Environmental Toxicology and Chemistry, Vol. 33, No. 5, pp. 1156–1162, 2014 # 2014 SETAC Printed in the USA

EFFECT OF STRAW LEACHATES FROM CRY1CA-EXPRESSING TRANSGENIC RICE ON THE GROWTH OF CHLORELLA PYRENOIDOSA JIAMEI WANG,yz XIUPING CHEN,*z YUNHE LI,z HAOJUN ZHU,yz JIATONG DING,y and YUFA PENGz

yCollege of Animal Science and Technology, Yangzhou University, Yangzhou, People’s Republic of China zState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China (Submitted 21 October 2013; Returned for Revision 7 January 2014; Accepted 27 January 2014) Abstract: Because of the prevalence of algae in rice paddy fields, they will be exposed to Bacillus thurigiensis (Bt) proteins released from Bt protein-expressing genetically engineered rice. To assess the effects of leachates extracted from Cry1Ca-expressing transgenic rice (T1C-19) straw on the microalga Chlorella pyrenoidosa, the authors added purified Cry1Ca (10 mg/L, 100 mg/L, and 1000 mg/L) and 5 concentrations of diluted extracts (5%, 10%, 20%, 40%, and 80%) from T1C-19 and the nontransformed control strain Minghui 63 (MH63) to the medium of C. pyrenoidosa. The authors found that the growth curves of C. pyrenoidosa treated with purified Cry1Ca overlapped with the medium control; that the order of C. pyrenoidosa growth rates for the T1C-19 leachate concentrations was 5% > 10% > 20% > control > 40% > 80%, and for the MH63 concentrations the order was 5% > 10% > control > 20% > 40% > 80%, but there were no statistical differences between the 20% T1C-19 or 20% MH63 leachate treatment and the medium control on day 8; and that after 7 d of culture, Cry1Ca could be detected in C. pyrenoidosa treated with different concentrations of T1C-19 leachate. The results demonstrated that Cry1Ca protein released from T1C-19 rice can be absorbed into C. pyrenoidosa but that purified Cry1Ca and leachates from T1C-19 rice have no obvious adverse effects on the growth of C. pyrenoidosa. Environ Toxicol Chem 2014;33:1156–1162. # 2014 SETAC Keywords: Bt-rice straw

Cry1Ca protein

Chlorella pyrenoidosa

Safety assessment

Nontarget effect

95  73 ng Cry1Ab protein/g dry weight in 28 (12.9%) of the biotopes examined [14]. However, only few studies have assessed the potential effects of Bt crops on aquatic organisms [11,13,15,16]. In traditional rice cultivation in China, rice byproducts are left on the planted surface after harvesting, where they can be deliberately shattered and returned to the soil to increase organic content, or they can be transported into the water system through weather events such as heavy rain, wind, snow, or hail. Once the Bt-rice biomass reaches an aquatic system, freely soluble protein may be released from the tissues into the surrounding water column. The potential exposure of aquatic organisms to Bt proteins in this scenario makes it necessary to assess potential nontarget impacts on aquatic organisms. To our knowledge, the environmental effects of Bt rice on algae have not yet been investigated. Algae are very prevalent in the paddy ecosystem [17], where they can absorb nitrogen and phosphorus, thereby increasing the organic content of the soil and improving its physical properties. Algae are also important primary producers, forming the basis of the entire food chain in open water and directly affecting the composition and function of aquatic ecosystems [18]. Water quality and nutrient changes affect the ecological distribution of algae [19]. In fact, the observance of floating algae can be a biological indicator of xenobiotic pollution and water quality decline [20]. Although transgenic crops can release Bt protein into water environments, it remains to be seen whether algae can absorb these Bt proteins and whether Bt proteins and Bt-rice straw can impact algal growth. The present study assessed the risk posed by straw leachates from Bt rice on the growth of the green alga Chlorella pyrenoidosa to provide further information about the environmental safety of genetically modified rice in China.

INTRODUCTION

Rice (Oryza sp.) is the most important staple food crop in China and many other Asian countries, and is a major food source for more than half of the world’s population. Unfortunately, rice is highly susceptible to many pests such as rice stem borers, planthoppers, leafhoppers, and thrips [1]. In 2003, annual losses caused by rice stem borers (e.g., the Asiatic rice borers Chilo suppressalis Walker and Scirpophaga incertulas Walker) alone were estimated to be 1.69 billion US dollars [2]. Use of chemical pesticides can resolve this problem, but excessive use of these compounds can have serious environmental consequences, threatening humans and beneficial creatures. Expression of a Bacillus thurigiensis (Bt) transgene in rice confers control to many insect pests, providing an alternative to chemical insecticides. The use of Bt rice is controversial, however, with heated debates in recent years focused on the potential development of resistance in the target pests [3] and the possible impact on nontarget organisms [4]. Discussion of the impact on nontarget organisms has focused primarily on terrestrial organisms such as natural enemies [5], economically important insects [6,7], and soil microorganisms [8]. It has been demonstrated that Bt toxins from transgenic corn (Zea mays) can be transferred from terrestrial to stream ecosystems through exudation from roots, dispersal of pollen, and movement of postharvest corn residues [9–13]. A comprehensive examination of 217 river biotopes neighboring transgenic crops 6 mo after harvest revealed corn byproducts in 86% of sites, and corn detritus sampled from the water contained an average of * Address correspondence to [email protected]. Published online 29 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2535 1156

Bt rice effects on Chlorella pyrenoidosa MATERIALS AND METHODS

Purified Cry1Ca protein

We purchased Cry1Ca proteins from Envirotest-China (agent for EnviroLogix-USA; www.envirotest-china.com). The protoxin from B. thuringiensis had been expressed as single-gene products in Escherichia coli at Case Western Reserve University (Cleveland, OH, USA). The protoxin inclusion bodies were then dissolved, isolated, and purified by ion exchange highperformance liquid chromatography followed by desalting and lyophilizing the pure fractions. Purity is approximately 94% to 96% (M.P. Carey, Case Western Reserve University, Cleveland, OH, USA, personal communication). Bioactivity of the Cry1Ca was confirmed in sensitive insect bioassays in our laboratory using neonate larvae of C. suppressalis that were fed for 7 d with artificial diet containing a range of protein concentrations. The toxin concentration resulting in 50% weight reduction compared with control (EC50) was estimated to be 18.07 ng/mL diet (Y. Li et al., unpublished data). Plants

The transgenic rice line T1C-19 and its corresponding nontransformed near isoline Minghui 63 (MH63) were used for all experiments. Transgenic T1C-19 plants express a gene encoding synthetic Cry1Ca under the control of the corn ubiquitin promoter and exhibit resistance to stem borers such as C. suppressalis, S. incertulas, and Cnaphalocrocis medinalis [21]. The MH63 line is an elite indica restorer line for cytoplasmic male sterility commonly grown in China. Both rice lines were obtained from Huazhong Agricultural University (Wuhan, China). The 2 rice lines were simultaneously planted in 2 adjacent plots in the experimental field station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (39.538N, 116.708E). Crops were cultivated according to commonly used local agricultural practices but without insecticide application. Rice straw collection

Rice was harvested at the end of October 2012, and rice stems of each line from 20 cm above the soil surface were collected and stored at 20 8C until use. Levels of Cry1Ca, selected amino acids, and conventional nutrient components of each rice line were analyzed by the Hangzhou Center for Inspection and Testing for Quality and Safety of Agricultural and Genetically Modified Products, Ministry of Agriculture, China. Degradation of Cry1Ca in water

A total of 100 g of rice straw samples were crushed into 1-cm pieces and randomly distributed in 3-L conical flasks containing 1500 mL sterile deionized water, and then the flasks were kept in a climate-controlled chamber at 28 8C with 80% relative humidity. After different intervals of incubation (1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 14 d, 21 d, and 28 d), 10 mL of each sample was taken and stored at 80 8C for determination of Cry1Ca content by enzyme-linked immunosorbent assay (ELISA). Preparation of rice straw leachates

Based on the observed rapid degradation of Cry1Ca in water, all leachate stock solutions were prepared from a 24-h incubation to maximize Cry1Ca content. A total of 100 g of rice straw samples were cut into 1-cm pieces and placed in 3-L conical flasks containing 1500 mL sterile deionized water. The flasks were placed in an incubator for 24 h at 28 8C. After 24 h, eluates

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were centrifuged at 15 700 g for 5 min at 4 8C, and the supernatants were sterilized by filtration through a 0.22-mm filter (Millipore). Water quality of the eluates was assessed by the Center for Environmental Quality Test, Tsinghua University (Beijing, China). C. pyrenoidosa testing

Chlorella pyrenoidosa was provided by the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The algae were grown in medium containing 1.5 mM (NH4)2SO4, 50 mM Ca(H2PO4)2 · H2O, 60 mM CaSO4 · 2H2O, 1.3 mM MgSO4 · 7 H2O, 1.0 mM NaHCO3, 0.3 mM KCl, 9 mM FeCl3, 20 mM H3BO3, 4.5 mM MnCl2 · 4H2O, 8 mM ZnSO4 · 7H2O, 8 mM Na2MoO4 · 2H2O, and 2 mM CuSO4 · 5H2O (pH 7.8). The algae were cultivated in a homoeothermic incubator (FPG3 model; Ningbo Saifu Scientific) at 25  1 8C with 4000 lux illumination and a light:dark period of 12:12 h and were manually shaken 3 times a day. Cells in the exponential growth phase were used in all experiments. Rice straw leachates were diluted to 5%, 10%, 20%, 40%, and 80% with culture medium in 200-mL flasks. Each dilution was prepared and replicated 4 times, and blank controls were prepared with medium alone and also replicated 4 times. The flasks were inoculated to achieve an initial algae concentration of 105 cells/mL, and then placed in an incubator with light at 3000 lux to 4000 lux. The temperature of the growth chamber was monitored, and the positions of the flasks were changed every day. Cell densities in 1-mL aliquots were measured daily using a Countstar automated cell counter (Shanghai Rui Yu Biotechnology). On day 8, C. pyrenoidosa cells were centrifuged from the supernatant and stored at 80 8C until use. Purified Cry1Ca was added to the culture medium at 0 mg/L, 10 mg/L, 100 mg/L, and 1000 mg/L, and atrazine (Sigma), a widely used herbicide, was added at 1.25 mg/L as a positive control. All of the culture conditions were the same as those described for the leachates above, and only the cell density was measured daily. Determination of enzyme activities

Chlorella pyrenoidosa cells were resuspended in 1.5 mL phosphate buffer (0.1 M, pH 7.8) and centrifuged at 1200 g for 10 min at 4 8C. The supernatants were used immediately to determine malondialdehyde (MDA) levels and total superoxide dismutase (T-SOD) activity with detection kits provided by the JianCheng Bioengineering Institute (Nanjing, China). Determination of Cry1Ca content by ELISA

The concentrations of Cry1Ca in rice straw, leachates, and C. pyrenoidosa cells were measured by using a Cry1C ELISAbased detection kit from EnviroLogix with a detection limit of 0.2 mg/L. Before analysis, rice straw and C. pyrenoidosa cells were washed in phosphate-buffered saline/Tween-20 (provided with the kit) to remove Bt toxin from their outer surfaces. Rice straw and C. pyrenoidosa were lyophilized and then homogenized in 1 mL phosphate-buffered saline/Tween-20 using a micropestle and mortar on ice. This step was not necessary for leachate samples. After centrifugation (1200 g, 5 min) and appropriate dilution of the supernatants, the ELISA detection was performed. Optical density values were read using a microplate spectrophotometer (PowerWave XS2, BioTek). Concentrations of Cry1Ca were calculated by comparison with a purified Cry1Ca standard curve.

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J. Wang et al. Table 1. Basic nutrient composition in T1C-19 and MH63 rice strawa

Test index Cry1Ca (mg/g) Crude protein (g/100 g) Crude fat (g/100 g) Crude ash (g/100 g) Moisture (g/100 g)

T1C-19

MH63

No.

p Value (t test)

2.15  0.76 4.81  0.35 1.36  0.26 10.86  0.33 16.85  0.51

0 5.13  0.20 1.40  0.17 10.58  0.32 16.33  0.05

8 4 4 4 4

– 0.167 0.669 0.109 0.083

a Data are means  standard deviation. T1C-19 ¼ Cry1Ca-expressing transgenic rice straw; MH63 ¼ nontransformed control strain Minghui 63.

Data analysis

Water quality of leachates

Statistically significant differences for test indices such as conventional nutrition composition levels, amino acid levels, and water quality measures were analyzed by Student’s t test; continuous variables such as number of C. pyrenoidosa cells, MDA levels, and T-SOD activity were analyzed using one-way analysis of variance analysis. All the results are presented as mean  standard deviation, unless indicated otherwise, with p < 0.05 considered to be significant.

The T1C-19 and MH63 leachates were collected after rice straw had been immersed in water for 24 h and were analyzed to assess water quality (Table 4). Total hardness, pH levels, chemical oxygen demand, total organic carbon, and NH4þ were nearly identical for the 2 rice lines (Table 4).

RESULTS

Nutrient composition and Cry1Ca content in rice straw

The average concentrations of Cry1Ca in transgenic T1C-19 rice straw and the nontransformed control strain MH63 were 2.15  0.76 mg/g and 0 mg/g, respectively (Table 1). Levels of moisture, crude protein, crude fat, and crude ash were very similar in T1C-19 and MH63 (Table 1), as were levels of 18 selected amino acids (Table 2). No statistical differences were observed between the 2 rice lines. Degradation dynamics of Cry1Ca in T1C-19 rice straw in water

Under laboratory conditions, Cry1Ca was rapidly released when T1C-19 rice straw was immersed in water (Table 3). The amount of Cry1Ca in the water peaked on day 1 (5.18 mg/L), and then decreased by approximately 50% by day 3 (2.71 mg/L). The levels continued to decrease steadily over the subsequent 4 d. At 14 d to 28 d, Cry1Ca protein was not detected.

Table 2. Amino acid composition in T1C-19 and MH63 rice straw (n ¼ 4)a Amino acid

T1C-19 (%)

MH63 (%)

Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Trp Phe Lys

0.40  0.01 0.46  0.01 0.21  0.01 0.22  0.01 0.34  0.01 0.16  0.00 0.20  0.00 0.24  0.01 0.19  0.01 0.09  0.01 0.22  0.01 0.06  0.01 0.07  0.01 0.16  0.01 0.30  0.01 0.03  0.00 0.17  0.01 0.22  0.01

0.38  0.02 0.45  0.02 0.19  0.01 0.19  0.01 0.32  0.01 0.16  0.01 0.18  0.01 0.22  0.01 0.17  0.01 0.08  0.01 0.20  0.01 0.04  0.00 0.10  0.01 0.14  0.01 0.27  0.02 0.03  0.01 0.15  0.01 0.20  0.01

a Data are means  standard deviation. T1C-19 ¼ Cry1Ca-expressing transgenic rice straw; MH63 ¼ nontransformed control strain Minghui 63.

Growth of C. pyrenoidosa treated with purified Cry1Ca

Growth curves of C. pyrenoidosa exposed to different concentrations of purified Cry1Ca and atrazine are shown in Figure 1. In the control groups exposed to culture medium alone, growth of C. pyrenoidosa increased in a linear manner from 1.1  106 cell/mL (day 0) to 9.2  106 cell/mL (day 8), with the trend described by the fitted curve y ¼ 1.1053x þ 0.7832 (r2 ¼ 0.9858). The growth curves of C. pyrenoidosa exposed to 10 mg/L, 100 mg/L, and 1000 mg/L Cry1Ca were almost overlapping with the blank control, whereas the growth of C. pyrenoidosa exposed to atrazine (positive control) was significantly inhibited by approximately 50% from day 2 to day 8. Growth of C. pyrenoidosa treated with straw leachates

Growth curves of C. pyrenoidosa exposed to different concentrations of leachates are shown in Figure 2. In the control group exposed to culture medium alone, growth of C. pyrenoidosa increased linearly from 6.5  105 cell/mL (day 0) to 3.5  106 cell/mL (day 8), with the trend described by the fitted curve y ¼ 3.7085x þ 6.5054 (r2 ¼ 0.9939). The order of C. pyrenoidosa growth rates for the T1C-19 leachate concentration groups was in a descending manner (5% > 10% > 20% > control > 40% > 80%), and the order for the MH63 leachate groups was 5% > 10% > control > 20% > 40% > 80%. In the 5% and 10% solutions of either leachate, growth of C. pyrenoidosa was significantly promoted compared with the control. No significant effect appeared in the growth rate of these leachates and control from 2 d to 8 d. In contrast, addition of 40% and 80% solutions of either leachate significantly suppressed growth of C. pyrenoidosa, with cell densities remaining below 2.6  106 cell/mL during the experimental period. Moreover, compared with the control, the addition of the 20% solution of leachate from T1C-19 rice straw promoted the growth of C. pyrenoidosa from 1 d to 1 wk, whereas addition of the 20% solution of leachate from MH63 rice straw almost inhibited the growth of C. pyrenoidosa. However, no significant differences among T1C-19, MH63, and the control treatment were seen on day 8 (37.70  3.34 vs 26.95  4.58 vs 35.18  1.12  105 cell/ mL, respectively). Cry1C content in C. pyrenoidosa cells and culture medium

The Cry1Ca concentration in the stock T1C-19 leachate was 1.36 ng/mL. Thus, we predicted the Cry1Ca content of the 5%, 10%, 20%, 40%, and 80% leachate solutions (Table 5). The

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Table 3. Degradation dynamics of Cry1Ca from T1C-19 rice straw in water (n ¼ 8) Cry1Ca content (mg/L) Sampling time (d)

Mean

Variance

Maximum

Minimum

1 2 3 4 5 6 7 14 21 28

5.18 4.34 2.71 1.23 0.66 0.46 0.37 ND ND ND

1.03 0.74 1.04 0.58 0.19 0.31 0.15 ND ND ND

6.51 5.36 3.88 1.97 1.32 1.00 0.56 ND ND ND

3.43 2.94 1.20 0.51 0.27 ND ND ND ND ND

T1C-19 ¼ Cry1Ca-expressing transgenic rice straw; ND ¼ not detectable.

Table 4. Water quality of T1C-19 and MH63 rice straw leachates (n ¼ 5)a Detection index

MH63

T1C-19

p Value (t test)

pH TH (mg/L) COD (O2, g/L) TOC (mg/L) NH4þ (mg/L)

4.56  0.23 93.60  7.19 1.61  0.05 504.80  19.23 11.56  0.80

4.72  0.20 91.76  4.95 1.67  0.06 501.00  32.12 10.96  0.55

0.279 0.650 0.116 0.826 0.204

a Data are means  standard deviation. T1C-19 ¼ Cry1Ca-expressing transgenic rice straw; MH63 ¼ nontransformed control strain Minghui 63; TH ¼ total hardness; COD ¼ chemical oxygen demand; TOC ¼ total organic carbon.

experimentally determined values were similar to the predicted values. After 7 d of culturing C. pyrenoidosa in the presence of leachate or control medium, we separated the cells from the culture medium by centrifugation and determined the Cry1Ca content in each sample (Table 5). No Cry1Ca was detected in the culture medium, except in the presence of 80% T1C-19 leachate. In C. pyrenoidosa cells, Cry1Ca was detectable in the presence of all T1C-19 leachates. No Cry1Ca was detected in C. pyrenoidosa cells or culture medium from control cultures containing leachate solutions from MH63 (Table 5).

with control; however, MDA levels in the 5% and 10% leachate groups from either strain were very similar to those of the control (Figure 3A). Malondialdehyde levels in MH63 leachate-treated cells appeared slightly higher than those in the T1C-19 leachatetreated cells, but significantly different only for the 10% treatment groups (Figure 3A). Similar results were observed for T-SOD. The T-SOD activities in MH63 leachate-treated cells were generally higher than those in the T1C-19 leachate-treated cells, but the difference was significantly different only for the 80% treatment groups (Figure 3B).

MDA level and T-SOD activity in C. pyrenoidosa

Levels of MDA and T-SOD activity were assayed in C. pyrenoidosa cells after all treatments. The MDA levels were significantly elevated following treatment with the 80%, 40%, and 20% solutions of leachates from either rice strain compared

Figure 1. Growth curves of Chlorella pyrenoidosa treated with different concentrations of purified Cry1Ca and atrazine (ATR) (mean  standard deviation).

DISCUSSION

In the present study, we evaluated the effects of purified Cry1Ca and leachates from T1C-19 rice straw on the growth of C. pyrenoidosa. We found that purified Cry1Ca and leachates from T1C-19 rice straw showed no obvious adverse effects on the growth of the algae. The maximum concentration of naturally occurring Bt protein in water environments is approximately 100 ng/L [13,22]. The levels of purified Cry1Ca that we used in the present study (10–1000 mg/L) were hundreds of times higher than those normally in the environment, yet we found that the growth curves of C. pyrenoidosa exposed to these levels were almost identical to blank controls, indicating that Cry1Ca has no suppressive or stimulative effect on the growth of this algae. Additional studies of the effects of leachate from rice carrying the gene encoding Cry1Ca on the algae will further identify other potential unintended effects [23]. Previous studies of effects of Bt gene products on aquatic nontarget organisms have revealed mixed results. Adverse effects on Daphnia magna (Diplostraca: Daphniida) were observed when organisms were fed flour from Bt maize containing Cry1Ab [15,16]. Another adverse effect occurred when Bt maize tissue or pollen was fed to larvae from 2 species

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Figure 2. Growth curves of Chlorella pyrenoidosa following treatment with 5%, 10%, 20%, 40%, and 80% straw leachate or culture medium (control) (mean  standard deviation). (A) Diluted exudates from Cry1Ca-expressing transgenic rice (T1C-19) straw; (B) diluted exudates from Minghui 63 (MH).

of caddisflies (Trichoptera) [11]. In both cases, however, the execution of the study and interpretation of the results have received strong and detailed criticism, and it is likely that the effects observed were caused by factors other than the Cry protein [24,25]. In addition, Jensen et al. [13] could not confirm the results for Trichoptera. Raybould and Vlachos [26] exposed D. magna to Vip3Aa20 and observed no effects on survival and fecundity but a small reduction on growth compared with unexposed controls. Jensen et al. [13] reported effects of Cry1Ab maize on 2 aquatic species, Tipula abdominalis (Diptera: Tipulidae) and Caecidotea communis (Isopoda: Asellidae). These effects, however, were unlikely to have occurred because of the Cry1Ab protein, because no effects were seen when the arthropods were fed with material from a stacked Bt maize variety that expressed Cry3Bb1 in addition to Cry1Ab.

Wang et al. [27] found that degradation of the Cry1Ab protein in rice paddy soil is significantly prolonged under flooded conditions compared with aerobic conditions. However, Douville et al. [28] reported that Bt-corn endotoxin is degraded more rapidly in water than in soil. Bai et al. [29] reported that the Cry1Ab concentration in exudates from mature rice stems and leaves submerged in water for >100 d ranged from 1.08 ng/mL to 1.52 ng/mL. Prihoda et al. [30] observed rapid dissipation of Cry3Bb1 from decomposing leaves, stalks, and roots of transgenic corn, with half-lives of 2.9 d, 0.57 d, and 0.42 d, respectively. In the present study, concentrations of Cry1Ca leached from T1C-19 rice straw into water were highest on the first day of immersion (3.06  2.67 mg/L), were substantially reduced within 7 d, and could not be detected after 14 d. The slight differences between our results and those reported previously may be attributable to differences in the accessibility of Bt proteins in the different tissues (leaves vs straw) used, water quality (e.g., sterility or pH) [27,28], leaching time, environmental conditions (such as temperature or humidity), or the detection limits for different Cry proteins. Despite these differences, we are in general agreement with the previous findings that the transgenic Bt protein exuded from plants appears to rapidly degrade in water. Several researchers have proposed that changes in nutrient availability or water quality can affect the geographical distribution of algae [19]. To investigate whether senesced Bttransgenic rice straw could affect the growth of C. pyrenoidosa, it was first necessary to characterize the basic nutritional composition and effects on water quality of rice straw. In the present study, we found that there were almost no differences between T1C-19 and MH63 in terms of basic nutrient composition, amino acid content, or effect on water quality indices. High concentrations of straw leachate from either rice strain (40% solution) significantly inhibited the growth of C. pyrenoidosa, whereas lower concentrations (20% solution) tended to promote growth. Allelopathy has been implicated in the ability of crude extracts of rice plants and decomposing rice residues to inhibit the growth of neighboring plant species [31]. Secondary metabolites such as phenolic acids, hydroxamic acids, fatty acids, terpenes, and indoles have been identified in extracts of rice plants [32], and 13 different phenolic acids have been found in decomposing rice straw [33]. Entry of these compounds into the environment may inhibit the germination and growth of neighboring plant species, acting as allelochemicals in natural ecosystems [31]. Furthermore, extracts from other plants can also inhibit the growth of C. pyrenoidosa [34]. On the basis of these previous researchers’ and our own results, we assume that the inhibitory effect of high concentrations of rice straw leachates on the growth of C. pyrenoidosa may be

Table 5. Cry1Ca content in Chlorella pyrenoidosa cells versus culture medium treated with T1C-19 rice straw leachates (n ¼ 4)a Day 0 (ng/mL) Group T1C-19 T1C-19 T1C-19 T1C-19 T1C-19

80% 40% 20% 10% 5%

Culture medium (Predicted)

Culture medium (Measured)

Culture medium (ng/mL)

C. pyrenoidosa (mg/g)

1.09  0.02 0.55  0.01 0.27  0.01 0.14  0.00 0.07  0.00

1.14  0.03 0.48  0.03 0.22  0.01 ND ND

0.31  0.10 ND ND ND ND

212.23  35.66 69.68  2.94 58.46  2.66 14.42  2.18 1.69  0.08

Data are means  standard deviation. T1C-19 ¼ Cry1Ca-expressing transgenic rice straw. a

Day 8

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MH63 rice straw leachates than in control samples and cells grown in low concentrations of leachate. This occurred because rice straw leachates may trigger the production of damaging reactive oxygen species in C. pyrenoidosa, which may be the mechanism by which leachates inhibit growth of the algae. The fact that there was very little difference in MDA and T-SOD activity between cells exposed to T1C-19 and MH63 leachates again suggests that the effects of rice straw leachates on C. pyrenoidosa are not the result of the presence of the Bt protein. CONCLUSIONS

The present study demonstrated that purified Cry1Ca has no suppressive or simulative effects on the growth of C. pyrenoidosa. We also found that although Cry1Ca released from T1C-19 rice straw can be absorbed by C. pyrenoidosa, T1C-19 rice straw leachates had no obvious adverse effects on the growth of the algae studied. Thus, we conclude that the entry of Cry1Ca into the environment from T1C-19 rice straw will not affect algae populations in aquatic ecosystems. Acknowledgment—We thank Y. Lin (Huazhong Agricultural University) for kindly providing transgenic rice seeds. We also thank J. Romeis (Agroscope Reckenholz-Tänikon Research Station ART, Zurich, Switzerland) for his constructive comments on an early draft of this manuscript. The present study was supported by the National GMO New Variety Breeding Program of the People’s Republic of China (grants 2012ZX08011-002 and 2014ZX08011001). REFERENCES

Figure 3. (A) Malondialdehyde (MDA) level and (B) total superoxide dismutase (T-SOD) activity (mean  standard deviation) in Chlorella pyrenoidosa after 7 d of culture. Columns marked with different letters indicate statistically significant differences. A one-way analysis of variance was conducted, followed by a least significant difference test. MH63 ¼ nontransformed control strain Minghui 63; T1C-19 ¼ Cry1Ca-expressing transgenic rice straw.

because of allelopathy and that the inhibitory effects of these allelochemicals may be mitigated by dilution. There were almost no significant differences between T1C-19 and MH63 leachate treatments on day 8, indicating that leachates from the Cry1Cacarrying rice strain are not obviously more toxic to C. pyrenoidosa. After 7 d of culture, Cry1Ca was almost undetectable in the culture media or in C. pyrenoidosa cells treated with MH63 leachate. However, Cry1Ca was detected in cells cultured with T1C-19 exudate, indicating that C. pyrenoidosa can absorb Bt proteins leached from Bt-containing rice straw. This is consistent with the observations that C. pyrenoidosa can absorb heavy metal ions (such as Au2þ, Pb2þ, and Zn2þ) and pesticides, by virtue of its unique cell wall structure [35]. The absorbtion of Cry protein by algae is a route through which aquatic invertebrates could be exposed to plant-produced insecticidal compounds that has so far not been considered. Future studies should investigate the fate of the Cry proteins after absorption by the algae. The final oxidation product in lipid peroxidation is MDA, which can cause cytotoxicity, in part by cross-linking protein and nucleic acid molecules [36]. Superoxide dismutase is a key enzyme that protects plant cells against oxidative stress [37] and is the main mechanism that protects plant chloroplasts against organic pollution [38]. The activity of SOD was significantly higher in C. pyrenoidosa cells grown in high concentrations of

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