Characterization of Synergistic Embryotoxicity of Nickel and

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Characterization of Synergistic Embryotoxicity of Nickel and Buprofezin in Zebrafish Tingting Ku, Wei Yan, Wuyao Jia, Yang Yun, Na Zhu, Guangke Li, and Nan Sang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506293t • Publication Date (Web): 19 Mar 2015 Downloaded from http://pubs.acs.org on March 23, 2015

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Environmental Science & Technology

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Title page

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Characterization of Synergistic Embryotoxicity of Nickel and Buprofezin in

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Zebrafish

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Tingting Ku, Wei Yan, Wuyao Jia, Yang Yun, Na Zhu, Guangke Li, Nan Sang*

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College of Environment and Resource, Research Center of Environment and Health, Shanxi University,

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Taiyuan, Shanxi 030006, PR China

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* Corresponding author. Tel.: +86-351-7011932

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Fax: +86-351-7011932

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E-mail: [email protected]

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Mailing address: Nan Sang

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College of Environment and Resource,

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Shanxi University, Taiyuan, Shanxi 030006

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People’s Republic of China

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ACS Paragon Plus Environment


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ABSTRACT: Multiple pollutants, usually at low levels, co-exist and may interact in the environment. It

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is therefore important to analyze the toxicity of mixtures of co-existing chemicals to evaluate the

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potential ecological risk. Concern regarding the co-occurrence and combined bio-effects of heavy

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metals and organic insecticides in aquatic settings has existed for many years, but a clear

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understanding of the interactions between and potential combined toxicity of these chemicals remains

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elusive. In the present study, the combined effects of the heavy metal nickel (NiSO4) and insect growth

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regulator buprofezin on the induction of embryo toxicity in zebrafish were assessed. By applying

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nonlinear regression to the concentration-response data with each of the chemicals using the Hill and

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Langmuir functions and computing the predictions using the model of concentration addition (CA), we

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confirmed that NiSO4 and buprofezin acted together to produce synergistic embryotoxicity in zebrafish.

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Subsequently, we further found that the combination of NiSO4 and buprofezin formed a complex that

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facilitated the uptake of nickel (Ni) and buprofezin by the embryos. Following this, we clarified that an

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oxidative mechanism of the complex might underlie the synergistic embryotoxicity of NiSO4 and

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buprofezin.

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INTRODUCTION

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Multiple pollutants, usually at low levels, coexist and may interact in the environment. For this

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reason, studies of the concentration-response relationship of individual chemicals cannot reflect the

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actual exposure risk to organisms. Therefore, a key question in the risk assessment for co-exposures is

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whether mixture effects occur when chemicals are combined at low doses that individually do not

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induce observable effects. Awareness of the need to address combined exposures is growing among

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experts, and mounting evidence indicates that there is a combined toxicity as a result of simultaneous

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co-exposure to chemicals. This is especially true for the combined bio-effects of heavy metals and

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organic insecticides in aquatic settings, although studies of these interactions are not new. However,

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current reports regarding harmful chemicals appear regularly in media outlets, and communication to

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the public of the risks of these interactions and the potential combined effects at low concentrations is

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still a significant problem.

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Buprofezin (2-tert-butylimino-3- isopropyl-5-phenyl-1,3,5-thiadiazinan-4-one) is an insect growth

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regulator characterized by a broad-spectrum, specific mode of action and a good record of

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environmental safety and has risen in popularity for agriculture use.

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buprofezin technical is approximately 4,500 tons/year. Due to its long-term retention, buprofezin

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residues can be detected in fruits, vegetables, crops, soil and aquatic environments,4-6 and their

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presence in the environment is a potential problem. Nickel (Ni) is a naturally occurring metallic element

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in the earth's crust, and its concentration in surface water and groundwater ranges between 1 and 10

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µg/L. Because Ni and its compounds are widely used in industry for various applications such as

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electroplating, welding, flame cutting, flame spraying and mold making,7 they are inevitably found at

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elevated levels in sediments, the water column, and biota, with concentrations of up to 100 to 1000

1-3


Currently, Chinese production of

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µg/L in freshwater and estuarine areas surrounding heavily developed urban areas.8 In addition, dietary

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intake through contaminated foodstuffs is another primary route of Ni exposure.9

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Environmental surveys have shown the co-occurrence of Ni and buprofezin in aquatic

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environments. In the Mekong River Delta of Vietnam, buprofezin in soils and sediments was

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omnipresent, and it was detected in all samples during both rice seasons with concentrations of up to

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521 µg/kg dm. Furthermore, buprofezin is the second most frequently detected insecticide in surface

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water and occasionally occurs in drinking water, with a quantification frequency of 58.7 % and 4.8 %

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and a median concentration of 0.19 and 0.12 µg/L, respectively. In contrast, in the Mekong River delta

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and its associated coastal zone, Ni has been detected as one of the trace elements in the dissolved

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phase with concentrations of 7.8 and 8.4 nM and has been found in suspended matter and superficial

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sediments in concentrations of 32 and 18 µg/g.10-12 In addition, preliminary studies have indicated that

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buprofezin and Ni co-exist in food and drink. Since buprofezin was recommended for use on exportable

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grapes in 2006, its residues have frequently been detected in wine. Whereas, Ni is typically one of the

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ultra microelements detected in wines from different regions.13,14 Among foodstuffs, relatively high

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concentrations of Ni are present in the leaves of the tea plant Camellia sinensis, whereas buprofezin

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residue has been found in tea samples.15,16 Furthermore, considering that Ni is an essential

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micronutrient and consistently found in organisms at low levels, even in the absence of elevated Ni

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exposure,

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hydrophobicity and stability in acid and alkali environments (the half-life is up to approximately 36−104

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days ), it is reasonable for us to expect that there is an increase in opportunities for co-exposure to

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buprofezin and Ni, and with this increase, the environmental safety of these chemicals, especially at

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low concentrations, are challenged.

17-19

and buprofezin residues are commonly detected in plants, soil and water

5,6

with high

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Despite the coexistence of these two substances in the environment, their ecological and health

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risk assessments have been primarily on the basis of the toxicological effects of the individual

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substances. The toxic effects of Ni in aquatic organisms, including oligochaetes, mollusks, fish and sea

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urchins,21-23 have been evaluated. These effects primarily include respiratory injuries to freshwater fish

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involving impaired gas exchange due to severely limiting the diffusive capabilities of the gills,

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increased morphological abnormality and mortality. Previous studies of buprofezin toxicity have mainly

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focused on the resultant dose-dependent reductions in the body length and spawning rates of Daphnia

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and decreases in the hatching and survival rates of African catfish embryos.

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interaction between Ni and buprofezin has not been investigated thus far, and their combined toxicity at

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low doses and the relevant mechanisms for such toxicity remain unclear.

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27,28

24-26

and

However, the

In the present study, the combined effects of NiSO4 and buprofezin on the induction of embryo

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toxicity

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concentration-response analyses with the individual chemicals and computing the prediction of the

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effects of a 1:1 mixture of the two chemicals by concentration addition (CA) model, we confirmed that

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NiSO4 and buprofezin acted together to cause synergistic embryo toxicity in zebrafish, even when each

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one existing at level below its no-observed-effect-concentration (NOEC). Subsequently, we used

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fluorescence spectrometry to investigate the interaction of these two chemicals and found that NiSO4

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and buprofezin formed a Ni-buprofezin complex. This complex facilitated uptake of Ni and buprofezin

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by the embryos after exposure to both and contributed to the combination effects. Finally, using

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pharmacological interference, we showed that the oxidative mechanism of the complex might underlie

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the synergistic embryotoxicity of NiSO4 and buprofezin.

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MATERIALS AND METHODS

in

zebrafish

were

assessed.

Applying

nonlinear

regression


to

the

extensive

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Chemicals. Nickel sulfate (NiSO4, purity>97%) and buprofezin (purity>99%) were purchased from

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Sigma-Aldrich Company (Saint Louis, Missouri, America) and Zhenbo Company (Jiangyin City, Jiangsu,

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China), respectively. All other chemicals used were commercially available appropriate grades. The

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buprofezin stock solution was prepared by dissolving a weighed amount of buprofezin in dimethyl

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sulfoxide (DMSO), and then, different exposure concentrations for the embryonic bioassay were

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obtained by diluting the buprofezin stock solutions into culture medium. The NiSO4 test solution was

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prepared by dissolving NiSO4 in distilled water to obtain a stock solution and then diluting the stock

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solution into culture medium immediately before use.

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Zebrafish Embryo Collection and Exposure. Mature wild-type (AB strain) zebrafish

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(approximately 8 months old) were maintained at 28 ± 0.5 ℃ in a 14 h light /10 h dark cycle in a

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continuous flow-through system in charcoal-filtered tap water. The fish were fed twice daily with

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Artemia nauplii. Zebrafish eggs were obtained from the spawning adults in groups of approximately 20

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males and 10 females held in tanks overnight. The zebrafish eggs were collected within 4 h of

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spawning from several breeding tanks, pooled, washed, and then randomly transferred into glass

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beakers. At 24 h post-fertilization (hpf), we randomly exposed normally developed embryos to Ni at 10,

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20, 40, 80, 200, 300, 400 and 600 mg/L or buprofezin at 2.5, 10, 40, 200, 300, 400, 500 and 600 mg/L,

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respectively. The exposure solution contained 294.0 mg/L CaCl22H2O, 123.3 mg/L MgSO47H2O, 63.0

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mg/L NaHCO3, and 5.5 mg/L KCl.

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buprofezin were both at 5, 10, 20, 40, 80 and 100 mg/L. To inhibit reactive oxygen species (ROS)

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production, the zebrafish embryos were exposed to Ni and buprofezin both at 10 mg/L in the absence

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and presence of a 2 h pre-treatment with the antioxidant agent Vitamin C (Vc, 250 µmol/L). Embryos

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were exposed to different treatments in a 24-well multi-plate at 3 embryos/well with 2 mL test solution,

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In the co-exposure treatments, the concentrations of Ni and



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and the plates were kept in a humidified incubator at 28 ± 0.5 ℃ under controlled lighting conditions.

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Both the vehicle control and the treated embryos received the maximum amount of 0.2 % DMSO (v/v),

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and three independent experiments for each treatment concentration were conducted in triplicate. For a

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single experiment, each concentration group contained 216 normally developed embryos. The

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embryos were examined at the end of the exposure period, the survival rate was recorded as the

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toxicity end point, and mortality was identified by a missing heartbeat, coagulation of the embryos,

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non-detached tail and failure to develop somites, as previously reported by Du et al.30

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Detection of ROS Production. According to a previously reported protocol,

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200 zebrafish

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embryos were washed twice with cold PBS (pH = 7.4) and then homogenized in 800 µL cold buffer.

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The homogenate was centrifuged at 12,000 × g for 10 min at 4 ℃, and the supernatant was collected.

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Next, 20 µL of the homogenates were added to the wells of a black 96-well plate(Costar 3925, Corning

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Incorporated, USA) and incubated at room temperature for 5 min. After the incubation, 100 µL PBS and

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8.3 µL dichlorofluorescein-diacetate (DCF-DA) stock solution (dissolved in DMSO, 10 mg/mL) were

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added to each well. The plate was incubated at 37 ℃ for 30 min in the dark. The fluorescence intensity

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was measured using a microplate reader (Varioskan Flash, Thermo Scientific, America) with excitation

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and emission at 485 and 530 nm, respectively. The ROS concentration was expressed in arbitrary units

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(fluorescence units/mg protein).

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Lipid Peroxidation and Protein Assay. Lipid peroxidation was detected by spectrofluorometric

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analysis as malondialdehyde (MDA) reacting with thiobarbituric acid to form a colored complex. The

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embryos were homogenized in cold PBS, and the homogenate was centrifuged at 12,000 × g for 10

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min at 4 ℃ to precipitate insoluble material. The supernatant was collected to analyze the MDA content

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using the corresponding reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The


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level of MDA is expressed as nanomoles per milligram protein. Protein levels were estimated by

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Bradford's method using bovine serum albumin as a standard.32

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Lactate Dehydrogenase (LDH) Release Assay. The culture medium was used to assess LDH

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release, and the activity was measured according to the protocol of Fontella et al.33 In detail, 100 µL of

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the medium was transferred to the reaction medium containing 7.5 mmol/L NAD , 260 mmol/L lactate,

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and

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1,10-phenanthroline was converted to a colored complex by reacting to NADH, which was determined

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at 490 nm.

+

1.6

mmol/L

1,10-phenanthroline.

Following

the

conversion

of

lactate

to

pyruvate,

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Fluorescence Spectral Analysis. A stock solution of buprofezin (10 g/L) was made by dissolving

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buprofezin in DMSO, sonicated for 5 min, and diluted by double distilled water. For the quenching

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experiments, the diluted buprofezin solutions and NiSO4 at various concentrations were applied into a

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measuring flask; and then the system was thoroughly mixed and quantified to 5 mL with double distilled

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water. The fluorescence intensity of mixtures was detected at 570-630 nm, with excitation wavelength

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of 300 nm (Cary Eclipse, Agilent Technologies, Australia).

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Determination of the Ni and Buprofezin Content of Embryos.

For Ni analysis, embryos of

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each treatment group were washed repeatedly with distilled water and digested overnight. The

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digestion was completed by microwave-assisted digestion at 185℃ for 20 min (Multiwave 3000, Anton

195

Paar, Australia). After cooling, the residues were transferred to 50 mL flasks and diluted with deionized

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water. Before analysis, the samples were filtered through a 0.45 µm membrane filter.34 The Ni contents

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were eventually measured by flame atomic absorption spectrometry (FAAS, AA240FS, Varian, USA).

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For the buprofezin determination, a gas chromatograph-mass spectrometer (GC-MS) was applied

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(TRACE-DSQ, Thermo Scientific, America). A DB-5 MS column of 30 m × 0.25 mm I.D × 0.25 µm film



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thickness was programmed at 100 ℃ for 1 min, and then the temperature was elevated to 300 ℃ at

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25 ℃/min and kept at 300 ℃ for 4 min. The temperatures of injection port and transfer line from GC to

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MS were kept at 250 ℃. The samples were automaticly introduced in splitless mode (t = 1 min), with a

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helium carrier gas flow of 1 mL/min. The EI was operated at 70 eV. The full scan mode ranged from m/z

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40 to 400. The ion source temperature was 250 ℃. The embryos were ultrasonically extracted (VWR

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ultrasonic bath, Filtrafine, Spain) with chloroform, and the extract was concentrated for 5 min in a rotary

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evaporator. Following this, the extract was dissolved in 1 mL of chloroform and dried under a gentle

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stream of N2. Finally, the extract was dissolved in 10 mL of chloroform, and 1 µL was injected into the

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GC-MS. Based on the data from the FAAS and GC-MS, we obtained the concentration of Ni and

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buprofezin of total embryos. The mass of Ni and buprofezin contained in each embryo was calculated

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from the measured concentration of the aliquots and the dilution factor according to previous

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literature.35 In detail, the Ni or buprofezin content of the embryo was calculated using the following

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equation: M

213

were the concentration of the Ni or buprofezin of total embryos from FAAS and GC-MS, the final

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volume and the total number of embryos, respectively.

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= CV N . In this equation, M was the Ni or buprofezin content of the embryo; C, V, and N

Concentration-response Analysis. Based on the scatter plots of embryo mortality ratio versus 36

216

concentration and previously reported “best fit” approach,

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analyses using suitable regression models, such as Hill and Langmuir models, etc. Also, we calculated

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the 95% confidence intervals of the estimated mean effects. Table S1 showed a complication of the

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nonlinear regression models applied in present study, and the NOEC of the individual chemicals and

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mixtures were estimated using the Dunnett test.36

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we conducted concentration-response

Calculation of Predicted Mixture Effects. We prepared mixtures at a mixture ratio of 1:1 and


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then treated the embryos with serial dilutions of a master solution. Then, we calculated the predicted

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combination effects over a range of concentrations using CA model. The model assumes that the

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combination effect of a mixture with n components is concentration additive, therefore, the following

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formula is used for calculating the mixture effects.37-42

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ECxmix = [∑ pi / ECxi ]−1 [1]

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In this equation, pi is the relative proportion of substance i expressed as a fraction of the total

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concentration of substances in the mixture. The effect concentrations ECxi were computed using the

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inverse expression of the proper regression functions from the concentration response function of

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individual components i (Table S1).

231

Statistical Analysis. A one-way analysis of variance (ANOVA) was used to analyze the mean

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differences among groups compared to the vehicle control. A two-tailed Student’s t-test was used to

233

analyze experimental data between two groups. The data are presented as the mean ± SE. p

Characterization of Synergistic Embryotoxicity of Nickel and

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