Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Contents lists available at ScienceDirect

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

Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna Yi Chena,b, Yanjie Gaoa, Haojun Zhua,c, Jörg Romeisa,b, Yunhe Lia, Yufa Penga, Xiuping Chena,

T ⁎

a

The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Agroscope, Research Devision Agroecology and Environment, 8046 Zurich, Switzerland c Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bt protein Environmental risk assessment Non-target effects Daphnia magna Chronic toxicity Catalase

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

1. Introduction Rice (Oryza sp.) is damaged by many different insect pests, e.g., rice leaf folders and stem borers, causing economic losses in the millions of U.S. dollars every year (Chen et al., 2011; Heinrichs et al., 2017). In general, chemical insecticides are used to control these pests. Expression of insecticidal cry genes from Bacillus thuringiensis (Bt) in transgenic rice lines provides an alternative way to pest control and a chance to minimize the use of insecticides (Chen et al., 2011; Huang et al., 2015). Many rice lines that produce Cry proteins from Bt have been successfully developed in China (Li et al., 2016). In 2009, safety certificates were granted to two Bt rice lines producing Cry1Ab/Ac for commercial planting in Hubei province, China (Lu, 2010), triggering a heated public debate. The core of this debate revolved around the effects that the insecticidal Cry proteins might have on food safety and the environment. Previous studies concerning the effects of Bt rice on the environment mainly focused on terrestrial non-target arthropods, the soil ecosystem, and gene flow (Li et al., 2016). Rice differs from dry-land crops such as corn and cotton in that it requires a layer of water, i.e., a paddy, during

⁎

most of its developmental stages. Notably, during traditional rice cultivation in China, rice byproducts are left on planted surfaces after harvest, so they might enter the local ground water via seepage into the soil following strong wind, rain, or hail. The plant-produced Cry protein may thus enter the aquatic system. Previous studies reported that transgenic rice released up to 30 ng/L of Cry1Ab/Ac into water (Liu et al., 2016; Wang et al., 2013a; Zhang, 2013). Therefore, the risk to non-target aquatic organisms by exposure to Bt proteins expressed in rice needs to be addressed. Zooplankton is an essential part of the aquatic food chain. They are the main consumer of single-cell algae and organic detritus, and serve as food for organisms at higher trophic levels. Hence, changes in their abundance, diversity, and distribution may have cascading effects throughout a water ecosystem (Takagaki, 2016). Moreover, zooplankton is very sensitive to many different types of contaminants and therefore can act as an indicator for changes in water quality (Xu et al., 2005; Jiang et al., 2012). Previous studies confirmed that pesticides used in paddy fields subsequently affect the zooplankton communities in adjacent lakes and ponds (Hanazato, 2001). Therefore, in case that Bt rice has an adverse impact on aquatic organisms in the paddy fields, the

Corresponding author. E-mail address: [email protected] (X. Chen).

https://doi.org/10.1016/j.ecoenv.2018.09.045 Received 4 June 2018; Received in revised form 28 August 2018; Accepted 9 September 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

consequences might also become evident in surrounding water ecosystems. The order Cladocera (water fleas) is a member of zooplankton and commonly found in the aquatic ecosystems of rice paddies (Bahaar and Bhat, 2011; Zhang, 2013). These organisms may potentially be exposed to plant-produced Cry proteins through ingestion of rice pollen, plant residues, or root exudates (Viktorov, 2011; Carstens et al., 2012). The Cladocera species Daphnia magna is widely used as a test species in environmental toxicology because of its short life cycle, fast reproduction rate, and high sensitivity to environmental contaminants (Hebert and Crease, 1983; Schindler, 1987; Kim et al., 2015). In addition, this organism is easy to obtain and to raise under laboratory conditions. Previous studies concerning the effect of Lepidoptera-active Cry proteins or Bt-transgenic crops on D. magna have produced inconsistent results. While most studies have reported no adverse effects (Mendelson et al., 2003; Oh et al., 2011; Wang et al., 2013b; Zhang et al., 2016), studies by one research group have repeatedly resulted in significant fitness effects (Bøhn et al., 2008, 2010, 2016; Holderbaum et al., 2015). The transgenic rice line T1C-19, which expresses the Cry1C protein, may be used as a commercial rice line in China because of its good resistance to lepidopteran rice pests including the stem borers Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrocis medinalis (Zheng et al., 2011; Wang et al., 2016). We previously assessed the effects of purified Cry1C on the development and reproduction of D. magna and found no adverse effects after 21 days of exposure (Chen et al., 2018). In the present study, we expanded our assessment by examining whether the straw leachate from T1C-19 rice affects the fitness of this aquatic arthropod.

Fig. 1. Cry1C concentrations (ng/L) in T1C-19 rice-straw leachate after different immersion time. Data are means ± SE (n = 4). Different lowercase letters mean significant differences between treatments at the same time point (one-way ANOVA followed by the Tukey HSD test).

(0.6–2.1 mg/L). 2.3. Leachate preparations and water quality analyses To assess to what extent the Cry protein will disperse into the leachate, T1C-19 or MH63 rice straw (cut into 1-cm pieces) was immersed in 1000 mL of M4 medium at different amounts i.e., 0.1, 0.2, or 0.4 g/L, and kept in a homoeothermic incubator (20 ± 1 °C, 70 ± 5% relative humidity). Water samples were taken after 24, 48, and 72 h of incubation to determine the Cry1C concentration by enzyme-linked immuno sorbent assay (ELISA) as described below. Rice straw leachates were produced by soaking straw samples of T1C-19 or MH63 (cut into 1-cm pieces) in 3-L conical flasks containing 1500 mL of M4 medium (0.4 g/L per sample). The flasks were placed into an incubator for 24 h at 20 °C. The amount of rice straw and the 24 h duration was chosen to maximize the Cry1C content in the M4 medium (Fig. 1). Subsequently, the extracts were centrifuged at 15,700×g for 5 min at 4 °C. The supernatants were sterilized by filtration through a 0.22-µm filter (Millipore) and stored for the subsequent bioassay (see below) or for the analysis of water quality which was performed at the Center for Environmental Quality Test, Tsinghua University (Beijing, China). The following six parameters were analyzed: pH, total hardness, chemical oxygen demand, total nitrogen, dissolved oxygen, ammonia nitrogen, in accordance with Chinese Standard methods (GB 6920-1986, GB/T5750.4-2006, HJ/T 399-2007, HJ 636-2012, GB/T 11913-1989, HJ 535-2009).

2. Materials and methods 2.1. Plants The transgenic rice line T1C-19 and its corresponding non-transgenic near-isoline Minghui 63 (MH63) were used for all experiments. The expression of the synthetic cry1C gene in T1C-19 rice is under the control of the corn ubiquitin promoter (Tang et al., 2006). The MH63 line is commonly grown in China and is an elite indica line for cytoplasmic male sterility. Both rice lines were obtained from Huazhong Agricultural University (Wuhan, China). The two rice lines were planted at the same time in adjacent plots in the experimental field station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (39.53°N, 116.70°E). Rice plants were cultivated according to commonly used local agricultural practices but without application of chemical insecticides. Rice was harvested at the end of October 2014, and rice straw (stems) of each line from 20 cm above the soil surface were collected, pooled, and stored at −20 °C until used. 2.2. Organisms

2.4. Effects of straw leachates on D. magna

The green alga Chlorella pyrenoidosa (Chlorococcale: Chlorellaceae) was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). D. magna was obtained from the Shanghai Ocean University (Shanghai, China) as a monoclonal strain in a state of parthenogenesis. Maternal D. magna were cultured in a 5-L beaker containing M4 medium in a homothermic incubator (20 ± 1 °C, 70 ± 5% relative humidity) under a 12-h light/12-h dark cycle. D. magna were fed daily a diet of C. pyrenoidosa at a concentration of 3 × 106 cells/mL. Neonates (6–24 h of age) from the same mother were used for the experiments following the test procedures of the International Organization for Standardization (ISO, 2012) and Organization for Economic Cooperation and Development (OECD, 2012). Prior to experimentation, D. magna sensitivity to potassium dichromate (K2Cr2O7) was tested, and the 24 h-EC50 value was found to be 0.76 mg/L, which is within the ISO 6341 requirement for the sensitivity of D. magna to K2Cr2O7

Newly hatched D. magna (acquired within 6–24 h of hatching) were kept individually in a 100-mL beaker containing 50 mL of M4 medium and C. pyrenoidosa at a concentration of 1.3 × 106 cells/mL. Twenty individuals (20 replicates) were exposed to each of the following seven treatments: (1−3) M4 medium plus T1C-19 rice straw leachate at 100%, 50%, or 25% (v/v) (denoted 100%, 50%, and 25% T1C-19 M4 medium, respectively); (4−6) M4 medium plus MH63 rice straw leachate at 100%, 50%, or 25% (v/v) (denoted 100%, 50%, and 25% MH63 M4 medium, respectively); and (7) pure M4 medium (negative control). Thus the total number of D. magna used in this experiment was 140. To minimize mechanical damage to D. magna caused by handling, the medium was replaced every 2 days. The number of D. magna surviving, the number of molts, and the number of offspring produced were recorded daily. Offspring were removed and stored at −70 °C for 631

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

measurement of catalase (CAT) activity and Cry1C content by ELISA (see below). The experiment was terminated after 21 days, at which time body length, body width, anal spine length, and body mass of the remaining living D. magna were measured individually. All living adults were then stored at −70 °C for subsequent measurement of CAT activity and Cry1C content by ELISA.

Table 1 Water quality of M4 medium incubated without or with MH63 or T1C-19 ricestraw leachate at a concentration of 0.4 g/L for 24 h. Data are means ± SE (n = 4).

2.5. Determination of CAT activity CAT is one of the key enzymes of biological defense system, which can promote the decomposition of hydrogen peroxide into oxygen and water, thus eliminates the cytotoxicity of hydrogen peroxide. The antioxidant activity of CAT was quantified using a commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Samples of D. magna adults and neonates were separately homogenized at 4 °C in physiological saline at a ratio of 1:9 (w/v). The homogenates were centrifuged at 2500–3000×g for 10 min at 4 °C, and the supernatants were then assayed for CAT activity according to the manufacturer's instructions. Optical density values were recorded using a microplate spectrophotometer XS2 (PowerWave XS2, BioTek, Winooski, VT, USA), and CAT activity was calculated using a calibration curve derived from standards provided with the kit.

Parameter

Pure M4 medium (control)

MH63

T1C-19

pH Dissolved oxygen (mg/L) Total hardness (mg/L) Chemical oxygen demand (mg/ L) Total nitrogen (mg/L) Ammonia nitrogen (mg/L)

7.75 ± 0.06a 7.07 ± 0.28a

6.58 ± 0.14b 0.45 ± 0.02b

6.78 ± 0.05b 1.08 ± 0.62b

253.00 ± 2.12a

248.50 ± 1.71a

246.00 ± 1.68a

27.50 ± 2.41a

36.43 ± 0.99a

33.05 ± 4.61a

0.40 ± 0.05a

1.65 ± 0.22b

2.30 ± 0.34b

0.06 ± 0.02a

0.35 ± 0.02b

0.62 ± 0.19b

Different lowercase letters within the same row indicate a significant difference among means (ANOVA followed by Tukey HSD-test; p < 0.05).

3. Results 3.1. Cry1C content and water quality of rice-straw leachates The ELISA results revealed that the Cry1C concentration in the leachates increased with increasing amounts of T1C-19 rice-straw added, but decreased within cubation time (Fig. 1). To assess the water quality, leachates were collected after T1C-19 and MH63 rice straws had been incubated in M4 medium for 24 h at a concentration of 0.4 g/L and compared to pure M4 medium (Table 1). No significant differences were observed among the three treatments in terms of total hardness and chemical oxygen demand (one-way ANOVA; total hardness: F2,9 = 3.68, p > 0.05; chemical oxygen demand: F2,9 = 2.17, p > 0.05). The pH, dissolved oxygen, total nitrogen, and ammonia nitrogen values of the leachates were significantly lower in the pure medium than in the medium containing rice leachates (one-way ANOVA followed by Tukey HSD test; pH: F2,9 = 43.78, p < 0.001; Mann-Whitney U-test; dissolved oxygen: χ2 = 7.54, p = 0.02; total nitrogen: χ2 = 8.80, p = 0.01; ammonia nitrogen: χ2 = 6.73, p = 0.03). For none of the parameters assessed were significant differences observed between the T1C-19 and MH63 leachates (all p > 0.05, Table 1).

2.6. Determination of Cry1C content by ELISA The concentration of Cry1C in D. magna and in T1C-19 rice straw leachates after a 24 h, 48 h, and 72 h exposure was measured by ELISA using a commercial Cry1C detection kit (Quanti-Plate, EnviroLogix). Prior to analysis, D. magna adults and neonates were washed in phosphate-buffered saline/Tween-20 (provided with the kit) to remove Bt toxin from their outer body surfaces. For Cry1C extraction, samples of D. magna were weighed and mixed with phosphate-buffered saline/ Tween-20 at ratios of 1:10–1:100 (mg/µL) in 1.5 mL centrifuge tubes. The samples were then homogenized using an electric grinding rod. This step was not necessary for leachate samples. After centrifugation and appropriate dilution, ELISA was performed using the supernatants according to the manufacturer's instructions. Optical density values were recorded using amicroplate spectrophotometer (PowerWave XS2, BioTek, Winooski, VT, USA), and concentrations of Cry1C were calculated using a calibration curve derived from the protein standards provided with the kit.

3.2. Effects of rice straw leachates on the fitness of D. magna 2.7. Data analysis After the 21-day exposure to pure M4 medium (control), the survival rate of the D. magna was 100%, while the survival rates in ricestraw leachate preparations were significantly lower (Kaplan-Meier procedure and log-rank test: all p < 0.05, Fig. 2). However, no significant differences were observed between the T1C-19 and MH63 leachate groups with the same volume percentage at the end of the experiment (all p ˃ 0.05, Fig. 2). The developmental parameters (body mass, body length, body width, anal spine length, and number of molts) of D. magna decreased with increasing rice-straw leachate volume percentages for both rice lines. At day 21, however, no significant differences were observed between the T1C-19 and MH63 leachate groups with the same volume percentage (Student's t-test; all p > 0.05, Table 2). The reproductive parameters, namely total number of broods, number of offspring in the first brood, average number of offspring per brood, and total number of offspring, decreased with increasing volume percentages of both types of rice-straw leachates; conversely, the number of days to first brood increased with the volume percentages of both types of rice-straw leachate preparations (Table 3). When comparing the two rice straw leachates, a significant difference was found

All data are presented as mean ± standard error (SE), unless otherwise indicated. The effects of the straw leachate preparations on D. magna survival were analyzed using the Kaplan-Meier procedure and log-rank test. Water quality parameters (pH value, total hardness, and chemical oxygen demand) and D. magna developmental parameters (body mass, body length, body width, and anal spine length), reproductive parameters (days to first brood, number of broods, and number of offspring in first brood), and Cry1C content and CAT activity in adults and neonates were analyzed by one-way ANOVA followed by the Tukey HSD test. In addition, water quality (dissolved oxygen, total nitrogen, and ammonia nitrogen concentrations), number of molts, average number of offspring per brood, and total number of offspring were analyzed by the Mann-Whitney U-test because of the associated hetero geneity of variance. In addition, Student's t-test was used to compare the developmental and reproduction parameters of D. magna that had been exposed to the same concentration of the T1C-19 and MH63 rice-straw leachates. Differences were considered significant at p < 0.05. All statistical analyses were conducted using the SPSS software package (version 13 for Windows, 2004). 632

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

Table 3 Reproductive parameters of D. magna after exposure to different volume percentages of 0.4 g/L rice-straw leachates in M4 medium for 21 days. Data are means ± SE (for 0 lechate group n = 20; for 25% MH63 leachate group n = 20; for 50% MH63 leachate group n = 18; for 100% MH63 leachate group n = 16; for 25% T1C-19 leachate group n = 19; for 50% T1C-19 leachate group n = 19; for 100% T1C-19 leachate group n = 19). Parameter

Volume percentage of ricestraw leachatea

MH63 leachateb

T1C-19 leachateb

Days to first brood

0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100%

6.95 ± 0.15a 7.35 ± 0.13ab 7.56 ± 0.17b 8.81 ± 0.34c 5.15 ± 0.08a 3.85 ± 0.20b 3.67 ± 0.21b 3.50 ± 0.18b 18.80 ± 0.81a 13.25 ± 0.51b 12.67 ± 0.70b 9.25 ± 0.77c 24.29 ± 0.86a 21.72 ± 0.83a 22.93 ± 1.27a 20.77 ± 1.92a 125.05 ± 4.72a 81.35 ± 2.48b 80.94 ± 3.28b 71.81 ± 5.96b

6.95 ± 0.15a 7.74 ± 0.24b 7.89 ± 0.07b* 8.47 ± 0.14c* 5.15 ± 0.08a 3.53 ± 0.18b 3.47 ± 0.21b 3.26 ± 0.18b 18.80 ± 0.81a 13.32 ± 0.80b 12.21 ± 0.79bc 10.00 ± 0.66c 24.29 ± 0.86a 22.99 ± 1.21a 21.88 ± 0.88a 21.72 ± 1.20a 125.05 ± 4.72a 77.63 ± 1.70b 76.32 ± 4.50b 70.11 ± 4.86b

Number of broods

Fig. 2. Survival of D. magna when exposed to 0%, 25%, 50%, or 100% T1C19 or MH63 rice-straw leachate added to M4 medium for 21 days. Data were analyzed with the Kaplan-Meier procedure followed by the log-rank test. An asterisk indicates that significant differences were observed between the control (pure M4 medium) and test groups (n = 20).

Number of offspring in first brood Average number of offspring per brood

Table 2 Developmental parameters of D. magna after exposure to different volume percentages of rice-straw leachates in M4 medium for 21 days. Data are means ± SE (for 0 lechate group n = 20; for 25% MH63 leachate group n = 13; for 50% MH63 leachate group n = 10; for 100% MH63 leachate group n = 8; for 25% T1C-19 leachate group n = 12; for 50% T1C-19 leachate group n = 9; for 100% T1C-19 leachate group n = 6; except for the parameter of number of molts, n = 20 for all treatments). Parameters

Volume percentage of rice-straw leachatea

MH63 leachateb

T1C-19 leachateb

Body mass (mg)

0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100% 0 25% 50% 100%

5.77 5.39 4.49 3.81 5.43 4.95 4.52 4.32 3.90 3.74 3.59 3.44 0.64 0.48 0.40 0.37 9.80 8.30 8.15 8.00

5.77 5.01 3.96 3.56 5.43 4.76 4.56 4.23 3.90 3.82 3.40 3.25 0.64 0.41 0.35 0.34 9.80 8.30 8.10 7.60

Body length (mm)

Body width (mm)

Anal spine length (mm) Number of molts

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.14a 0.23ab 0.38bc 0.20c 0.10a 0.17b 0.16bc 0.14c 0.09a 0.10a 0.17a 0.24a 0.02a 0.02b 0.04b 0.03b 0.14a 0.26b 0.33b 0.38b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Total number of offspring

a

The percentage volume indicates the amount of rice-straw leachate (0.4 g/ L) that was mixed with M4 medium (v: v). b Different lowercase letters within the same column indicate a significant difference among means (ANOVA followed by Tukey HSD-test; p < 0.05). An Asterisk denotes a significant difference between the two rice-straw leachates at the same volume percentage (Student's t-test, p < 0.05).

0.14a 0.36a 0.15b 0.14b 0.10a 0.11b 0.13b 0.25b 0.09a 0.09ab 0.16b 0.29b 0.02a 0.03b 0.03b 0.03b 0.14a 0.23b 0.30b 0.28b

Fig. 3. Cry1C concentration detected in D. magna adults and neonates eafter exposure to 25%, 50%, or 100% T1C-19 medium. Data are means ± SE (n = 4). Different lowercase letters indicate significant differences between treatments (one-way ANOVA followed by the Tukey HSD test). FW = fresh weight.

a The percentage volume indicates the amount of rice-straw leachate (0.4 g/ L) that was mixed with M4 medium (v: v). b Different lowercase letters within the same column for a parameter indicate a significant difference among means (ANOVA followed by Tukey HSD-test; p < 0.05). Means did never differ between the two rice-straw leachatesat at the same volume percentage (Student's t-test; p > 0.05).

was determined by ELISA (Fig. 3). The protein was detected in adults and neonates from all three T1C-19 groups, and the Cry1C concentrations were significantly greater in adults than in neonates (one-way ANOVA followed by the Tukey HSD test; all p < 0.05). Furthermore, the Cry1C concentration in adults and neonates increased with increasing amounts of leachate volume percentages. Cry1C was not detected in any of D. magna from the MH63 treatments (data not shown in Fig. 3) or the blank control (Fig. 3).

for the days to the first brood. The results, however, were not consistent as the duration was increased in the T1C-19 leachate compared to the MH63 leachate at the 50% volume percentage (t = 1.91, df = 35, p < 0.001) and the opposite effect was found at the 100% leachate volume percentage (t = 0.97, df = 33, p = 0.01) (Table 3). For none of the other parameters and any of the volume percentages tested did we observe a difference between the two leachates types (Student's t-test; all p ˃ 0.05, Table 4). After exposure to both rice-straw leachates at each volume percentage, the concentration of Cry1C in D. magna adults and neonates

3.3. CAT activity in D. magna adults and neonates CAT activity in adults and neonates from the control group (0% rice 633

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

incubation with the rice straws, whereas the total nitrogen and ammonia nitrogen concentrations were significantly increased. The differences in water quality between M4 medium incubated with either of the rice straws and the untreated control medium maybe the reason for the decrease in D. magna fitness when exposed to the rice-straw leachates. Some researchers have demonstrated that poor water quality such as low pH and low dissolved oxygen level and/or high nitrogen level can significantly affect the survival and development of D. magna (Alibone and Fair, 1981). Moreover, decomposing rice straw is known to release secondary metabolites, e.g., hydroxamic acids, fatty acids, terpenes, and indoles, and 13 different types of phenolic acids (Kuwatsuka and Shindo, 1973). D. magna is recommended as an indicator of water quality (ISO, 2012; OECD, 2012). Different studies that exposed daphnids to Bt rice flour or stalks found no significant changes in the studied life-table parameters, suggesting that Bt rice causes no toxic effects (Oh et al., 2011; Wang et al., 2013b; Zhang et al., 2016). In addition, our previous study has shown that purified Cry1C has no adverse effects on the development and reproduction of D. magna (Chen et al., 2018). The results from the present study agree with those findings and support the conclusion that Bt rice T1C-19 producing Cry1C is safer for D. magna than is its non-transformed isoline MH63 because of the decreased need for pesticide application (Li et al., 2014a). Our results are also in accordance with a number of studies on different Lepidopetra-active Cry proteins (Mendelson et al., 2003; Oh et al., 2011; Wang et al., 2013b; Zhang et al., 2016). Previously, adverse effects of the Lepidoptera-active Cry1Ab protein or material from Cry1Ab-producing Bt maize on D. magna were reported by one research group (Bøhn et al., 2008, 2010, 2016; Holderbaum et al., 2015). However, the studies were suffering from some problems in design and uncertainty remains as to whether these effects were e.g. caused by nutritional deficiencies related to the maize-based diet, the genetic/varietal background of the conventional counterpart used for comparison, or an artefact of high doses of purified toxins used (EFSA 2012, 2016; Romeis et al., 2013; Raybould et al., 2014). Bt protein specifically binds receptors that localize to gut epithelial cells of target insects which results in perforation of the cell membrane, cell swelling and lysis, and ultimately death of the target insect (Hofmann et al., 1988). Exposure to this protein should not affect its non-target organinsms due to the fact that their intestinal epithelial cells do not have the protein's binding site. Therefore, based on the known spectrum of activity of Cry1C against lepidopteran species (Zheng et al., 2011; Wang et al., 2016) and the phylogenetic distances between D. magna and the target species, susceptibility of D. magna to Cry1C was not expected. Various non-target-effect studies have reported a lack of effect of purified Cry1C protein on non-Lepidoptera species. This includes the springtails Folsomia candida (Collembola: Isotomidae; Yang et al., 2015), and the fruit fly Drosophila melanogaster (Diptera: Drosophilidae; Haller et al., 2017). In addition, the T1C-19 rice line was found to cause no adverse effects on the following terrestrial arthropods: the green lacewing Chrysoperla nipponensis (Neuroptera: Chrysopidae; Li et al., 2014b), the honey bee Apis mellifera (Hymenoptera: Apidae; Wang et al., 2015), the parasitoid Pseudogonatopus flavifemur (Hymenoptera: Dryinidae; Tian et al., 2017), the lady beetle Propylea japonica (Coleoptera: Coccinellidae; Li et al., 2015), the stinkbug Cyrtorhinuslividipennis (Hemiptera: Miridae; Han et al., 2017), and the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae; Lu et al., 2015). Furthermore, different non-arthropod species were found to be insensitive to Cry1C. These include the green algae C. pyrenoidosa (Wang et al., 2014), the mice Mus musculus (Rodentia: Muridae; Cao et al., 2010), the African clawed frog Xenopus laevis (Anura: Pipidae; Chen et al., 2015), and the zebrafish Danio rerio (Cypriniformes: Cyprinidae; Gao et al., 2018). In our study, we detected Cry1C in T1C-19 rice-straw leachate and in adult and larval D. magna exposed to the leachate which confirms that the test organisms were exposed to the insecticidal protein. This

Fig. 4. Catalase activity in adults and neonates D. magna after exposure to different volume percentages of 0.4 g/L rice straw leachates in M4 medium. Data are means ± SE (n = 4). Means with different letters are significantly different at p < 0.05 according to a one-way ANOVA followed by a Tukey HSD test.

leachates) was significantly lower than that measured in adults and neonates treated with either of the rice-straw leachates, and the CAT activity in adults treated with 100% rice straw leachates was significantly greater than that of adults treated with 25% and 50% rice straw leachates (one-way ANOVA followed by theTukey HSD; all p < 0.05, Fig. 4). In addition, no significant differences were found between adults and neonates from the T1C-19 and MH63 treatments when their volume percentages were the same (Student's t-test; all p > 0.05, Fig. 4). 4. Discussion We found an inverse dose-response relationship between the volume percentages of rice-straw leachate in M4 medium preparations and the fitness of D. magna, i.e., survival, development, and reproduction. With the exeption of one parameter, no significant differences were found for the fitness of D. magna between the leachate from Bt and non-Bt rice (Fig. 2, Tables 2 and 3). The significant difference seen for the days to firs brood at two volume percentages was inconsistent. Thus, our results demonstrate that it was the leachates and not the presence of Cry1C that significantly reduced the fitness of D. magna. Prior to assessing the safety of the T1C-19 and MH63 rice-straw leachates, we analyzed the water quality of the M4 medium that had been incubated with T1C-19 or MH63 rice straw at a concentration of 0.4 g/L (Table 1). Compared with the control medium, the pH values and dissolved oxygen concentrations were significantly decreased by 634

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

exposure may have been achieved by D. magna consuming C. pyrenoidosa. Green alga that had accumulated nanoparticles or heavy metals has been used as a vector to transfer these toxicants into D. magna (McTeer et al., 2014; De Schamphelaere and Janssen, 2004). Moreover, our previous study showed that C. pyrenoidosa adsorb Cry toxins (Wang et al., 2014) and thus could serve as a vector for transporting Cry toxins dissolved in the water to the gut of a consumer (Venter and Bøhn, 2016). The ELISA results also showed that the Cry1C concentration was significantly greater in D. magna adults than in neonates (Fig. 2). This finding is consistent with our previous report (Chen et al., 2018) and is likely due to the fact that exposure time for the neonates was shorter, neonates were collected on the day they emerged, while the adults had been exposed for 21 days. We also detected CAT activity in the D. magna adult and neonates exposed to the rice-straw leachates. CAT is a terminal oxidase found in all organisms, and it neutralizes the toxicity of hydrogen peroxide, a toxic metabolic waste product, into oxygen and water, thereby eliminating its toxicity (Chelikani et al., 2004). In our study, CAT activity in the medium containing rice-straw leachates was significantly greater than in the control medium (pure M4). Exposure to the leachates may have produced damaging reactive oxygen species in the D. magna, thereby reducing the fitness of D. magna. We found no difference in CAT activity between D. magna exposed to the T1C-19 and MH63 leachates, again suggesting that the effects of the leachates on D. magna are not the result of the presence of Cry1C.

thuringiensis Cry1C protein for Daphnia magna based on different functional traits. Ecotoxicol. Environ. Saf. 147, 631–636. De Schamphelaere, K.A., Janssen, C.R., 2004. Effects of chronic dietary copper exposure on growth and reproduction of Daphnia magna. Environ. Toxicol. Chem. 23, 2038–2047. EFSA (European Food Safety Authority), 2012. Scientific opinion on a request from the European Commission related to the emergency measure notified by France on genetically modified maize MON810 according to Article 34 of regulation (EC) No 1829/2003. EFSA J. 10 (5), 2705. https://doi.org/10.2903/j.efsa.2012.2705. EFSA (European Food Safety Authority), 2016. Relevance of a new scientific publication (Bøhn et al. 2016) for previous environmental risk assessment conclusions on the cultivation of Bt -maize events MON810 and Bt11. EFSA Support. Publ. 13 (7). Gao, Y.J., Zhu, H.J., Chen, Y., Li, Y.H., Peng, Y.F., Chen, X.P., 2018. Safety assessment of Bacillus thuringiensis insecticidal proteins Cry1C and Cry2A with a zebrafish embryotoxicity test. J. Agric. Food Chem. 66, 4336–4344. Haller, S., Romeis, J., Meissle, M., 2017. Effects of purified or plant-produced Cry proteins on Drosophila melanogaster (Diptera: drosophilidae) larvae. Sci. Rep. 7, 11172. https://doi.org/10.1038/s41598-017-10801-4. Han, Y., Ma, F., Nawaz, M., Wang, Y., Cai, W., Zhao, J., He, Y., Hua, H., Zou, Y., 2017. The tiered-evaluation of the effects of transgenic cry1c rice on Cyrtorhinus lividipennis, a main predator of Nilaparvata lugens. Sci. Rep. 7, 42572. https://doi.org/10.1038/ srep42572. Hanazato, T., 2001. Pesticide effects on freshwater zooplankton: an ecological perspective. Environ. Pollut. 112, 1–10. Hebert, P.D.N., Crease, T., 1983. Clonal diversity in populations of Daphnia pulex reproducing by obligate parthenogenesis. Heredity 51, 353–369. Heinrichs, E.A., Nwilene, F.E., Stout, M., Hadi, B.A.R., Freitas, T., 2017. Rice Insect Pests and Their Management. Burleigh Dodds Science Publishing Limited, Cambridge, UK. Hofmann, C., Vanderbruggen, H., Höfte, H., Van Rie, J., Jansens, S., Van Mellaert, H.,, 1988. Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midgets. Proc. Natl. Acad. Sci. USA 85, 7844–7848. Holderbaum, D.F., Cuhra, M., Wickson, F., Orth, A.I., Nodari, R.O., Bøhn, T., 2015. Chronic responses of Daphnia magna under dietary exposure to leaves of a transgenic (event MON810) Bt–maize hybrid and its conventional near- isoline. J. Toxicol. Environ. Health A 78, 993–1007. Huang, J., Hu, R., Qiao, F., Yin, Y., Liu, H., Huang, Z., 2015. Impact of insect-resistant GM rice on pesticide use and farmers' health in China. Sci. China Life Sci. 58, 466–471. ISO (International Organization for Standardization), 2012. Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) —acute toxicity test. (ISO, Switzerland 〈〈https://doi.org/10.3403/ 30213505〉〉, Accessed 15 October 2012). Jiang, Y., Xu, H., Zhang, W., Zhu, M., Al-Rasheid, K.A., 2012. Can body-size patterns of ciliated zooplankton be used for assessing marine water quality? A case study on bioassessment in Jiaozhou Bay, northern Yellow Sea. Environ. Sci. Pollut. Res. Int. 19, 1747–1754. Kim, H.J., Koedrith, P., Seo, Y.R., 2015. Ecotoxico genomic approaches for under standing molecular mechanisms of environmental chemical toxicity using aquatic invertebrate, Daphnia model organism. Int. J. Mol. Sci. 16, 12261–12287. Kuwatsuka, S., Shindo, H., 1973. Behavior of phenolic substances in the decaying process of plants: i. Identification and quantitative determination of phenolic acids in rice straw and its decayed product by gas chromatography. Soil. Sci. Plant Nutr. 19, 219–227. Li, G.S., Wang, Y.M., Liu, B., Zhang, G., 2014a. Transgenic Bacillus thuringiensis (Bt) rice is safer to aquatic ecosystems than its non-transgenic counterpart. PLoS One 9, e104270. Li, Y.H., Chen, X.P., Hu, L., Romeis, J., Peng, Y.F., 2014b. Bt rice producing Cry1C protein does not have direct detrimental effects on the green lacewing Chrysoperlasinica (Tjeder). Environ. Toxicol. Chem. 33, 1391–1397. Li, Y.H., Hallerman, E.M., Liu, Q.S., Wu, K.M., Peng, Y.F., 2016. The development and status of Bt rice in China. Plant Biotechnol. J. 14, 839–848. Li, Y.H., Zhang, X.J., Chen, X.P., Romeis, J., Yin, X.M., Peng, Y.F., 2015. Consumption of Bt rice pollen containing Cry1C or Cry2A does not pose a risk to Propylea japonica (Thunberg) (Coleoptera: coccinellidae). Sci. Rep. 5, 7679. https://doi.org/10.1038/ srep07679. Liu, Y.B., Li, J.S., Luo, Z.L., Wang, H.R., Liu, F., 2016. The fate of fusion Cry1Ab/1Ac proteins from Bt-transgenic rice in soil and water. Ecotoxicol. Environ. Saf. 124, 455–459. Lu, C., 2010. The first approved transgenic rice in China. GM Crops 1, 113–115. Lu, Z.B., Liu, Y.E., Han, N.S., Tian, J.C., Peng, Y.F., Hu, C., Guo, Y.Y., Ye, G.Y., 2015. Transgenic cry1C or cry2A rice has no adverse impacts on the life-table parameters and population dynamics of the brown planthopper, Nilaparvata lugens (Hemiptera: delphacidae). Pest Manag. Sci. 71, 937–945. McTeer, J., Dean, A.P., White, K.N., Pittman, J.K., 2014. Bioaccumulation of silver nanoparticles into Daphnia magna from a freshwater algal diet and the impact of phosphate availability. Nanotoxicology 8, 305–316. Mendelson, M., Kough, J., Vaituzis, Z., Matthews, K., 2003. Are Bt crops safe? Nat. Biotechnol. 21, 1003–1009. Oh, S.D., 2011. Evaluation and assessment of biosafety for Bacillus thuringiensis (Bt)transgenic rice: responses of Daphnia magna fed on Bt-transgenic rice variety. J. Appl. Biol. Chem. 54, 296–302. OECD (Organization for Economic and Development), 2012. Guidelines for testing chemicals: Daphnia magna reproduction test. OECD, Paris. https://doi.org/10.1787/ 9789264185203-en. (Accessed 2 October 2012). Raybould, A., Burns, A., Hamer, M., 2014. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study

5. Conclusions The survival, developmental, and reproductive parameters of D. magna were reduced when exposed to rice-straw leachates. However, almost no consistent significant differences were found for exposure to the T1C-19 containing Cry1C and MH63 rice-straw leachate treatments with the same volume percentages. We thus conclude that the presence of Cry1C released into the environment from T1C-19 rice straw is unlikely to pose a risk to Cladocera populations in aquatic ecosystems. Acknowledgments We thank Professor Yongjun Lin (Huazhong Agricultural University) for kindly providing transgenic rice seeds. This study was supported by the National GMO New Variety Breeding Program of the People's Republic of China (2016ZX08011-001). References Alibone, M.R., Fair, P., 1981. The effects of low pH on the respiration of Daphnia magna Straus. Hydrobiologia 85, 185–188. Bahaar, S.W.N., Bhat, G.A., 2011. Aquatic biodiversity in the paddy fields of Kashmir valley (J & K) India. Asian J. Agric. Res. 5, 269–276. Bøhn, T., Primicerio, R., Hessen, D.O., Traavik, T., 2008. Reduced fitness of Daphnia magna fed a Bt-transgenic corn variety. Arch. Environ. Contam. Toxicol. 55, 584–592. Bøhn, T., Rover, C.M., Semenchuk, P., 2016. Daphnia magna negatively affected by chronic exposure to purified cry-toxins. Food Chem. Toxicol. 91, 130–140. Bøhn, T., Traavik, T., Primicerio, R., 2010. Demographic responses of Daphnia magna fed transgenic Bt corn. Ecotoxicology 19, 419–430. Cao, S.S., He, X.Y., Xu, W.T., Ran, W.J., Liang, L., Luo, Y.B., Yuan, Y.F., Zhang, N., Zhou, X., Huang, K.L., 2010. Safety assessment of Cry1C protein from genetically modified rice according to the national standards of PR China for a new food resource. Regul. Toxicol. Pharmacol. 58, 474–481. Carstens, K., Anderson, J., Bachman, P., De Schrijver, A., Dively, G., Federici, B., Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Rauschen, S., Ridley, G., Romeis, J., Waggoner, A., 2012. Genetically modified crops and aquatic ecosystems: considerations for environmental risk assessment and nontarget organism testing. Transgenic Res. 21, 813–842. Chelikani, P., Fita, I., Loewen, P.C., 2004. Diversity of structures and properties among catalases. Cell. Mol. Life Sci. 61, 192–208. Chen, M., Shelton, A., Ye, G., 2011. Insect-resistant genetically modified rice in China: from research to commercialization. Annu. Rev. Entomol. 56, 81–101. Chen, X., Wang, J., Zhu, H., Li, Y., Ding, J., Peng, Y., 2015. Effects of transgenic cry1Ca tice on the development of Xenopuslaevis. PLoS One 10, e0145412. https://doi.org/ 10.1371/journal.pone.0145412. Chen, Y., Yang, Y., Zhu, H., Romeis, J., Li, Y., Peng, Y., Chen, X.P., 2018. Safety of Bacillus

635

Ecotoxicology and Environmental Safety 165 (2018) 630–636

Y. Chen et al.

laboratory studies on the impact of two Bt rice lines expressing a fusion protein Cry1Ab/1Ac on aquatic organisms. Ecotoxicol. Environ. Saf. 92, 87–93. Wang, Y.N., Ke, K.Q., Li, Y.H., Han, L.Z., Liu, Y.M., Hua, H.X., Peng, Y.F., 2016. Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis (Lepidoptera: crambidae). Insect Sci. 23, 78–87. Wang, Y.Y., Li, Y.H., Huang, Z., Chen, X.P., Romeis, J., Dai, P.L., Peng, Y.F., 2015. Toxicological, biochemical, and histopathological analyses demonstrating that Cry1C and Cry2A are not toxic to larvae of the honey bee, Apis mellifera. J. Agric. Food Chem. 63, 6126–6132. Xu, M.Q., Cao, H., Xie, P., Deng, D.G., Feng, W.S., Xu, J., 2005. The temporal and spatial distribution, composition and abundance of protozoa in Chaohu Lake, China: relationship with eutrophication. Eur. J. Protistol. 41, 183–192. Yang, Y., Chen, X.P., Cheng, L.S., Cao, F.Q., Romeis, J., Li, Y.H., Peng, Y.F., 2015. Toxicological and biochemical analyses demonstrate no toxic effect of Cry1C and Cry2A to Folsomia candida. Sci. Rep. 5, 15619. https://doi.org/10.1038/srep15619. Zhang, L., 2013. Study on Environmental Persistence of Exogenous Protein of Transgenic Cry1ab/c Rice and Its Effects on the Hydrobiont (Dissertation). Nanjing Agricultural University, China (In Chinese with an English abstract). Zhang, L., Guo, R.Q., Fang, Z.X., Liu, B., 2016. Genetically modified rice Bt-Shangyou63 expressing Cry1Ab/c protein does not harm Daphnia magna. Ecotoxicol. Environ. Saf. 132, 196–201. Zheng, X., Yang, Y., Xu, H., Chen, H., Wang, B., Lin, Y., Lu, Z., 2011. Resistance performances of transgenic Bt rice lines T2A-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: pyralidae). J. Econ. Entomol. 104, 1730–1735.

designs and ecological risk assessments for GM crops. GM Crops Food 5, 296–301. Romeis, J., McLean, M.A., Shelton, A.M., 2013. When bad science makes good headline: bt corn and regulatory bans. Nat. Biotechnol. 31, 386–387. Schindler, D.W., 1987. Detecting ecosystem responses to anthropogenic stress. Can. J. Fish. Aquat. Sci. 44, 6–25. Takagaki, S., 2016. The Distribution and Abundance of Surface Macro-zooplankton in the Waters off the Otago Coast (Thesis, Master of Science). University of Otago. Tang, W., Chen, H., Xu, C., Li, X., Lin, Y., Zhang, Q., 2006. Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene. Mol. Breed. 18, 1. https://doi. org/10.1007/s11032-006-9002-9. Tian, J.C., Romeis, J., Liu, K., Zhang, F.C., Zheng, X.S., Xu, H.X., Chen, G.H., He, X.C., Lu, Z.X., 2017. Assessingthe effects of Cry1C rice and Cry2A rice to pseudogonatopus flavifemur, a parasitoid of rice planthoppers. Sci. Rep. 7, 7838. https://doi.org/10. 1038/s41598-017-08173-w. Venter, H.J., Bøhn, T., 2016. Interactions between Bt crops and aquatic ecosystems: a review. Environ. Toxicol. Chem. 35, 2891–2902. Viktorov, A.G., 2011. Transfer of Bt corn byproducts from terrestrial to stream ecosystems. Russ. J. Plant Physiol. 58, 543–548. Wang, J.M., Chen, X.P., Li, Y.H., Su, C.Q., Ding, J.T., Peng, Y.F., 2014. Green algae (Chlorella pyrenoidosa) adsorbs Bacillus thuringiensis (Bt) toxin, Cry1Ca insecticidal protein, without an effect on growth. Ecotoxicol. Environ. Saf. 106, 6–10. Wang, Y.M., Hu, H.W., Huang, J.C., Li, J.H., Liu, B., Zhang, G.A., 2013a. Determination of the movement and persistence of Cry1Ab/1Ac protein released from Bt transgenic rice under field and hydroponic conditions. Soil Biol. Biochem. 58, 107–114. Wang, Y.M., Huang, J.C., Hu, H.W., Li, J.H., Liu, B., Zhang, G.A., 2013b. Field and

636