Ecotoxicology and Environmental Safety 147 (2018) 631–636

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on MARK
different functional traits
Yi Chena,1, Yan Yanga,1, Haojun Zhua,b, Jörg Romeisa,c, Yunhe Lia, Yufa Penga, Xiuping Chena,⁎
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
b
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
c
Agroscope, Research Division Agroecology and Environment, 8046 Zurich, Switzerland

A R T I C L E I N F O A B S T R A C T

Keywords: Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed
Bt protein in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins pro-
Water flea duced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia
Environmental risk assessment magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to
Aquatic organisms
this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different
Non-target effects
concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known
toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used
as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D.
magna were not significantly different between Cry1C and negative control treatments but were significantly
inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

1. Introduction L in samples downstream of Bt corn fields. Strain and Lydy (2015)

Transgenic rice expressing insecticidal proteins from the soil bac-
terium Bacillus thuringiensis (Bt) can effectively prevent and control
insect pests but has not yet been approved for commercial cultivation in
China (Li et al., 2016). A major concern is that the planting of Bt rice
may adversely affect biodiversity owing to gene flow, non-target ef-
fects, effects on the soil ecosystem, and the risk of transgenic rice be-
coming a new weed (Li et al., 2016). Discussions have thus far focused
primarily on terrestrial ecosystems, and the potential effect of Bt crops
on aquatic organisms has rarely been addressed, in part because there is
only a small chance of aquatic organisms being exposed to transgenic
plant material or plant-produced Bt proteins (Viktorov, 2011; Carstens
et al., 2012).
Only a few studies have reported that Bt proteins or transgenic crop
byproducts can enter stream ecosystems adjacent to agricultural fields
through dispersal of pollen, exudation from roots, and movement of
post-harvest corn residues (Viktorov, 2011; Carstens et al., 2012). For
example, Tank et al. (2010) found that 86% of active stream sites
contained corn leaves, husks, cobs, and/or stalks, and the average
concentration of Cry1Ab in water samples that tested positive for the
protein reached 14 ± 5 ng/L, with a maximum concentration of 32 ng/
found that Cry1Ab protein in surface water runoff increased during the
growing season, peaking during corn flowering at 130 ng/L. Several
studies have been conducted to test whether insect-resistant transgenic
crops affect aquatic organisms, including larvae of caddisflies (Tri-
choptera), a crane fly (Diptera: Tipulidae), an amphipod (Amphipoda),
and an isopod (Isopoda) (Rosi-Marshall et al., 2007; Chambers et al.,
2010; Jensen et al., 2010). However, these studies have been limited to
Bt-transgenic corn. Rice, unlike dry-land crops, requires water during
most of its developmental stages, and Bt rice is known to release at least
small amounts of Bt protein into irrigation water. Previous studies re-
ported that transgenic rice can release up to 30 ng/L Cry1Ab/Ac protein
into water (Liu et al., 2016; Wang et al., 2013; Zhang, 2013). Therefore,
the risk from the release of Bt protein from rice to aquatic organisms
should be addressed.
Zooplankton play an important role in the aquatic food chain be-
cause they are both the main consumer of bacteria, single-cell algae,
and organic detritus and the main food source for higher trophic levels,
including fish. Changes in their abundance, diversity, and distribution
can thus have cascading effects throughout an entire water ecosystem
(Takagaki, 2016). Zooplankton are also very sensitive to many con-
taminants and are thus used as an indicator to monitor changes in water

⁎
Corresponding author.
E-mail address: xpchen@ippcaas.cn (X. Chen).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ecoenv.2017.08.065
Received 8 May 2017; Received in revised form 22 August 2017; Accepted 24 August 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Y. Chen et al.
quality (Xu et al., 2005). For example, zooplankton are commonly used
to evaluate the ecotoxicology of Bti sprays that are applied to control
mosquito larvae (Duchet et al., 2008; Lagadic et al., 2014; Olmo et al.,
2016).
Cladocera (water fleas) are zooplankton that are commonly found in
rice paddy aquatic ecosystems and thus can potentially be exposed to Bt
insecticidal proteins produced by transgenic rice through ingestion of
rice pollen, plant residues, or root exudates in the water. The Cladocera
Daphnia magna is widely used in environmental toxicology because of
its short life cycle, fast reproduction, and high sensitivity to external
contaminants such as chemical pesticides, heavy metals, nanoparticles,
and other manmade toxins (Brausch and Salice, 2011; Meyer et al.,
2015; Xiao et al., 2015). This organism is easy to obtain and raise under
laboratory conditions. Previous studies concerning the effect of Bt crops
on D. magna have yielded inconsistent results. Although most reports
have shown no obvious adverse effects of transgenic Bt crops on D.
magna based on developmental and reproduction analyses (Mendelson
et al., 2003; De Schrijver et al., 2016), others have reported that Bt corn
or Bt protein expressed by Bt corn can reduce the fitness of D. magna
(Bøhn et al., 2008, 2010, 2016).
Cry1C is a Bt protein that provides good resistance to lepidopteran
pests (Zheng et al., 2011; Wang et al., 2016) and has shown no adverse
effects on rats (Cao et al., 2010) and some terrestrial insects such as
green lacewing (Neuroptera: Chrysopidae) (Li et al., 2014a), honey bees
(Hymenoptera: Apidae) (Wang et al., 2015), lady beetles (Coleoptera:
Coccinellidae) (Li et al., 2015b), and springtails (Collembola: Iso-
tomidae) (Yang et al., 2015). However, no report is available about its
effect on aquatic organisms. In the present study, D. magna was exposed
to purified Cry1C protein for 21 d according to the guidelines of ISO
6341 (ISO, 2012) and OECD 211 (OECD, 2012) to assess the aquatic
environmental safety of this particular Bt protein.
2. Materials and methods
2.1. Reagents and insecticidal compounds
The composition and preparation of M4 medium for the culture of
D. magna were in accordance with the guidelines of OECD 211 and ISO
6341, and all reagents for the medium were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Potassium dichromate (K2Cr2O7) was
purchased from Xilong Chemical Ltd. Co. (Nanjing, China). Purified
Cry1C protein was purchased from Envirotest-China (agent for
EnviroLogix Inc., Portland, ME, USA; www.envirotest-china.com). The
protoxins from Bt were expressed as single-gene products in Escherichia
coli at Case Western Reserve University (Cleveland, OH, USA). The
protoxin inclusion bodies were dissolved and trypsinized, then isolated
and purified by ion-exchange HPLC, followed by desalting and lyo-
philizing of the pure fractions. The purity ranged from 94% to 96%
(Marianne P. Carey, Case Western Reserve University, personal com-
munication). Cry1C from the same source has been used in previous
toxicological studies (Li et al., 2014b; Yang et al., 2015).
2.2. Organisms
The green alga Chlorella pyrenoidosa (Chlorellales: Oocystaceae) was
obtained from the Fresh Water Algae Culture Collection of the Institute
of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Chilo
suppressalis (Lepidoptera: Crambidae) was obtained from a laboratory
colony that had been maintained on an artificial diet (Li et al., 2015a)
for > 60 generations with yearly introduction of individuals from field
populations.
D. magna was obtained from the Shanghai Fisheries 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 homoeothermic incubator (25 ± 1 °C, 70 ± 5% RH) under
a 12-h light/12-h dark cycle. D. magna were fed daily a diet of C.
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Ecotoxicology and Environmental Safety 147 (2018) 631–636
pyrenoidosa at a concentration of 3 × 106 cells/mL. Neonates (6–24 h
old) from the same mother were used for this experiment following test
procedures of ISO 6341 and OECD 211 (ISO, 2012; OECD, 2012). Prior
to our experiment, D. magna sensitivity to K2Cr2O7 was tested according
to the ISO 6341 standard. The 24 h-EC50 for D. magna to K2Cr2O7 was
0.76 mg/L, which is within the ISO 6341 requirement for the sensitivity
of D. magna (0.6–2.1 mg/L) (ISO, 2012).
2.3. Insecticidal activity of purified Cry1C in M4 medium
Purified Cry1C was added to 50 mL of M4 medium at the nominal
concentrations of 125, 250, and 500 µg/L and named mixed culture
medium (MCM). The actual Cry1C content in MCM after 0 h (D0) and
24 h (D1) exposure was quantified by enzyme-linked immunosorbent
assay (ELISA, see below). Bt-susceptible C. suppressalis larvae were used
as known sensitive insects to verify the bioactivity of Cry1C protein in
MCM after 0 h and 24 h feeding exposure. D0 and D1 MCM were stored
at –70 °C until they were mixed with the artificial diet (Li et al., 2015a)
for C. suppressalis (0.5 mL/g diet). To avoid degradation of Cry proteins
during the required heating stage of diet preparation, D0 and D1 MCM
were mixed into the diet after cooling to < 60 °C. Once the mixed diet
had solidified, it was cut into slices that were placed individually in
Petri dishes (90 mm diameter, 15 mm height). Neonates of C. sup-
pressalis were transferred individually to the Petri dishes, which were
subsequently sealed with Parafilm. Forty neonates divided into four
replicates were tested for each treatment. The mortality and weight of
C. suppressalis were recorded for each treatment after 7 days.
2.4. Effects of purified Cry1C on D. magna
Newly hatched D. magna larvae (within 6–24 h of hatching) were
kept individually in 100-mL beakers containing 50 mL M4 medium and
C. pyrenoidosa at a concentration of 1.3 × 106 cells/mL. Twenty in-
dividuals were tested (20 replicates) for each of the following treat-
ments: (1) M4 medium containing 125, 250, or 500 µg/L Cry1C
(treatment groups); (2) M4 medium containing 500 µg/L K2Cr2O7
(positive control); and (3) M4 medium alone (negative control). Thus,
the total number of insects used in this experiment was 100. K2Cr2O7
was selected as the positive control because it has a strong effect on the
reproduction of D. magna (Gopi et al., 2012); the concentration was
based on the results of a preliminary experiment. To address the known
degradation of the test compound (Cry1C) in water (Wang et al., 2014),
fresh Cry1C was added every day. Meanwhile to minimize manual
mechanical damage to D. magna, medium (with or without Cry1C or
K2Cr2O7) was renewed every 2 days. The survival, number of molts, and
number of offspring produced were recorded every day. All larvae
offspring were collected each day after counting and stored at −70 °C
for subsequent determination of enzyme activities and ELISA (see
below). The experiment was terminated after 21 days, at which time
four variables were measured: body length, body width, anal spine
length, and body weight. Pictures of the test adult insects were taken by
photomicroscope, and length measurements were then conducted using
a gauge. Living adult insects were then dried by removing their surface
water, and body weight was measured using an electronic microbalance
(CPA224S, Sartorius, Germany). All living adults were collected and
stored at −70 °C for subsequent determination of enzyme activities and
ELISA (see below).
2.5. Determination of enzyme activities
The activities of the antioxidant-related enzymes superoxide dis-
mutase (SOD), peroxidase (POD), and catalase (CAT) were quantified
using ELISA kits purchased from Nanjing Jiancheng Ltd. Co. (Nanjing,
China). Samples containing D. magna adults and larvae were homo-
genized 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,
Y. Chen et al. Ecotoxicology and Environmental Safety 147 (2018) 631–636

and the supernatants were used for enzyme analysis following the
manufacturer's instructions. Optical density values were read with a
microplate spectrophotometer XS2 (PowerWave XS2, BioTek,
Winooski, VT, USA), and enzyme activities were calculated by cali-
bration to a range of concentrations of standards provided with the kits.

2.6. Determination of Cry1C protein content by ELISA

The concentrations of Cry1C in D. magna and in MCM at D0 and D1

were measured by ELISA using a Cry1C detection kit (Quanti-Plate,
EnviroLogix). Before analysis, D. magna adults and larvae were washed
in phosphate-buffered saline/Tween-20 (PBST, provided with the kit) to
remove Bt toxins from the outer surfaces. For Cry1C extraction, samples
of D. magna, D0 MCM, and D1 MCM were weighed and mixed with
PBST at a ratio of 1:10 to 1:100 (mg sample/µL PBST) in 1.5-mL cen-
trifuge tubes. The samples were fully ground using an electric grinding
rod. After centrifugation and appropriate dilution of the supernatants,
ELISA was performed according to the manufacturer's instructions.
Optical density values were read with a microplate spectrophotometer
XS2, and concentrations of Cry1C were calculated by calibration to a
range of concentrations of a Cry1C standard provided with the kit.
2.7. Data analysis
All data are presented as mean ± standard error (SE) unless
otherwise indicated. The survival rates of C. suppressalis and D. magna
among different treatment and control groups were compared using the
Chi-square test. Body weights of C. suppressalis were compared by one-
way ANOVA with Duncan analysis. The effect of Cry1C and control
treatments on D. magna survival was analyzed using the Kaplan-Meier
procedure and log rank test. Body weight, body length, body width,
anal spine length, molting time, days to first brood, number of broods,
offspring number in the first brood, average number of offspring per
brood, and total number of offspring of D. magna were compared by
one-way ANOVA followed by the Tukey HSD test. Cry1C concentrations
and enzyme activities in D. magna adults and larvae were also analyzed
by one-way ANOVA with the Tukey HSD test. Differences were con-
sidered significant at P < 0.05. All statistical analyses were conducted
using the SPSS software package (version 13 for Windows, 2004).
3. Results
3.1. Insecticidal activity of Cry1C in M4 medium
The mean concentrations (± SE) of Cry1C detected by ELISA in
MCM on D0 and D1 were significantly lower than their nominal con-
centration (Table 1). Although the sensitive-insect bioassays revealed
that the survival rate of C. suppressalis larvae exposed to Cry1C was not
significantly different from that of negative control larvae (Chi-square
test, adjusted α = 0.008; Table 1), the body weight of survivors in the
Fig. 1. Survival rate of Daphnia magna when fed a pure diet (negative control) or diet
containing different concentrations of Cry1C protein or K2Cr2O7 (positive control) for 21
days. Data were analyzed with the Kaplan-Meier procedure and log rank test. *Significant
difference from negative control (P < 0.05).
125 µg/L-D0, 250 µg/L-D0, 500 µg/L-D0, and 500 µg/L-D1 groups was
significantly reduced compared with the negative control (one-way
ANOVA with Tukey HSD test; F = 17.24, df = 7, P < 0.05; Table 1),
demonstrating that Cry1C dissolved in M4 medium had insecticidal
activity.
3.2. Toxicity of purified Cry1C to D. magna
The survival rate of D. magna was 100% when cultured in negative
control or medium containing Cry1C at concentrations of 125, 250, or
500 µg/L (Fig. 1). The survival rate was significantly reduced when D.
magna was cultured in positive control medium containing 500 µg/L
K2Cr2O7 (Kaplan-Meier and log rank test; χ2 = 6.90, P < 0.05).
The developmental parameters (body weight, body length, body
width, anal spine length, and number of molts) of D. magna were not
affected by Cry1C (one-way ANOVA with Tukey HSD test; body weight:
F = 47.80, df = 4, P > 0.05; body length: F = 63.11, df = 4, P > 0.05;
body width: F = 69.48, df = 4, P > 0.05; anal spine length: F = 7.89,
df = 4, P > 0.05; number of molts: F = 19.10, df = 4, P > 0.05;
Table 2); however, all these parameters were significantly reduced in
the positive control (all P < 0.05).
The reproductive parameters (days to first brood, total number of
broods, number of offspring in first brood, average number of offspring
per brood, and total number of offspring) were not affected by Cry1C
(one-way ANOVA with Tukey HSD test; days to first brood: F = 12.72,
df = 4, P > 0.05; total number of broods: F = 19.10, df = 4, P > 0.05;
number of offspring in first brood: F = 6.66, df = 4, P > 0.05; average
number of offspring per brood: F = 88.92, df = 4, P > 0.05; total
number of offspring: F = 103.76, df = 4, P > 0.05; Table 3). Sig-
nificant differences were observed for all parameters in the positive

Table 1
Survival and body weight of Chilo suppressalis when fed a pure diet or diet containing different concentrations of Cry1C protein for 7 days.

Treatment Cry1C content in M4 medium (µg/L)a,b Cry1C content in diet (ng/g)a,b Survival rate (%, n)c Body weight (mg)a, §

Negative control-D0 0 0 100.0 (40) 3.55 ± 0.25 a

Negative control-D1 0 0 95.0 (38) 3.49 ± 0.31 a
125 µg/L-D0 1.16 ± 0.10 8.24 ± 0.36 100.0 (40) 2.42 ± 0.15 b
125 µg/L-D1 0.62 ± 0.07 6.77 ± 0.63 95.0 (38) 3.28 ± 0.27 a
250 µg/L-D0 3.12 ± 0.36 10.21 ± 1.21 100.0 (40) 2.25 ± 0.18 b
250 µg/L-D1 1.84 ± 0.17 9.73 ± 1.11 92.5 (37) 3.34 ± 0.28 a
500 µg/L-D0 4.86 ± 0.75 71.14 ± 5.59 97.5 (39) 1.18 ± 0.12 c
500 µg/L-D1 2.75 ± 0.30 48.01 ± 1.45 97.5 (39) 1.61 ± 0.12 c

a
Values represent mean ± SE.
b
n = 4.
c
Chi-square test with Bonferroni corrections (adjusted α = 0.008).
§
Different letters within the same column indicate significant difference (P < 0.05; one-way ANOVA with Duncan analysis).

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Table 2
Developmental parameters of Daphnia magna when fed a pure diet (negative control) or diet containing different concentrations of Cry1C protein or K2Cr2O7 (positive control) for 21 days.

Treatment Body weight (mg) Body length (mm) Body width (mm) Anal spine length (mm) Number of molts

Negative control 6.92 ± 0.22 a 4.15 ± 0.03 a 3.08 ± 0.02 a 0.81 ± 0.02 a 9.20 ± 0.09 a
Cry1C 125 μg/L 7.07 ± 0.22 a 4.19 ± 0.04 a 3.03 ± 0.02 a 0.81 ± 0.01 a 9.20 ± 0.09 a
Cry1C 250 μg/L 6.73 ± 0.19 a 4.06 ± 0.03 a 2.98 ± 0.03 a 0.79 ± 0.02 a 9.30 ± 0.10 a
Cry1C 500 μg/L 7.10 ± 0.20 a 4.07 ± 0.03 a 3.01 ± 0.03 a 0.76 ± 0.02 a 9.15 ± 0.08 a
K2Cr2O7 500 μg/L 3.44 ± 0.15 b 3.40 ± 0.04 b 2.39 ± 0.02 b 0.67 ± 0.02 b 7.25 ± 0.41 b

Values represent mean ± SE. For control and Cry1C treatments, n = 20; for K2Cr2O7 treatment, n = 14.
†
For each parameter, different letters within the same column indicate significant difference (one-way ANOVA with Tukey HSD test).

control (all P < 0.05).

After Cry1C exposure, the mean concentration (± SE) of Cry1C
protein in D. magna adults and larvae was determined by ELISA (Fig. 2).
Cry1C protein was detected in both adults and larvae in all three Cry1C
test groups, and the Cry1C level was significantly higher in adults than
in larvae (Tukey HSD test; all P < 0.05). Furthermore, the Cry1C
concentration increased with increasing amounts of added Cry1C pro-
tein. No Cry1C protein was detected in the negative control.

3.3. Enzyme activity in adults and larvae of D. magna

After exposure to the positive control, the activities of SOD and POD
in adults and larvae and the activity of CAT in larvae were significantly
higher than in the negative control and Cry1C treatment groups (Tukey
HSD test; all P < 0.05; Fig. 3). In contrast, there were no obvious dif-
ferences between the negative control and any Cry1C treatment groups
(all P > 0.05). The activity of CAT in adults was also not significantly
different from that of the negative control or any Cry1C treatment
groups (all P < 0.05).
4. Discussion
In this 21-day experiment, the survival rate was 100% when D.
magna was cultured in untreated M4 medium (negative control) or
medium containing Cry1C up to a concentration of 500 µg/L, whereas
the survival rate was reduced to 70% when D. magna was cultured in
medium with K2Cr2O7 (positive control). Similarly, there were no sig-
nificant differences in any of the developmental or reproductive para-
meters between the negative control and the Cry1C treatment groups,
whereas all parameters were significantly reduced in the positive con-
trol. Overall, our feeding bioassays revealed that D. magna was not
affected by exposure to Cry1C.
Bt proteins produced by transgenic plants can be dispersed in
aquatic environments adjacent to crop fields (Carstens et al., 2012).
Whether this would affect aquatic organisms needs to be taken into
consideration before large-scale cultivation of Bt-transgenic plants. The
maximum concentration of Bt protein produced by Bt maize that has
been detected in natural aquatic environments is less than 130 ng/L
(Douville et al., 2007; Strain and Lydy, 2015; Tank et al., 2010).
Likewise, the reported maximum of Bt protein released by Bt rice into
aquatic environments is 30 ng/L (Liu et al., 2016; Wang et al., 2013;
Zhang, 2013). In the current study, we establish a Tier-1 laboratory
Fig. 2. ELISA results for Cry1C protein concentration in Daphnia magna adults and larvae
when fed a pure diet (negative control) or diet containing different concentrations of
Cry1C protein. Fresh weight (FW). Different lowercase letters indicate significant differ-
ences (one-way ANOVA with Tukey HSD test, P < 0.05, n = 4 per group).
assay to explore the safety of Cry1C protein in D. magna. The nominal
protein exposure concentrations (125, 250 and 500 µg/L) were thus
1000-fold higher than the level D. magna would encounter in the nat-
ural aquatic environment. Owing to the rapid degradation of Cry1C
protein in water, the actual exposure concentrations of Cry1C decreased
to 0.62–4.86 µg/L (Table 1) but were still up to 10-fold higher than the
environmental concentration and thus met the requirements for a Tier-
1 assay (Li et al., 2014c).
Some studies have indicated that Bt crops that have no significant
effect on growth indices of non-target organisms may still have a sig-
nificant effect on physiological and biochemical indices (Zhou et al.,
2014). Thus, we tested the influence of purified Cry1C on SOD, POD,
and CAT activities of D. magna under laboratory conditions. SOD, POD,
and CAT are important antioxidant enzymes that protect organisms
against the peroxidation system and maintain the redox state of the cell.
SOD is the first line of cell defense, as it neutralizes superoxide anions
(O2•–) to yield hydrogen peroxide and molecular oxygen. Inside the cell,
the level of hydrogen peroxide is maintained by CAT and/or POD,
which catalyze its decomposition into molecular oxygen and water
(Ismaiel et al., 2016). In the present study, the activities of SOD and
POD increased significantly only in the positive control, suggesting a
cooperative role for these enzymes to detoxify reactive oxygen species
(Kumar et al., 2002). In contrast, there were no significant differences

Table 3
Reproductive parameters of Daphnia magna when fed a pure diet (negative control) or diet containing different concentrations of Cry1C protein or K2Cr2O7 (positive control) for 21 days.

Treatment Days to first brood Number of broods Number of offspring in first brood Average number of offspring per brood Total number of offspring

Negative control 7.10 ± 0.07 a 5.00 ± 0.00 a 22.75 ± 1.08 a 31.97 ± 1.08 a 159.85 ± 5.40 a
Cry1C 125 μg/L 7.25 ± 0.10 a 5.00 ± 0.00 a 21.60 ± 1.23 a 34.54 ± 0.90 a 172.70 ± 4.52 a
Cry1C 250 μg/L 7.15 ± 0.08 a 5.00 ± 0.00 a 20.25 ± 1.36 ab 32.95 ± 0.97 a 164.95 ± 4.79 a
Cry1C 500 μg/L 7.05 ± 0.05 a 5.00 ± 0.00 a 21.55 ± 1.55 a 34.50 ± 0.71 a 172.50 ± 3.56 a
K2Cr2O7 500 μg/L 8.00 ± 0.19 b 3.70 ± 0.37 b 14.60 ± 0.95 b 15.08 ± 0.67 b 55.45 ± 6.10 b

Values represent mean ± SE. For control and Cry1C treatments, n = 20; for K2Cr2O7 treatment, n = 14.
†
For each parameter, different letters within the same column indicate significant difference (one-way ANOVA with Tukey HSD test).

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Y. Chen et al.
Fig. 3. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in
Daphnia magna adults and larvae when fed a pure diet (negative control) or diet con-
taining different concentrations of Cry1C protein or K2Cr2O7 (positive control).
*Significant difference from other groups (one-way ANOVA with Tukey HSD test, P <
0.05, n = 8 per group).
between the Cry1C treatment groups and the negative control. Inter-
estingly, the positive control treatment did not alter CAT activity in
adult D. magna, possibly owing to the insensitive CAT response of adult
D. magna exposed to K2Cr2O7. The biochemical analysis thus provides
further evidence that Cry1C causes no sublethal effects in D. magna. To
our knowledge, the current study is the first to measure the activities of
these antioxidant enzymes (SOD, POD, and CAT) in D. magna as a re-
sponse to Cry1C protein uptake.
In the current study, Cry1C protein was detected in D. magna adults
and larvae in all three Cry1C test groups, but the Cry1C level was sig-
nificant higher in adults than in larvae. The lower levels in larvae may
be attributable to the short exposure time: larvae were collected after
emergence each day, whereas adults were exposed for 21 days.
Furthermore, the Cry1C concentration increased over time, as in-
creasing amounts of protein were added to the medium. Overall, these
data confirm that adults and larvae of D. magna were exposed to Cry1C
in our feeding bioassays. This exposure may have been achieved by
consumption of C. pyrenoidosa because the algae have been shown to
adsorb Cry proteins (Venter and Bøhn, 2016; Wang et al., 2014), na-
noparticles (McTeer et al., 2014), and heavy metals (De Schamphelaere
and Janssen, 2004), which can thus serve as a vector for transporting
such compounds to algae-consuming aquatic organisms.
D. magna is a standard organism for testing chemicals and water
quality (ISO, 2012; OECD, 2012) and is an important species for fish
aquaculture (Chen et al., 1997). Many researchers have used D. magna
as an aquatic test organism to assess the environmental safety of
transgenic crops, but the findings have been inconsistent. Laboratory
studies and field surveys conducted by Mendelson et al. (2003) and
Zhang et al. (2016) demonstrated that Bt crops had no obvious effects
on D. magna. Li et al. (2014a) even showed that Bt rice is safer for D.
magna than its non-transgenic counterpart because it eliminated the
need for application of pesticides. In contrast, Bøhn et al., (2008, 2010,
635
Ecotoxicology and Environmental Safety 147 (2018) 631–636
2016) reported significant differences in lethal and sublethal para-
meters in D. magna that were exclusively fed Bt (Cry1Ab) transgenic
corn material (leaf powder) compared with organisms fed control maize
material or organisms exposed to purified Cry protein. These studies,
however, have received much criticism (Ricroch et al., 2010; Romeis
et al., 2013). First, because the Bt and non-Bt corn materials used were
not comparable (no near-isoline, produced in a different environment),
it is highly likely that the observed effects were caused by plant para-
meters other than the Cry protein. Second, the experiments are not
reliable based on the overall high control mortality and the fact that
three experimental runs yielded very different results. Third, the effects
of the different treatments were only notable later than the proposed
21-day duration of the bioassay. Fourth, there was no verification that
the test organisms were actually exposed to biologically active Cry
protein. These issues have been addressed in our study. First, we ver-
ified the bioactivity of Cry1C dissolved in M4 medium and determined
that the test organism was actually exposed to Cry1C protein—two
essential steps in the risk assessment of Bt crops (Romeis et al., 2011).
Second, we conducted our experiment strictly according to the protocol
for the testing of reproduction in D. magna (ISO, 2012; OECD, 2012),
and we increased the number of test organisms from the required 10
individuals to 20. Finally, we tested three different concentrations of
purified Cry1C on D. magna and obtained consistent results within the
21-day period of the bioassay.
Prior to cultivation, any Bt-transgenic plant must pass an environ-
mental risk assessment. This assessment typically follows a tiered ap-
proach starting in the laboratory, where the impact of the stressor of
concern (here: the insecticidal compound) on valued non-target or-
ganisms can be studied under controlled, worst-case exposure condi-
tions. Higher-tier studies (i.e., semi-field and field tests) are subse-
quently conducted if a detrimental effect is identified or the remaining
uncertainty is high (Romeis et al., 2008; Li et al., 2014c). Since the
substantially equivalence of the Cry1C protein to the protein released
from transgenic cry1C rice was previously confirmed by Cao et al.
(2010), we think this particular transgenic rice is safe to D. magna.
5. Conclusions
Ours is the first study to assess the effects of Cry1C protein on the
development and reproduction of D. magna. None of the investigated
developmental, reproductive, and biochemical parameters of D. magna
were significantly altered by exposure to Cry1C protein. The results
demonstrate that Cry1C protein has no lethal or sublethal effects on this
non-target species.
Acknowledgments
This study was supported by the National GMO New Variety
Breeding Program of PRC (2016ZX08011-001).
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