may be incomplete. Cd is a commonly pollutant that accumulates in aquatic organism. Its toxicity is caused by the production of reactive oxygen species that disrupt the normal physiological processes (Stohs and Bagghi, 1995). The key detoxification process of Cd implies a metalloprotein, the metallothioneins (MTs), which are non-enzymatic proteins with a low molecular weight, high cysteine content, no aromatic amino acids and heat stability. MTs have been considered playing an important role to detoxify or control the internal availability of trace metals in many aquatic invertebrates (Roesijadi, 1992; Wallace et al., 2003). Some studies have proved that MTs are the main detoxification mechanism for Daphnia magna to handle Cd stress (Klaassen et al., 1999; Fraysse et al., 2006). The current study was undertaken to assess the toxicity of pulsed Cd exposure to D. magna. The changes of tolerance and MT levels of two successive generations D. magna were measured as the test endpoint. The questions to be answered were, firstly how does

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were conducted with “recovered” daphnia on the day 12. The MT measurements were conducted immediately after exposure and every three days. In single pulsed Cd exposure, there were no significant changes in MT levels three days after exposure. So the MT measurements were conducted until the day 15, that is, three days after second exposure. The 21-day mortality of first pulse and second pulse were recorded. The changes of 48-h EC50s and MT induction in two successive generations: The 48 h immobilization tests (OECD, 2004) were conducted with F1 daphnia whose parents (F0) were exposed by 40, 60, 80 and 100 mg/l Cd pulse for 6 h, respectively. The concentrations used in immobilization tests were: control, 10, 20, 40, 80 and 100 mg/l. 3-day-old F1 daphnia were pulsed by the same cadmium exposure as their parents. The MT levels in F0, pulsed F1 (F1p) and no pulsed F1 (F1n) were measured. The 21-day mortality of F0, F1p and F1n were recorded. The whole flow diagram of the experimental procedure was shown in Fig. 1.

recovery happen during pulsed exposure in a generation? Secondly, whether trans-generational effect exists during pulsed exposure?

2. Materials and methods 2.1. Experimental organisms The cladoceran D. magna clone (from the Chinese Academy of Sciences, Wuhan, PR, China) has been cultured in our laboratory for more than ten years. They were maintained at a density of 10 adults/l in M7-Elendt medium at 20 71 °C in a light: dark regime of 16:8 h (OECD, 1997). Culture medium was renewed and the offspring were discarded twice a week. Brood daphnia were replaced after four to six weeks in culture. D. magna were fed a suspension of the unicellular green alga Pseudokirchneriella subcapitata three times a day. The feeding rate was 2
105 cells/ml/d. Algae were grown in the medium described in the International Organization for Standardization guideline 8692 (IOS, 1989).

2.3. Metallothioneins measurement 2.2. Testing methods The MT concentrations in the daphnia were quantified using a modified Ag saturation method (Shi and Wang, 2004; Scheuhammer and Cherain, 1991). 20 daphnia were dabbed dry with a paper and the wet weights were determined. The animals then were frozen at 80 °C until MT quantification. 0.5 ml of 0.25 M sucrose buffer were added to daphnia and then the mixture were homogenized in an ice bath. After ultrasonic treatment, the homogenates were centrifuged at 16,000g (20 min at 4 °C) to obtain the supernatants. 0.3 ml of 0.5 M glycine buffer (Sigma) and 0.5 ml of 20 mg Ag ml (Sigma) spiked with 110mAg (Risø National Laboratory) at 3.7 kBq/ml were added to supernatants. After MT binding sites were saturated with Ag within 10 min at room temperature, and excess Ag was removed by three additions of 0.1 ml of rabbit red blood cell hemolysate. After heating (5 min at 100 °C) and centrifuging (5 min at 5000g), the supernatant was centrifuged again for 5 min at 16,000g. The amount of Ag left in the supernatant was determined following the radio-assay for 110m Ag. MT concentrations were calculated as 3.55
of the Ag concentrations and expressed as μg/g wet weight of the daphnia.

3-day-old daphnia were used at the beginning of the test. Pulsed exposure immediately started after transferring D. magna to the beakers which were filled with 400-ml test solutions. Exposed time was 6 h and concentrations were 40, 60, 80 and 100 mg/l, respectively. After exposure, the daphnia were transferred to clean media. Five D. magna were exposed per beaker, with each treatment for MT measurement having 20 replicates and other treatments having 4 replicates. The changes of 48-h EC50s and MT induction in a generation: The 48 h immobilization tests referred to OECD guidance (OECD, 2004) were conducted with daphnia pre-exposed to100 mg/l, 6 h Cd pulse every three days until the day 21. The concentrations used in immobilization tests were: control, 10, 20, 40, 80 and 100 mg/l. There were no significant difference of 48-h EC50s between the treated group and the control group on the day 12. The daphnia could recover from100 mg/l, 6 h pulsed exposure on the day 12 and, of course, could recover from a 6 h Cd pulsed exposure whose concentrations were lower than 100 mg/l. So the second pulses

Recovery and MT induction in a generation First pulsed exposure rd

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Recovery and MT induction in two successive generations Pulsed exposure F0 (3rd day)

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F1p (3rd day)

Fig. 1. The flow diagram of the experimental procedure. In a generation test, the 48 h immobilization tests were conducted from 3rd day to 21st day. The MT measurement was conducted from 3rd to 15th day. Second pulsed exposure was conducted on 12th day. In two successive generations test, the 48 h immobilization tests and MT measurement were all conducted for F0, F1n and F1p. 21-day mortality of all treatments were recorded.

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2.4. Water chemistry

50 C P

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6d

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Fig. 2. The 48-h EC50 values with standard deviation (error bars) from acute toxicity test of pulsed 100 μg/l F0 and control. Asterisk represents statistically significant differences at P o 0.05.

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Pulsed Cadmium concentration of F0 (µg/l) Fig. 3. The 48-h EC50 values with standard deviation (error bars) from acute tests with Cd of F1. Asterisk represents statistically significant differences at Po 0.05. Table 1 21-day mortality of F0 and F1 for each generation and concentration. (%, mean 7 standard deviation, n¼ 4).

3.1. Acute toxicity tests and 21-days mortality Fig. 2 displayed the comparison between the 48-h EC50 values of 100 μg/l pulsed Cd exposure (P) and controls (C) from day 6 of test to the end of test (day 21). A clear tolerance variability of the P could be documented, with that of day 6 being the lowest point of test and then increasing over time. On the day 6 and 9, that is 3 and 6 days after pulsed exposure, the EC50 values of pulsed treatments were significantly lower than that of controls. But from the day 12, there was no difference between the two groups until the test ended. The EC50 values of F1 generation were illustrated in Fig. 3. On the scale of test, the 48-h EC50 of F1 decreased with increasing pulsed concentrations of F0. All 48-h EC50 values were lower than that of control. Specially, the EC50 values of 80 and 100 mg/l treatments significantly decreased compared with the control. There were no differences of 21-day mortality between first pulse and second pulse of double pulses, 5 72% and 9 74%

30

Time (days)

2.5. Statistics The 48-h EC50 and their 95% confidence limits were calculated with Origin software (8.0 SR4, Origin Lab Corporation, USA). Datasets were tested for normality using the Shapiro–Wilk W test. Statistical differences between the treatments were tested by Friedmann ANOVA. EC50 values which met assumptions of normal distribution and homogeneity of variance were compared using one way analysis of variance (ANOVA) followed by Dunnett's test. The MT levels were analyzed using the nonparametric Wilcoxon's rank test. A significant difference was accepted at P o0.05.

*

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20

48h EC50(µg/l)

The flame atom absorption spectrophotometry (F-AAS, Perkin– Elmer M1100, Waltham, Massachusetts, USA) using a Tritisol Cd standard (1 g/l, Merck Darmstadt, Germany) was used to analyze Cd concentrations in test media which came from acidified (0.2%, HNO3) cadmium chloride (CdCl2.H2O, Merck, Germany). Artificial, simplified M7-Elendt medium, without EDTA, was used as exposure water. Water hardness, alkalinity, pH, dissolved oxygen and DOC were characterized at the start and end of exposures and weekly during each test according to standard methods (Lewis et al., 1994, APHA et al., 1995). Hardness and alkalinity were determined by titration with 0.01 M ethylenediamine tetra acetate (EDTA) and 0.02 N H2SO4, respectively. A Thermo Orion pH meter (model 525, Orion Research, Beverly, MA, USA) and an YSI meter (model 85, YSI Incorporated, Yellow Springs, OH, USA) were used to measure the pH and dissolved oxygen respectively. DOC was determined by combustion of 0.45-μm filtered water (Model TOC 5000A, Shimadzu Scientific Instruments, Columbia, MD, USA). The pH and dissolved oxygen concentration, hardness, and alkalinity in all test waters were 7.28 70.27, 8.067 0.22 mg/l, 189 713 mg/l as CaCO3, and 68 78 mg/l as CaCO3, 0.53 70.14 mg/l, respectively. We performed QA/QC analyses of Cd and DOC that included sample duplicates and spikes, initial and continuing verification standards, initial and continuing calibration blanks, and external QC samples according to USEPA (1994) guidelines. Analyses of certified Cd reference standards averaged 111% of the certified value (S.D. ¼6.2%, n ¼36), recovery of analytical spikes of Cu averaged 92% (S.D. ¼ 10.9%, n ¼ 10). The instrument detection limit for Cd was about 0.01 μm. Measured concentrations never differed more than 10% from the nominal values and nominal concentrations were used throughout the study.

73

F0 F1n F1p

40 μg Cd l  1

60 μg Cd l  1

80 μg Cd l  1

100 μg Cd l  1

17 0a 37 1b 47 0.5b

3 70.5a 4.5 72a 10 74b

57 1a 97 1b 207 3c

57 2a 177 3b 247 3c

Different letters in the superscripts represent statistically significant differences between treatments within each concentratrion (Po 0.05).

respectively. Other results of 21-day mortality were shown in Table 1. On the whole, there were significant difference between four treatments of F0 and F1p, three treatments of F0 and F1n, except 60 mgCdl  1 treatment, two higher concentration treatments, 80 and 100 mg/l of F1n and F1p. 3.2. MT induction in single pulse and the changes over time The different MT levels in D. magna exposed to four cadmium pulses and the changes over time were presented in Fig. 4. After 6 h pulse, there were significant differences between four

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Fig. 4. The different MT levels in D. magna exposed to four cadmium pulses and changes over time. (A) the 3rd day; (B) the 6th day; (C) the 9th day; (D) the 12th day. Different letter represents statistically significant differences at P o 0.05.

treatments and the control. The MT levels of 60, 80 and 100 mg/l treatments were clearly higher than that of 40 mg/l treatment, but there were not differences among them (Fig. 4a). On 6th day, three days after pulses, the MT levels of 80 and 100 mg/l treatments increased again and were significant higher than that of 60 mg/l treatment (Fig. 4b). Thereafter, there were no changes in relations among treatments on the 9th day (Fig. 4c) and 12th day (Fig. 4d). 3.3. The comparisons of MT levels between single and double pulses over time As shown in Fig. 5, on the 12th day, the MT in double pulses treatments were introduced by the second cadmium pulse and the levels were significant higher than that in D. magna which only experienced the single one. On the 15th day, three days after the second pulses, the MT levels of all double pulses increased again and were about twice than that of the single one. 3.4. The MT levels in F0 and F1 D. magna The MT levels obtained after exposure of the F0 and F1 D. magna are presented in Fig. 6. MT was induced in all pulsed animals, being significantly different from control and the levels all increased with increasing Cd concentrations. The difference between F1n and control was not obvious. Compared to F1p, MT levels of F0 were lower when treated with the same pulsed concentrations and the differences between them were significant. 4. Discussion 4.1. The effect of exposure duration and concentration on induction

of MT MT levels have a close relationship with metal accumulation concentration (Roesijadi, 1992). Toxicant accumulation is dependent on exposure concentration and duration, so MT levels should relatively dependent on exposure concentration and duration (Guan and Wang, 2004). After pulsed Cd exposure for 6 h, significant induction of MT was present in D. magna, but there were no significant differences among the three higher concentrations. The higher concentration did not increase MT levels. However, this result was inconsistent with some previous studies. For instance, significant MT induction was observed on the first day in T. brevicornis exposed to Cd, especially it had a dose-effect relation, without threshold (Barka et al., 2001). Pan and Zhang (2006) observed the increased levels of MT in gills and hepatopancreas in C. japonita after 3 days of exposure to Cd, and also there was a dose– effect relation. One main difference between our study and mentioned above was exposure duration. Because the duration of pulsed exposure was relatively shorter (6 h in our study) and the concentrations were relatively higher, the duration became the limiting factor of MT induction. This may be explained by the induction of MT during double cadmium pulses, twice duration formed about twice MT levels compared to single pulse. In some laboratory tests, a feature of organism exposed to high concentration of metals was the reduction of MT levels, probably as a result of a toxic effect preventing detoxification processes from being fully functional. For instance, in amphipod E. echinosetosus exposed to Cd for 24 h, MT was significantly induced over the range of exposure concentrations from 100 to 1000 μg/l. For the highest dose tested (2000 μg/l), MT concentration was higher than that in control but not as high as in specimen exposed to

S. Li et al. / Ecotoxicology and Environmental Safety 126 (2016) 71–77

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Fig. 5. The comparison of MT levels between single pulses (S-pulse) and double pulses (D-pulse) over time. (A) 40 mg/l; (B) 60 mg/l; (C) 80 mg/l; (D) 100 mg/l. Different letters represent statistically significant differences at P o0.05.

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MT is key protein of detoxification in D. magna under stress environment, but not the only one. The “spill over” of MT induction has not been observed at the highest concentration, 100 mg/l, but this phenomenon of hsp70 has been reported in our previous study (Li et al., 2014). This result showed toxicity threshold of hsp70 was lower than that of MT. This may explain why MT is more important than hsp70 in detoxification of metals. In general, in the experimental range, exposure duration also played a decisive role in MT induction. The conclusion of dosedependent relation came from single exposure duration may be inappropriate, especially when the MT level was used to be biomarker of metal contamination.

control

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4.2. Delayed effect and shortcoming of evaluation for recovery

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Pulsed Cadmium concentration (µg/l) Fig. 6. The MT levels of F0, F1n and F1p. Different letters represent significant differences at Po 0.05.

1000 μg/l (Martinez et al., 1996). Such a phenomenon has previously been termed “spill over” (Brown and Parsons, 1978). This phenomenon has not been observed in our study even the highest concentration, 100 mg/l, which was about 2.5 times 48 h EC50. This concentration was higher than 17 day LC50, but the latter caused the absence of MT induction or even a depletion of MT levels when a amphipod, O. gammarellus exposed to Cu and Zn (Mouneyrac et al., 2002). This may demonstrate it is inappropriate to explain the test results only using exposure concentration, especially for pulsed exposure.

For single pulse, the MT levels of 80 and 100 mg/l treatments were still rising on the 6th day which was three days after exposure. For double pulses, the MT levels of all treatments were still rising on the 15th day which was three days after exposure. It was clear that the two increases of MT levels caused by previous pulsed cadmium exposure and there was a delayed time between exposure and induction. Guan and Wang (2004) have mentioned a delay in MT synthesis with the Cd accumulation in the animals and they defined it as initial delay. There was a certain trigger level for MT induction in response to Cd, the delay was time organism needed to accumulate Cd to reach the trigger level. In combination of our results, the delay of MT induction might include two parts, the initial and the last. The initial was the threshold of MT synthesis and indicated the limited function of MT as a protection mechanism under condition of metal stress. The last was the increasing of MT level after exposure. This process help organism

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improve the detoxification capability for cadmium and also may be the reason for recovery. Maybe the recovery for organism is to improve the toxic threshold after exposure and the recovery period is time which organism needed to improve toxic threshold to some extent. The delay of hsp70 induction has been observed in our previous study, a day after exposure the levels of hsp70 still increased (Li et al., 2014). That is, there were two detoxification mechanisms after exposure at least and they all help organism recover from exposure. 80–100% recoveries of Ach-E and Cb-E activities in D. magna took place 12 and 96 h after exposure to organophosphorous and carbamate pesticides, respectively (Carlos et al., 2004). More importantly, the recovery of population needs more attention. The effect of pulsed exposure on offspring however, remains largely unknown. Limited data showed that the average cumulated weight of offspring produced by control animals was more than twice that of the group exposed to 30 mg l  1 of dimethoate for 3 h (Andersen et al., 2006). The hatchability and incidence of abnormalities in the offspring of flagfish (jordanella floridae) were all adversely affected by a single 2-h exposure of 8-D-old juveniles to concentrations of methoxychlor 40.25 mg/l (Holdway and Dixon, 1986). In our previous study, the growth and reproduction were not affected by pulsed cadmium exposure (Li et al., 2014). But the tolerance of offspring came from 80 and 100 mg/l treatments reduced significantly. The effect on 21-day mortality was more apparently. The 21-day mortality of the 80 and 100 mg/l treatments of F1n were clearly higher than that of F0 even without exposure. After exposure, the responses of all treatments (F1p) were significant difference with F0. During the range of the test, 9 days after the exposure, the exposed organism recovered, but this recovery was only toxic effect on individuals in a laboratory test. When the results were used to explain and evaluate the consequences of pulsed exposure in the field, the risk may be underestimated. The effects of temporary chemical exposures on future ecosystem should be taken into account. Such “recovered” population maybe fragile and the fate of it is unknown in complex stress environments. 4.3. Tolerance and trans-generational effect For multiple pulses, the previous pulse can be viewed as preexposure of the latter. After pre-exposure, D. magna were more tolerant to some metals, including Cu (LeBlanc, 1982), Cd (Guan and Wang, 2004; Bodar et al., 1990) and Hg (Tsui and Wang, 2005a; 2005b). But in our study, the tolerance significantly decreased after the first pulse and nine days were needed to recover to a similar level in control. An important factor for tolerance may be the ratio of the pre-exposure concentration to the sensitivity (48 h EC50) of control organisms as described by Chapman (1985). The ratio ranged from 0.003 to 0.014 could significantly increase tolerance. The concentrations of pulsed exposure are generally greater than the 48 h EC50 of the organism and this ratio ranged from 1.039 to 2.597 in our study. After the same pulse, the MT levels of F1p were significant higher than that of F0. In general, there are two reasons for that: 1) the weight of F1p was lower than that of F0. So the MT level of F1p will be higher under the same induction of MT. Andersen et al. (2006) reported that the average cumulated weight of offspring produced by control animals was more than double that of the group exposed to 30 mg/l dimethoate pulse for 3 h. But there was no difference between F1p and F0 in our study (data in the Supplementary material). 2) The induction of F1p was stronger than that of F0. The reason of it was attributed to trans-generational effect which made the MT in F1p more sensitive to cadmium. But

what contradicted with this was the MT levels had no difference in F1n and control. Guan and Wang (2006) have speculated the transgenerational effect of Cd exposure on the MT induction came from increased Cd body burden in neonatal daphnia. There was a certain trigger level for MT induction in response to Cd body burden. The Cd body burden in F1n came from maternal exposure may be lower than that and did not introduce significant MT synthesis. But after a pulse, in F1p, the Cd body burden increased and was higher than the trigger level. Following the induction, the MT increased with increasing Cd body burden. A 10-day intra-peritoneal injection of Cd could result in maternal transfer of the messenger ribonucleic acid (mRNA) of MT in fish to their eggs (Lin et al., 2000), but if this effect exists in D. magna is unknown. However, it was shown that one generation exposure to a sub-lethal Zn concentration caused an overall reduction in DNA methylation in the F1 offspring (Vandegehuchte et al., 2009). There was a clear difference between the induction of MT and hsp70 in F1p exposed to the same pulse. Compared to F0, MT level increased, but hsp70 decreased (Li et al., 2014). The reasons need further study and so we need to be more careful when use them as biomarkers. It was worth noting that detoxification of increased MT levels in F1p to Cd exposure has not appeared. For F1p, the tolerance decreased and the 21-day mortality increased. This may be because partially inactive of some detoxification mechanism, for instance, decreased hsp70 induction.

5. Conclusion The present study demonstrates the changes of tolerance and induction of MT in two successive generations of D. magna during pulsed Cd exposure. The reduced tolerance of exposed organism get back to levels similar to that in control after a certain period of time, but the tolerance of their offspring decreased with increasing pulsed concentrations. Exposure duration also played a decisive role in MT induction. The increase of MT levels after exposure may be the reasons for recovery. Exposed to same pulse, although the MT levels of F1 were higher than that of F0, the detoxification effect of them have not appeared and the reasons need to be further explored. Our results suggest that just consider recovery in a generation is inappropriate. The risk assessment for pulsed exposure should take trans-generational effect into account.

Acknowledgments This research was funded by the Beihua University (NO. 2015-01).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at 10.1016/j.ecoenv.2015.10.015.

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