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Selection of organosilicone surfactants for tank-mixed pesticides considering the balance between synergistic effects on pests and environmental risks
Accepted Manuscript Selection of organosilicone surfactants for tank-mixed pesticides considering the balance between synergistic effects on pests and environmental risks
Bei-xing Li, Yang Liu, Peng Zhang, Xiao-xu Li, Xiu-yu Pang, Yun-he Zhao, Hua Li, Feng Liu, Jin Lin, Wei Mu PII:
S0045-6535(18)32168-4
DOI:
10.1016/j.chemosphere.2018.11.061
Reference:
CHEM 22545
To appear in:
Chemosphere
Received Date:
11 October 2018
Accepted Date:
09 November 2018
Please cite this article as: Bei-xing Li, Yang Liu, Peng Zhang, Xiao-xu Li, Xiu-yu Pang, Yun-he Zhao, Hua Li, Feng Liu, Jin Lin, Wei Mu, Selection of organosilicone surfactants for tank-mixed pesticides considering the balance between synergistic effects on pests and environmental risks, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.11.061
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ACCEPTED MANUSCRIPT
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Selection of organosilicone surfactants for tank-mixed pesticides
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considering the balance between synergistic effects on pests and
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environmental risks
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Bei-xing Li,1,3 Yang Liu,3 Peng Zhang,2 Xiao-xu Li,3 Xiu-yu Pang,4 Yun-he Zhao,3
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Hua Li,3,5 Feng Liu,3 Jin Lin,1,3 Wei Mu1,3*
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1. Research Center of Pesticide Environmental Toxicology, Shandong Agricultural
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University, Tai’an 271018, China
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2. College of Environmental Science and Engineering, Nankai University, Tianjin
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300350, China
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3. Key Laboratory of Pesticide Toxicology & Application Technique, College of
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Plant Protection, Shandong Agricultural University, Tai’an 271018, China
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4. Department of Nutrition and Food Hygiene, College of Public Health, Harbin
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Medical University, 157 Baojian Road, Harbin 150081, China
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5. Zaozhuang No. 1 High School of Shandong Province, Zaozhuang 277300, China
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*Corresponding
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Protection, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong
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271018, P.R. China. Tel: +086-0538-8242611.
authors: Prof. Wei Mu ([email protected]), College of Plant
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1
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Abstract: In this study, the bioactivities of binary mixtures of organosilicone
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surfactants and indoxacarb against two Lepidopteran pests were investigated along
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with their environmental risks. All of the tested organosilicone surfactants had
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obvious synergistic effects on the contact toxicity of indoxacarb against Spodoptera
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exigua and Agrotis ipsilon. However, all of the organosilicone surfactants exhibited
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certain antagonism for indoxacarb against S. exigua in terms of stomach & contact
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toxicity; both Silwet-408 and Silwet-806 exhibited additivity against A. ipsilon,
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whereas Silwet-618 and Silwet-DRS-60 exhibited synergism and slight antagonism,
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respectively. All of the tested chemicals were highly toxic to Daphnia magna, among
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which Silwet-DRS-60 had the lowest acute toxicity (EC50 of 94.91 µg/L). However,
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these chemicals were less toxic to Brachydanio rerio. Silwet-DRS-60 had a low
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toxicity to B. rerio, while Silwet-408, Silwet-806 and Silwet-618 were moderately
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toxic to B. rerio. For the joint toxicity evaluation of organosilicone surfactants and
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indoxacarb to D. magna and B. rerio, the additive index method, concentration
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addition method and toxicity unit method were robust in judging synergism or
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antagonism, whereas other methods were more conservative; the V-value method and
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equilibrium curve method exhibited high robustness and viability in evaluating the
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combined effects of binary mixtures. Overall, we should carefully select
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organosilicone surfactants for premixed or tank-mixed pesticides in agriculture to
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obtain a balance between synergistic effects on pests and environmental risks.
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Keywords: organosilicone surfactants; indoxacarb; combined effect; joint toxicity;
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Daphnia magna; Brachydanio rerio 2
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1. Introduction
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Pesticides are one of the most crucial procreative materials that ensure the
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production and quality of agricultural products (Zhao et al., 2018). The active
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ingredients in pesticides cannot work unless they are contacted or absorbed by pests
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or protected targets. However, the compact wax coating of crop surfaces and target
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organisms may hamper the efficacy of pesticides, as they are hard to adhere to or
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infiltrate (Antonious and Saito, 1983; Querejeta et al., 2012). Tank-mix surfactants
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are always added for foliar spraying to help with the adhesion and penetration of
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active ingredients in pesticides and, thus, promote the utilization efficiency and
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efficacy of pesticides (Ryckaert et al., 2008; Hunsche et al., 2011; Abdelgaleil et al.,
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2018). Among them, organosilicone surfactants, especially trisiloxane surfactants,
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have been rapidly developed and applied in agricultural production due to their
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favorable wettability, spreadability, adhesivity and penetrability characteristics (G.,
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1993; Hill, 2002; Michel et al., 2016). To date, studies on organosilicone surfactants
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mainly focus on their physicochemical properties, such as super-spreading and
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dispersive behaviors (Wagner et al., 1999; Peng et al., 2010; Lin et al., 2016), super
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dispersive mechanisms (Chengara et al., 2002; Nikolov et al., 2002; Kumar et al.,
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2006), phase behaviors (And et al., 2004) and more (Churaev et al., 2001). There are
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also studies reporting their application in pest management. Single Silwet L-77 (0.1%
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solution) was found to have high lethal bioactivity against various piercing-sucking
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mouthparts pests, such as Aphis gossypii, Frankliniella occidentalis, and Tetranychus
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pacificus (Imai et al., 1995; Tipping et al., 2003). The addition of Silwet L-77 was 3
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reported to maintain the efficacy of azinphos methyl, chlorpyrifos, and captan for
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insect and disease control on apples despite halving the pesticide dosage (Stevens et
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al., 1994).
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Studies of serial Silwet organosilicone surfactants in China have also increased
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rapidly since the cooperation of Momentive Performance Materials with the Ministry
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of Agricultural of China (the current name is the Ministry of Agriculture and Rural
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Affairs of the People’s Republic of China) in 2006. Organosilicone is widely used as a
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tank-mix synergist in the management of Homoptera and Acariformes pests on cereal
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and horticultural plants (Qiu et al., 2006). Although single organosilicone surfactants
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have certain lethal bioactivity against small pests (such as aphids and mites) via
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mechanical respiratory inhibition or interference with important physiological
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processes, whether single organosilicone surfactants have bioactivities against larger
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pests (such as Lepidopteran pests) or their combined effects with pesticides are rarely
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reported.
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The long-term extensive use of organosilicone in agricultural production may
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also lead to environmental risks. Toxicological studies of organosilicone on aquatic
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flora and fauna have become popular research topics. Li et al. (Li et al., 2013)
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reported that Breakthru S240 had moderate acute toxicity to Daphnia magna, and the
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chronic toxicity test indicated that it can significantly affect the growth, breeding, and
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sex differentiation of D. magna, even at 0.2 mg/L. Wu et al. (Wu et al., 2009)
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evaluated the acute toxicity of four commercial organosilicone surfactants to
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Brachydanio rerio. Pesticides may also be released into the environment and persist 4
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for a long period along with surfactants and, thus, threaten the health and survival of
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various environmental organisms (Fenoll et al., 2014). The potential environmental
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risks of the binary mixtures of pesticides and surfactants should not be neglected.
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However, there have been few articles reporting the joint toxicity of organosilicone
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and pesticides to environmental organisms.
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In the current study, indoxacarb, which is a widely used oxadiazine pesticide in
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the management of Lepidopteran pests (Zlotkin, 1999), was used as a model to reveal
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the synergism of organosilicone surfactants in pest control. Then, D. magna and B.
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rerio, which are two kinds of typical aquatic organisms recommended by the OECD
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guidelines to assess the potential toxicity risks of chemicals in aquatic
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ecosystems(Barmentlo et al., 2015), were used as model organisms to determine the
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acute toxicity of single organosilicone and indoxacarb. The joint toxicity of
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organosilicone and indoxacarb to D. magna and B. rerio were also fully evaluated
99
using seven commonly used methods.
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2. Materials and methods
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2.1 Test chemicals
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Four kinds of organosilicone surfactants (Silwet-408, Silwet-806, Silwet-618, and
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Silwet-DRS-60) were purchased from Momentive Performance Materials Inc.
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(Connecticut, USA). Potassium dichromate (K2Cr2O7, AR) was purchased from the
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Beijing Chemical Reagent Research Institute (Beijing, China). Indoxacarb (with a
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purity of 98%) was purchased from Rainbow Chemical Co., Ltd. (Shandong, China)
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and fabricated into a 15% stock suspension using Tween-80 aqueous solution (0.1%, 5
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w/v) for convenient use.
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2.2 Bioactivity of organosilicone+indoxacarb against pests
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The two model Lepidopteran pests used in the bioassay were selected from
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populations raised for a long period in our laboratory. The bioactivities of single
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chemicals and mixtures against Spodoptera exigua and Agrotis ipsilon were evaluated
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using standardized 3rd instar larvae. Both the contact toxicity and stomach & contact
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toxicity of the chemicals were investigated in this study.
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The contact toxicity of the chemicals was determined in accordance with the
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protocols of the immersion test by dipping the insect body (Busvine, 1980). Five or
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more concentrations of indoxacarb adopted in the official tests were determined based
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on preliminary tests. To evaluate the bioactivity of organosilicone+indoxacarb, the
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concentration of organosilicone surfactants was maintained at 0.05%, which is a
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common concentration used for tank mixing in agricultural systems. The bioactivity
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of single organosilicone against pests was also investigated. Water treatment served as
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a negative control. Briefly, twenty-four larvae were transferred to cell culture plates
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after they were dipped into the dilutions for 7 s and dried with filter paper. Then, fresh
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cabbage leaves without any contaminants were used to feed the larvae. The mortality
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of the larvae was calculated 48 h after treatment (Zhang et al., 2016). All experiments
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were repeated three times for each concentration. The synergistic ratios were
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calculated as the LC50 value of indoxacarb to that of surfactants+indoxacarb (Liu et
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al., 2011).
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Slightly different from the measurement of contact toxicity, the stomach & 6
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contact toxicity of the chemicals was determined according to the leaf immersion test
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(Zhou et al., 2011). After the preparation of serial dilutions, fresh cabbage leaves were
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dipped into the solutions for 7 s and then allowed to dry with filter paper. Then, they
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were transferred to cell culture plates, and the larvae were added to the plates. All
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experiments consisted of 24 larvae and were repeated in triplicate. Water treatment
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served as the negative control. The mortality of the larvae was calculated 48 h after
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treatment. The synergistic ratios were also calculated as the LC50 value of indoxacarb
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to that of surfactants+indoxacarb.
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2.3 Measurements of the surface tension and retention of the surfactant solutions
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The surface tension of the 0.05% organosilicone surfactant solutions was measured
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using a BZY-1 automatic surface tension meter (Shanghai Hengping Instrument and
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Meter Factory, Shanghai, China), which was repeated three times. The retention of the
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dilutions on the cabbage leaves was determined according to a previously reported
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protocol (Yuan et al., 2000). In brief, two pieces of cabbage leaves (Φ = 1 cm) were
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dipped into the solutions for 7 s and allowed to hang vertically. The weights of the
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cabbage leaves before and after immersion were measured to calculate the weight of
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the dilutions retained on the leaves. The retention of the dilutions was calculated as
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the ratio of increased weight to the dilutions to the surface area for all of the leaves.
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The experiments were repeated six times.
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2.4 Acute single toxicity of indoxacarb and organosilicone surfactants to
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environmental organisms
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The environmental organisms, B. rerio and D. magna, belong to two typical trophic 7
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levels and, therefore, were used in the current study to evaluate the acute toxicity of
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the chemicals.
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B. rerio was cultivated in accordance with the OECD Guidelines (OECD) and
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held in groundwater (Tai’an, China; 36°15′17″N, 117°06′15″E), with a pH of 6-8, a
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total hardness of 140-250 mg/L and a dissolved oxygen concentration of
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approximately 4.0 mg/L, for seven days before testing. In the acute toxicity test, ten
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healthy B. rerio, with body lengths of 2.3±0.3 cm, were exposed to serial dilutions
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(more than five concentrations based on a preliminary test) containing indoxacarb or
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organosilicone surfactants. The exposure duration was 96 h, and the mortality of B.
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rerio was recorded at 24, 48, 72 and 96 h. The groundwater treatment served as a
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negative control. The toxicity of potassium dichromate to B. rerio was also
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investigated as a reference test. Subsequently, the chemical concentrations that killed
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fifty percent of the fish were calculated by the probit regression of the B. rerio
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mortality against the log10 values of the chemical concentrations using SPSS. All
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experiments were repeated in triplicate.
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D. magna is a pure strain introduced by the National Institute of Environmental
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Health at the Chinese Center for Disease Control and Prevention (Beijing, China) and
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is cultivated in accordance with the protocols recommended in the OECD guidelines
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(OECD, 2004). Briefly, these organisms were held in groundwater and fed daily with
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1 mL of algae at a concentration of approximately 3×105 cells/ml (Fan et al., 2017).
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Similar to the toxicity test of B. rerio, ten neonatal D. magna (ages less than 24 h)
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were exposed to these serial dilutions containing toxicants. The immobilization of D. 8
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magna was identified as the inability of D. magna to swim after 15 s of gentle
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agitation and recorded at 48 h to calculate the EC50 value. Groundwater without
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contamination served as the control. The toxicity of potassium dichromate to D.
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magna was also investigated as a reference test. All treatments were repeated three
178
times.
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2.5 Acute joint toxicity of indoxacarb and organosilicone surfactants to
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environmental organisms
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The joint toxicities of organosilicone surfactants and indoxacarb to B. rerio and D.
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magna were fully evaluated by seven commonly used methods, including the additive
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index method (Marking, 1977; Li et al., 2018b; Wu et al., 2018), concentration
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addition method (W, 2000; Loureiro et al., 2009; Li et al., 2018a), V-value method
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(He and Xiong, 1994), toxicity unit method (Brown, 1968; Sprague, 1970;
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Nirmalakhandan et al., 1997), Smyth’s method (Keplinger and Deichmann, 1967),
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mixture toxicity index method (Könemann, 1981; March, 1987; Mori et al., 2015) and
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equilibrium curve method (also known as the isobologram method) (Altenburger et
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al., 1990; De Liguoro et al., 2009). The concentrations of each toxicant used in the
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joint toxicity test were designed based on their acute single toxicities to B. rerio and
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D. magna. Joint concentrations were determined based on one toxic unit, which was
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defined as the 1:1 ratio of EC50 to (A) indoxacarb or (B) organosilicone surfactants.
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The serial toxicant concentrations were determined as 0.125 × (EC50-A + EC50-B), 0.25
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× (EC50-A + EC50-B), 0.50 × (EC50-A + EC50-B), 0.75 × (EC50-A + EC50-B) and 1.00 ×
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(EC50-A + EC50-B) to calculate the joint toxicity of the mixtures (Li et al., 2018a). 9
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2.6 Data analysis
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The LC50 or EC50 values were calculated by the probit regression of the mortality of S.
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exigua, A. ipsilon, B. rerio or the D. magna immobilization rates against the log10
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values of the toxicant concentrations. All of the statistical analyses were carried out
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using the SPSS statistical package (v17.0, USA). The surface tension and retention of
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the surfactant solutions were compared via Tukey’s test at the p < 0.05 level and
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displayed as the mean ± standard deviation.
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3. Results and analysis
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3.1 Bioactivity of organosilicone+indoxacarb against S. exigua and A. ipsilon
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The bioactivity of single organosilicone surfactants against S. exigua and A. ipsilon
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was assessed prior to that of organosilicone+indoxacarb. The results showed that all
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of the tested organosilicone surfactants had no lethal bioactivities against S. exigua or
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A. ipsilon, even at concentrations two folds greater than the fortified concentrations in
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the tested dilutions. Their physical activities were also not visually affected. In terms
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of the immersion of the insect bodies, all of the tested organosilicone surfactants had
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obvious synergistic effects on the contact toxicity of indoxacarb to S. exigua and A.
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ipsilon, although the synergistic ratios for the control of the two pests were partially
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inconsistent (Figure 1a and c). For the bioassay, which was measured by exposing
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pests to poisonous leaves, both stomach toxicity and contact toxicity worked. As
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depicted in Figure 1b, all of the organosilicone surfactants exhibited certain
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antagonism for indoxacarb against S. exigua, with the lowest synergistic ratios of 0.25
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and 0.24 for Silwet-806 and Silwet-DRS-60, respectively. However, the surfactants 10
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against A. ipsilon were much different (Figure 1d). Both Silwet-408 and Silwet-806
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exhibited additive effects, whereas Silwet-618 exhibited synergism and Silwet-DRS-
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60 exhibited slight antagonism.
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3.2 Single toxicities of organosilicone surfactants and indoxacarb to D. magna
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The acute toxicity of potassium dichromate to D. magna was investigated as a
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reference prior to the toxicity test of the organosilicone surfactants and indoxacarb.
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As shown in Table 1, the 24-h EC50 value of potassium dichromate against D. magna
225
was 1.282 mg/L, which indicated the favorable physiological status of D. magna and
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suitability for the toxicity test (Boillot and Perrodin, 2008). Then, single toxicities of
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organosilicone surfactants and indoxacarb to D. magna were evaluated. As displayed
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in Table 2, all of the tested chemicals were highly toxic to D. magna. Among them,
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Silwet-DRS-60 had the lowest acute toxicity to D. magna, with an EC50 value of
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94.91 µg/L, whereas Silwet-618 had the highest acute toxicity (EC50 value of 4.53
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µg/L). The typical toxic symptoms included motility reduction and bleached bodies of
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D. magna.
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3.3 Joint toxicities of organosilicone surfactants and indoxacarb to D. magna
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The joint toxicities of organosilicone surfactants and indoxacarb to D. magna were
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fully evaluated by the additive index method, concentration addition method, V-value
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method, toxicity unit method, Smyth’s method, mixture toxicity index method and
237
equilibrium curve method, as depicted in Figure 2. The additive index method
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(Figure 2a), toxicity unit method (Figure 2d), and mixture toxicity index method
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(Figure 2f) showed highly consistent results in assessing the combined effects of 11
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organosilicone surfactants and indoxacarb, where all of the combined effects
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exhibited synergism. Silwet-DRS-60 had the lowest synergism when enhancing the
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toxicity of indoxacarb to D. magna, while the synergisms of Silwet-408 and Silwet-
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618 were much higher. However, in terms of the concentration addition method
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(Figure 2b) and V-value method (Figure 2c), the combined effects were much
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different. Silwet-408 showed the highest synergism, followed by Silwet-806 and
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Silwet-618, whereas Silwet-DRS-60 exhibited slight antagonism or simple addition
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characteristics. Smyth’s method (Figure 2e) and the equilibrium curve method
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(Figures 2g, h, i) exhibited similar trends when evaluating the joint toxicity of
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organosilicone surfactants and indoxacarb. Both of these methods were more
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conservative than the other methods when identifying synergism, as they had much
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larger confidence intervals. The combined effects of Silwet-806+indoxacarb and
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Silwet-DRS-60+indoxacarb were both identified as additive effects (Figures 2e, h, j),
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whereas Silwet-408+indoxacarb (Figures 2e, g) and Silwet-618+indoxacarb (Figures
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2e, i) exhibited synergism.
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3.4 Single toxicities of organosilicone surfactants and indoxacarb to B. rerio
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The B. rerio juvenile fish were also used as a model to assess the toxicities of
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organosilicone surfactants and indoxacarb at a higher trophic level for comprehensive
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consideration. The acute toxicity of potassium dichromate to B. rerio was initially
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investigated as a reference. As displayed in Table 1, the EC50 value of potassium
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dichromate against B. rerio was 283.5 (259.4-309.2) mg/L, which indicated the
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favorable physiological status of B. rerio and suitability for the toxicity test. As 12
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displayed in Figure 3a, Silwet-DRS-60 was slightly toxic to B. rerio (LC50 values of
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60.61-96.51 mg/L), whereas the other tested organosilicone surfactants were
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moderately toxic to B. rerio, with LC50 values of 5.61-6.93, 3.89-4.45 and 6.47-7.20
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mg/L for Silwet-408, Silwet-806 and Silwet-618, respectively, when the
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measurements were conducted 24, 48, 72 and 96 h after chemical exposure.
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Apparently, the single toxicities of all tested organosilicone surfactants calculated at
268
different time nodes had no significant differences. In addition, the single toxicities of
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the chemicals did not change greatly, even for doses of LC20 and LC10 (Figures 3b,
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c). However, indoxacarb was highly toxic to B. rerio, regardless of the test or doses
271
(LC10, LC20 or LC50) used.
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3.5 Joint toxicities of organosilicone surfactants and indoxacarb to B. rerio
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The joint toxicities of organosilicone surfactants and indoxacarb to B. rerio were
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largely different those to D. magna. As depicted in Figure 4, all of the combined
275
effects of organosilicone surfactants and indoxacarb against B. rerio were determined
276
as exhibiting antagonism when using the additive index method (Figure 4a),
277
concentration addition method (Figure 4b), and toxicity unit method (Figure 4d).
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The evaluation methods with confidence intervals were much different in identifying
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antagonism. In terms of the V-value method, Silwet-408+indoxacarb and Silwet-
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618+indoxacarb displayed additive effects against B. rerio, whereas Silwet-
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806+indoxacarb and Silwet-DRS-60+indoxacarb still exhibited antagonism (Figure
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4c). However, only the combined effect of Silwet-618 and indoxacarb against B. rerio
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was determined as an additive effect (Figure 4i) via the equilibrium curve method, 13
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while others were identified as exhibiting antagonism (Figures 4g, h, j). In addition,
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evaluation methods with larger confidence intervals, such as Smyth’s method (Figure
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4e) and the mixture toxicity index method (Figure 4f), were much more conservative
287
in identifying antagonism, where all of the combined effects of organosilicone
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surfactants and indoxacarb against B. rerio were identified as the additive effect.
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Overall, Silwet-806 and Silwet-DRS-60 were more likely to cause antagonism than
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Silwet-618 and Silwet-408 when combined with indoxacarb.
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4. Discussion
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In recent years, the development of synergistic techniques and synergists has paid
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extensive attention to yielding the high use efficiency of pesticides and other
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agrochemicals (Chang et al., 2015). The key approaches to increasing the use
295
efficiency of pesticides lie in the promotion of pesticide deposition on the interfaces
296
of targets, the improvement in the uniform distribution of pesticide dilutions, the
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regulation of the release profiles of pesticide formulations, and the enhancement of
298
pesticide transport into the inner parts of targets (Yuan et al., 2000; Cao et al., 2014).
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Surfactants with favorable wettability, spreadability, and penetrability are always
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added as premixed or tank-mixed agents to assist in depositing pesticides onto targets,
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adhering to biological interfaces, and retaining and penetrating compact cell
302
membrane barriers (Baur et al., 1997; Baur et al., 1999; Burton and Bhushan, 2006).
303
With the help of surfactants, the use efficiency and control efficacy of pesticides have
304
become highly encouraged in pest management (Antonious and Saito, 1981;
305
Querejeta et al., 2012). 14
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Among them, organosilicone surfactants are one of the most highly consumed
307
synergists in agricultural production due to their synergistic effects on pesticides in
308
the management of agricultural pests. In the current study, we found that
309
organosilicone surfactants of the same class had apparently different synergistic
310
effects when controlling Lepidopteran pests (e.g., S. exigua and A. ipsilon), among
311
which Silwet-618 and Silwet-408 had much higher synergistic effects, and Silwet-
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DRS-60 had very small synergistic effects (via the contact toxicity test; Figure 1a, c).
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However, there were certain antagonistic effects for all of the mixtures against S.
314
exigua and partial mixtures against A. ipsilon in terms of the stomach & contact
315
toxicities, which were measured by dipping cabbage leaves into the toxicant solutions
316
and then inoculating them with pests. Indeed, the surface tension of the organosilicone
317
solutions significantly decreased compared with that of the water treatment, as
318
displayed in Figure 5a. The retention of dilutions containing indoxacarb on the
319
cabbage leaves also increased for all organosilicone treatments (Figure 5b). This
320
indicated that there were more toxicants on the cabbage leaves. However, why were
321
there certain antagonistic effects for all of the mixtures against S. exigua and partial
322
mixtures against A. ipsilon? We hypothesized that the decreased mobility of pests and
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the reduced consumption of the poisonous cabbage leaves could be key factors. We
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observed that the pests were less likely to move on the leaves after toxication,
325
especially for the organosilicone+indoxacarb group; thus, their exposure to pesticides
326
on the surfaces of the cabbage leaves was reduced. In addition, the average poisonous
327
leaf consumption of S. exigua and A. ipsilon in the organosilicone+indoxacarb group 15
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was much smaller than that in the single indoxacarb groups. We have used the
329
average leaf consumption of S. exigua and A. ipsilon for a dose of LC50 for each
330
mixture as verification. As shown in Figure 5c, the average poisonous leaf
331
consumptions of S. exigua and A. ipsilon in the organosilicone+indoxacarb groups
332
were both significantly lower than those of the single indoxacarb treatments. This has
333
great significance for pest management. For instance, imagine that we use the mixture
334
of Silwet-618 and indoxacarb to control A. ipsilon; we would use less indoxacarb to
335
achieve the same efficacy. In addition, damage on the crops would be reduced, as A.
336
ipsilon pests in the binary mixture group consume fewer leaves.
337
Organosilicone surfactants exhibited much different toxicity to typical aquatic
338
organisms at different trophic levels. All of the tested chemicals were highly toxic to
339
D. magna, whereas these chemicals were less toxic to B. rerio. Silwet-DRS-60 had a
340
low toxicity to B. rerio, while Silwet-408, Silwet-806 and Silwet-618 were
341
moderately toxic to B. rerio. The integument of D. magna was very thin and it could
342
hardly delay the penetration of organosilicone surfactants into the body whereas B.
343
rerio had protective fish scales and thicker epidermis. This may be the main reason
344
accounting for the fact that these chemicals were much toxic to D. magna than B.
345
rerio. It was also consistent with a previous review which reported that acute toxicity
346
tests with D. magna respond to a larger variety of chemicals with a higher sensitivity
347
than the acute toxicity tests with Danio rerio (Martins et al., 2007).
348
In this study, seven statistical methods were integrated to obtain comprehensive
349
evaluations on the combined effects of binary mixtures of organosilicone surfactants 16
ACCEPTED MANUSCRIPT 350
and pesticides. Overall, these methods showed similar trends in evaluating the joint
351
toxicity of binary mixtures, excluding the differences in the identification of
352
synergism, additivity or antagonism. Until now, many quantitative methods have been
353
proposed to evaluate the joint toxicity of chemicals (Lydy and Austin, 2004; Belden et
354
al., 2007; Li et al., 2018b); however, extensively applicable models and high
355
prediction accuracies were always scarce. In the current study, the additive index
356
method, concentration addition method and toxicity unit method were robust in
357
identifying synergism or antagonism, as they had no buffers (except for a critical
358
decision point), whereas the other methods, which had large buffers or confidence
359
intervals, were more conservative because quite a large proportion of the joint toxicity
360
was identified as additivity. Several previous studies have proposed the use of
361
endpoints equal to 0.5 and 2.0 to classify the toxicity unit and concentration addition
362
methods (Deneer, 2000; Boillot and Perrodin, 2008). However, we suggested the use
363
of a toxicity unit of 0.5-1.5 based on the additivity results in this study, which was
364
consistent with the recommendation of the European Inland Fisheries Advisory
365
Commission (EIFAC, 1980). In terms of the additive index method, we proposed a
366
cut-off of 0±0.2 to define the additive effect and reduce deviation or bias. In
367
summary, the V-value method and equilibrium curve method exhibited high
368
robustness and viability in evaluating the combined effects of binary mixtures.
369
Therefore, they deserve more attention in the assessment of joint toxicity.
370
In addition, the synergism of chemical mixtures against pests tends to be
371
consistent with their ecotoxicological risks to environmental organisms. In other 17
ACCEPTED MANUSCRIPT 372
words, surfactants with high wettability, spreadability and penetrability are more
373
likely to synergize pesticides in terms of pest management, and they may also cause
374
high toxicological risks or toxicity to other creatures. Thus, we should carefully select
375
organosilicone or other surfactants for premixed or tank-mixed pesticides in
376
agricultural production. Setting isolation belts and implementing other strategies are
377
imperative to reduce the discharge of various agrochemicals to nontarget organisms.
378
5. Conclusion
379
All of the tested organosilicone surfactants had obvious synergistic effects on the
380
contact toxicity of indoxacarb to S. exigua and A. ipsilon. However, all of the
381
organosilicone surfactants exhibited certain antagonistic effects for indoxacarb against
382
S. exigua in terms of stomach & contact toxicity; both Silwet-408 and Silwet-806
383
exhibited additive effects against A. ipsilon, whereas Silwet-618 and Silwet-DRS-60
384
exhibited synergism and slight antagonism, respectively. All of the tested chemicals
385
were highly toxic to D. magna, among which Silwet-DRS-60 had the lowest acute
386
toxicity (EC50 value of 94.91 µg/L). Silwet-DRS-60 was slightly toxic to B. rerio
387
(LC50 values of 60.61-96.51 mg/L), whereas other tested organosilicone surfactants
388
were moderately toxic to B. rerio, with LC50 values of 5.61-6.93, 3.89-4.45 and 6.47-
389
7.20 mg/L for Silwet-408, Silwet-806 and Silwet-618, respectively. The additive
390
index method, concentration addition method and toxicity unit method were robust in
391
identifying the synergism or antagonism of joint toxicity organosilicone surfactants
392
and indoxacarb to D. magna and B. rerio, whereas other methods were more
393
conservative. The V-value method and equilibrium curve method exhibited high 18
ACCEPTED MANUSCRIPT 394
robustness and viability in evaluating the combined effects of binary mixtures.
395
Overall, the current study would deepen the concept to assess the introduction of
396
chemicals into agriculture and the environment from all angles. And we should
397
carefully select organosilicone or other surfactants for premixed or tank-mixed
398
pesticides in agricultural production to obtain a balance between synergistic effects on
399
pests and environmental risks.
400
Acknowledgments
401
This work was supported by grants from the National Key R&D Program of China
402
(2017YFD0200307) and the National Natural Science Foundation of China
403
(31772203).
404
Declaration of interest statement
405
The authors declare no competing financial interests.
406
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ACCEPTED MANUSCRIPT 573
Table 1 The acute toxicity of potassium dichromate to Daphnia magna and
574
Brachydanio rerio EC50 and 95% confidence Organism
Time (h)
Regression
R2
interval (mg/L) D. magna
24
1.282 (0.943-1.874)
Y=2.832X-0.306
0.955
B. rerio
24
283.5 (259.4-309.2)
Y=10.32X-25.32
0.936
575 576 577 578
Table 2 Single toxicity of indoxacarb or organosilicone surfactants to Daphnia
579
magna EC50 and 95% confidence Chemicals
Regression
R2
Toxicity
interval (µg/L) Indoxacarb
23.72 (12.53-38.68)
Y=1.105X+1.791
0.941
Highly toxic
Silwet-408
31.84 (12.19-100.7)
Y=0.537X+0.804
0.854
Highly toxic
Silwet-806
15.04 (5.888-41.16)
Y=0.561X+1.022
0.907
Highly toxic
Silwet-618
3.598 (1.312-8.874)
Y=0.586X+1.426
0.948
Highly toxic
Silwet-DRS-60
94.91 (27.56-603.1)
Y=0.681X+0.697
0.961
Highly toxic
28
ACCEPTED MANUSCRIPT 581
Figure captions
582
Figure 1 Contact toxicity (a) and stomach+contact toxicity (b) of indoxacarb and
583
organosilicone surfactants to Spodoptera exigua. Contact toxicity (c) and
584
stomach+contact toxicity (d) of indoxacarb and organosilicone surfactants to Agrotis
585
ipsilon. The synergistic ratios below the lower dotted lines (synergistic ratio<0.80),
586
above the upper dotted lines (synergistic ratio>1.20) and between the dotted lines
587
(0.80
588
effect, respectively.
589 590
Figure 2 Joint toxicity (at the EC50 level) of indoxacarb and organosilicone
591
surfactants to Daphnia magna evaluated by the (a) additive index method, (b)
592
concentration addition method, (c) V-value method, (d) toxicity unit method, (e)
593
Smyth’s method, (f) mixture toxicity index method, and (g-i) equilibrium curve
594
method. The term Ind represents indoxacarb, while S408, S806, S618 and DRS-60
595
represent Silwet-408, Silwet-806, Silwet-618, and Silwet-DRS-60, respectively.
596 597
Figure 3 Single toxicity of indoxacarb or organosilicone surfactants to Brachydanio
598
rerio. (a) LC50 values, (b) LC20 values and (c) LC10 values.
599 600
Figure 4 Joint toxicity (at the LC50 level) of indoxacarb and organosilicone
601
surfactants to Brachydanio rerio evaluated by the (a) additive index method, (b)
602
concentration addition method, (c) V-value method, (d) toxicity unit method, (e) 29
ACCEPTED MANUSCRIPT 603
Smyth’s method, (f) mixture toxicity index method, and (g-i) equilibrium curve
604
method. The term Ind represents indoxacarb, while S408, S806, S618 and DRS-60
605
represent Silwet-408, Silwet-806, Silwet-618, and Silwet-DRS-60, respectively.
606 607
Figure 5 (a) Surface tension of the organosilicone solutions. (b) Retention of the
608
dilutions containing indoxacarb on cabbage leaves. (c) The average leaf consumption
609
of Spodoptera exigua and Agrotis ipsilon at a dose of LC50 for each mixture. The data
610
are displayed as the mean ± standard deviation, and those with different lowercase
611
letters are significantly different at the p < 0.05 level by Tukey’s test.
30
ACCEPTED MANUSCRIPT Highlights
Organosilicone were synergistic on the contact toxicity of indoxacarb.
Organosilicone were highly toxic to D. magna but less toxic to B. rerio.
Additive index, concentration addition and toxicity unit were robust.
V-value and equilibrium curve were conservative in the joint toxicity evaluation.
Bei-xing Li, Yang Liu, Peng Zhang, Xiao-xu Li, Xiu-yu Pang, Yun-he Zhao, Hua Li, Feng Liu, Jin Lin, Wei Mu PII:
S0045-6535(18)32168-4
DOI:
10.1016/j.chemosphere.2018.11.061
Reference:
CHEM 22545
To appear in:
Chemosphere
Received Date:
11 October 2018
Accepted Date:
09 November 2018
Please cite this article as: Bei-xing Li, Yang Liu, Peng Zhang, Xiao-xu Li, Xiu-yu Pang, Yun-he Zhao, Hua Li, Feng Liu, Jin Lin, Wei Mu, Selection of organosilicone surfactants for tank-mixed pesticides considering the balance between synergistic effects on pests and environmental risks, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.11.061
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ACCEPTED MANUSCRIPT
1
Selection of organosilicone surfactants for tank-mixed pesticides
2
considering the balance between synergistic effects on pests and
3
environmental risks
4
Bei-xing Li,1,3 Yang Liu,3 Peng Zhang,2 Xiao-xu Li,3 Xiu-yu Pang,4 Yun-he Zhao,3
5
Hua Li,3,5 Feng Liu,3 Jin Lin,1,3 Wei Mu1,3*
6
1. Research Center of Pesticide Environmental Toxicology, Shandong Agricultural
7
University, Tai’an 271018, China
8
2. College of Environmental Science and Engineering, Nankai University, Tianjin
9
300350, China
10
3. Key Laboratory of Pesticide Toxicology & Application Technique, College of
11
Plant Protection, Shandong Agricultural University, Tai’an 271018, China
12
4. Department of Nutrition and Food Hygiene, College of Public Health, Harbin
13
Medical University, 157 Baojian Road, Harbin 150081, China
14
5. Zaozhuang No. 1 High School of Shandong Province, Zaozhuang 277300, China
15 16
*Corresponding
17
Protection, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong
18
271018, P.R. China. Tel: +086-0538-8242611.
authors: Prof. Wei Mu ([email protected]), College of Plant
19
1
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Abstract: In this study, the bioactivities of binary mixtures of organosilicone
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surfactants and indoxacarb against two Lepidopteran pests were investigated along
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with their environmental risks. All of the tested organosilicone surfactants had
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obvious synergistic effects on the contact toxicity of indoxacarb against Spodoptera
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exigua and Agrotis ipsilon. However, all of the organosilicone surfactants exhibited
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certain antagonism for indoxacarb against S. exigua in terms of stomach & contact
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toxicity; both Silwet-408 and Silwet-806 exhibited additivity against A. ipsilon,
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whereas Silwet-618 and Silwet-DRS-60 exhibited synergism and slight antagonism,
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respectively. All of the tested chemicals were highly toxic to Daphnia magna, among
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which Silwet-DRS-60 had the lowest acute toxicity (EC50 of 94.91 µg/L). However,
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these chemicals were less toxic to Brachydanio rerio. Silwet-DRS-60 had a low
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toxicity to B. rerio, while Silwet-408, Silwet-806 and Silwet-618 were moderately
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toxic to B. rerio. For the joint toxicity evaluation of organosilicone surfactants and
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indoxacarb to D. magna and B. rerio, the additive index method, concentration
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addition method and toxicity unit method were robust in judging synergism or
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antagonism, whereas other methods were more conservative; the V-value method and
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equilibrium curve method exhibited high robustness and viability in evaluating the
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combined effects of binary mixtures. Overall, we should carefully select
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organosilicone surfactants for premixed or tank-mixed pesticides in agriculture to
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obtain a balance between synergistic effects on pests and environmental risks.
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Keywords: organosilicone surfactants; indoxacarb; combined effect; joint toxicity;
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Daphnia magna; Brachydanio rerio 2
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1. Introduction
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Pesticides are one of the most crucial procreative materials that ensure the
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production and quality of agricultural products (Zhao et al., 2018). The active
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ingredients in pesticides cannot work unless they are contacted or absorbed by pests
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or protected targets. However, the compact wax coating of crop surfaces and target
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organisms may hamper the efficacy of pesticides, as they are hard to adhere to or
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infiltrate (Antonious and Saito, 1983; Querejeta et al., 2012). Tank-mix surfactants
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are always added for foliar spraying to help with the adhesion and penetration of
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active ingredients in pesticides and, thus, promote the utilization efficiency and
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efficacy of pesticides (Ryckaert et al., 2008; Hunsche et al., 2011; Abdelgaleil et al.,
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2018). Among them, organosilicone surfactants, especially trisiloxane surfactants,
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have been rapidly developed and applied in agricultural production due to their
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favorable wettability, spreadability, adhesivity and penetrability characteristics (G.,
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1993; Hill, 2002; Michel et al., 2016). To date, studies on organosilicone surfactants
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mainly focus on their physicochemical properties, such as super-spreading and
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dispersive behaviors (Wagner et al., 1999; Peng et al., 2010; Lin et al., 2016), super
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dispersive mechanisms (Chengara et al., 2002; Nikolov et al., 2002; Kumar et al.,
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2006), phase behaviors (And et al., 2004) and more (Churaev et al., 2001). There are
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also studies reporting their application in pest management. Single Silwet L-77 (0.1%
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solution) was found to have high lethal bioactivity against various piercing-sucking
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mouthparts pests, such as Aphis gossypii, Frankliniella occidentalis, and Tetranychus
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pacificus (Imai et al., 1995; Tipping et al., 2003). The addition of Silwet L-77 was 3
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reported to maintain the efficacy of azinphos methyl, chlorpyrifos, and captan for
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insect and disease control on apples despite halving the pesticide dosage (Stevens et
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al., 1994).
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Studies of serial Silwet organosilicone surfactants in China have also increased
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rapidly since the cooperation of Momentive Performance Materials with the Ministry
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of Agricultural of China (the current name is the Ministry of Agriculture and Rural
70
Affairs of the People’s Republic of China) in 2006. Organosilicone is widely used as a
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tank-mix synergist in the management of Homoptera and Acariformes pests on cereal
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and horticultural plants (Qiu et al., 2006). Although single organosilicone surfactants
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have certain lethal bioactivity against small pests (such as aphids and mites) via
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mechanical respiratory inhibition or interference with important physiological
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processes, whether single organosilicone surfactants have bioactivities against larger
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pests (such as Lepidopteran pests) or their combined effects with pesticides are rarely
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reported.
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The long-term extensive use of organosilicone in agricultural production may
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also lead to environmental risks. Toxicological studies of organosilicone on aquatic
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flora and fauna have become popular research topics. Li et al. (Li et al., 2013)
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reported that Breakthru S240 had moderate acute toxicity to Daphnia magna, and the
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chronic toxicity test indicated that it can significantly affect the growth, breeding, and
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sex differentiation of D. magna, even at 0.2 mg/L. Wu et al. (Wu et al., 2009)
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evaluated the acute toxicity of four commercial organosilicone surfactants to
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Brachydanio rerio. Pesticides may also be released into the environment and persist 4
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for a long period along with surfactants and, thus, threaten the health and survival of
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various environmental organisms (Fenoll et al., 2014). The potential environmental
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risks of the binary mixtures of pesticides and surfactants should not be neglected.
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However, there have been few articles reporting the joint toxicity of organosilicone
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and pesticides to environmental organisms.
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In the current study, indoxacarb, which is a widely used oxadiazine pesticide in
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the management of Lepidopteran pests (Zlotkin, 1999), was used as a model to reveal
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the synergism of organosilicone surfactants in pest control. Then, D. magna and B.
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rerio, which are two kinds of typical aquatic organisms recommended by the OECD
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guidelines to assess the potential toxicity risks of chemicals in aquatic
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ecosystems(Barmentlo et al., 2015), were used as model organisms to determine the
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acute toxicity of single organosilicone and indoxacarb. The joint toxicity of
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organosilicone and indoxacarb to D. magna and B. rerio were also fully evaluated
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using seven commonly used methods.
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2. Materials and methods
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2.1 Test chemicals
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Four kinds of organosilicone surfactants (Silwet-408, Silwet-806, Silwet-618, and
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Silwet-DRS-60) were purchased from Momentive Performance Materials Inc.
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(Connecticut, USA). Potassium dichromate (K2Cr2O7, AR) was purchased from the
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Beijing Chemical Reagent Research Institute (Beijing, China). Indoxacarb (with a
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purity of 98%) was purchased from Rainbow Chemical Co., Ltd. (Shandong, China)
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and fabricated into a 15% stock suspension using Tween-80 aqueous solution (0.1%, 5
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w/v) for convenient use.
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2.2 Bioactivity of organosilicone+indoxacarb against pests
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The two model Lepidopteran pests used in the bioassay were selected from
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populations raised for a long period in our laboratory. The bioactivities of single
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chemicals and mixtures against Spodoptera exigua and Agrotis ipsilon were evaluated
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using standardized 3rd instar larvae. Both the contact toxicity and stomach & contact
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toxicity of the chemicals were investigated in this study.
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The contact toxicity of the chemicals was determined in accordance with the
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protocols of the immersion test by dipping the insect body (Busvine, 1980). Five or
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more concentrations of indoxacarb adopted in the official tests were determined based
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on preliminary tests. To evaluate the bioactivity of organosilicone+indoxacarb, the
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concentration of organosilicone surfactants was maintained at 0.05%, which is a
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common concentration used for tank mixing in agricultural systems. The bioactivity
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of single organosilicone against pests was also investigated. Water treatment served as
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a negative control. Briefly, twenty-four larvae were transferred to cell culture plates
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after they were dipped into the dilutions for 7 s and dried with filter paper. Then, fresh
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cabbage leaves without any contaminants were used to feed the larvae. The mortality
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of the larvae was calculated 48 h after treatment (Zhang et al., 2016). All experiments
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were repeated three times for each concentration. The synergistic ratios were
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calculated as the LC50 value of indoxacarb to that of surfactants+indoxacarb (Liu et
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al., 2011).
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Slightly different from the measurement of contact toxicity, the stomach & 6
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contact toxicity of the chemicals was determined according to the leaf immersion test
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(Zhou et al., 2011). After the preparation of serial dilutions, fresh cabbage leaves were
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dipped into the solutions for 7 s and then allowed to dry with filter paper. Then, they
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were transferred to cell culture plates, and the larvae were added to the plates. All
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experiments consisted of 24 larvae and were repeated in triplicate. Water treatment
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served as the negative control. The mortality of the larvae was calculated 48 h after
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treatment. The synergistic ratios were also calculated as the LC50 value of indoxacarb
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to that of surfactants+indoxacarb.
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2.3 Measurements of the surface tension and retention of the surfactant solutions
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The surface tension of the 0.05% organosilicone surfactant solutions was measured
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using a BZY-1 automatic surface tension meter (Shanghai Hengping Instrument and
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Meter Factory, Shanghai, China), which was repeated three times. The retention of the
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dilutions on the cabbage leaves was determined according to a previously reported
143
protocol (Yuan et al., 2000). In brief, two pieces of cabbage leaves (Φ = 1 cm) were
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dipped into the solutions for 7 s and allowed to hang vertically. The weights of the
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cabbage leaves before and after immersion were measured to calculate the weight of
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the dilutions retained on the leaves. The retention of the dilutions was calculated as
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the ratio of increased weight to the dilutions to the surface area for all of the leaves.
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The experiments were repeated six times.
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2.4 Acute single toxicity of indoxacarb and organosilicone surfactants to
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environmental organisms
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The environmental organisms, B. rerio and D. magna, belong to two typical trophic 7
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levels and, therefore, were used in the current study to evaluate the acute toxicity of
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the chemicals.
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B. rerio was cultivated in accordance with the OECD Guidelines (OECD) and
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held in groundwater (Tai’an, China; 36°15′17″N, 117°06′15″E), with a pH of 6-8, a
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total hardness of 140-250 mg/L and a dissolved oxygen concentration of
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approximately 4.0 mg/L, for seven days before testing. In the acute toxicity test, ten
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healthy B. rerio, with body lengths of 2.3±0.3 cm, were exposed to serial dilutions
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(more than five concentrations based on a preliminary test) containing indoxacarb or
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organosilicone surfactants. The exposure duration was 96 h, and the mortality of B.
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rerio was recorded at 24, 48, 72 and 96 h. The groundwater treatment served as a
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negative control. The toxicity of potassium dichromate to B. rerio was also
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investigated as a reference test. Subsequently, the chemical concentrations that killed
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fifty percent of the fish were calculated by the probit regression of the B. rerio
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mortality against the log10 values of the chemical concentrations using SPSS. All
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experiments were repeated in triplicate.
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D. magna is a pure strain introduced by the National Institute of Environmental
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Health at the Chinese Center for Disease Control and Prevention (Beijing, China) and
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is cultivated in accordance with the protocols recommended in the OECD guidelines
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(OECD, 2004). Briefly, these organisms were held in groundwater and fed daily with
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1 mL of algae at a concentration of approximately 3×105 cells/ml (Fan et al., 2017).
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Similar to the toxicity test of B. rerio, ten neonatal D. magna (ages less than 24 h)
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were exposed to these serial dilutions containing toxicants. The immobilization of D. 8
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magna was identified as the inability of D. magna to swim after 15 s of gentle
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agitation and recorded at 48 h to calculate the EC50 value. Groundwater without
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contamination served as the control. The toxicity of potassium dichromate to D.
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magna was also investigated as a reference test. All treatments were repeated three
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times.
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2.5 Acute joint toxicity of indoxacarb and organosilicone surfactants to
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environmental organisms
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The joint toxicities of organosilicone surfactants and indoxacarb to B. rerio and D.
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magna were fully evaluated by seven commonly used methods, including the additive
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index method (Marking, 1977; Li et al., 2018b; Wu et al., 2018), concentration
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addition method (W, 2000; Loureiro et al., 2009; Li et al., 2018a), V-value method
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(He and Xiong, 1994), toxicity unit method (Brown, 1968; Sprague, 1970;
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Nirmalakhandan et al., 1997), Smyth’s method (Keplinger and Deichmann, 1967),
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mixture toxicity index method (Könemann, 1981; March, 1987; Mori et al., 2015) and
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equilibrium curve method (also known as the isobologram method) (Altenburger et
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al., 1990; De Liguoro et al., 2009). The concentrations of each toxicant used in the
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joint toxicity test were designed based on their acute single toxicities to B. rerio and
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D. magna. Joint concentrations were determined based on one toxic unit, which was
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defined as the 1:1 ratio of EC50 to (A) indoxacarb or (B) organosilicone surfactants.
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The serial toxicant concentrations were determined as 0.125 × (EC50-A + EC50-B), 0.25
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× (EC50-A + EC50-B), 0.50 × (EC50-A + EC50-B), 0.75 × (EC50-A + EC50-B) and 1.00 ×
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(EC50-A + EC50-B) to calculate the joint toxicity of the mixtures (Li et al., 2018a). 9
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2.6 Data analysis
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The LC50 or EC50 values were calculated by the probit regression of the mortality of S.
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exigua, A. ipsilon, B. rerio or the D. magna immobilization rates against the log10
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values of the toxicant concentrations. All of the statistical analyses were carried out
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using the SPSS statistical package (v17.0, USA). The surface tension and retention of
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the surfactant solutions were compared via Tukey’s test at the p < 0.05 level and
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displayed as the mean ± standard deviation.
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3. Results and analysis
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3.1 Bioactivity of organosilicone+indoxacarb against S. exigua and A. ipsilon
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The bioactivity of single organosilicone surfactants against S. exigua and A. ipsilon
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was assessed prior to that of organosilicone+indoxacarb. The results showed that all
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of the tested organosilicone surfactants had no lethal bioactivities against S. exigua or
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A. ipsilon, even at concentrations two folds greater than the fortified concentrations in
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the tested dilutions. Their physical activities were also not visually affected. In terms
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of the immersion of the insect bodies, all of the tested organosilicone surfactants had
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obvious synergistic effects on the contact toxicity of indoxacarb to S. exigua and A.
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ipsilon, although the synergistic ratios for the control of the two pests were partially
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inconsistent (Figure 1a and c). For the bioassay, which was measured by exposing
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pests to poisonous leaves, both stomach toxicity and contact toxicity worked. As
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depicted in Figure 1b, all of the organosilicone surfactants exhibited certain
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antagonism for indoxacarb against S. exigua, with the lowest synergistic ratios of 0.25
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and 0.24 for Silwet-806 and Silwet-DRS-60, respectively. However, the surfactants 10
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against A. ipsilon were much different (Figure 1d). Both Silwet-408 and Silwet-806
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exhibited additive effects, whereas Silwet-618 exhibited synergism and Silwet-DRS-
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60 exhibited slight antagonism.
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3.2 Single toxicities of organosilicone surfactants and indoxacarb to D. magna
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The acute toxicity of potassium dichromate to D. magna was investigated as a
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reference prior to the toxicity test of the organosilicone surfactants and indoxacarb.
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As shown in Table 1, the 24-h EC50 value of potassium dichromate against D. magna
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was 1.282 mg/L, which indicated the favorable physiological status of D. magna and
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suitability for the toxicity test (Boillot and Perrodin, 2008). Then, single toxicities of
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organosilicone surfactants and indoxacarb to D. magna were evaluated. As displayed
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in Table 2, all of the tested chemicals were highly toxic to D. magna. Among them,
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Silwet-DRS-60 had the lowest acute toxicity to D. magna, with an EC50 value of
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94.91 µg/L, whereas Silwet-618 had the highest acute toxicity (EC50 value of 4.53
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µg/L). The typical toxic symptoms included motility reduction and bleached bodies of
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D. magna.
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3.3 Joint toxicities of organosilicone surfactants and indoxacarb to D. magna
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The joint toxicities of organosilicone surfactants and indoxacarb to D. magna were
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fully evaluated by the additive index method, concentration addition method, V-value
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method, toxicity unit method, Smyth’s method, mixture toxicity index method and
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equilibrium curve method, as depicted in Figure 2. The additive index method
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(Figure 2a), toxicity unit method (Figure 2d), and mixture toxicity index method
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(Figure 2f) showed highly consistent results in assessing the combined effects of 11
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organosilicone surfactants and indoxacarb, where all of the combined effects
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exhibited synergism. Silwet-DRS-60 had the lowest synergism when enhancing the
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toxicity of indoxacarb to D. magna, while the synergisms of Silwet-408 and Silwet-
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618 were much higher. However, in terms of the concentration addition method
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(Figure 2b) and V-value method (Figure 2c), the combined effects were much
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different. Silwet-408 showed the highest synergism, followed by Silwet-806 and
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Silwet-618, whereas Silwet-DRS-60 exhibited slight antagonism or simple addition
247
characteristics. Smyth’s method (Figure 2e) and the equilibrium curve method
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(Figures 2g, h, i) exhibited similar trends when evaluating the joint toxicity of
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organosilicone surfactants and indoxacarb. Both of these methods were more
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conservative than the other methods when identifying synergism, as they had much
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larger confidence intervals. The combined effects of Silwet-806+indoxacarb and
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Silwet-DRS-60+indoxacarb were both identified as additive effects (Figures 2e, h, j),
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whereas Silwet-408+indoxacarb (Figures 2e, g) and Silwet-618+indoxacarb (Figures
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2e, i) exhibited synergism.
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3.4 Single toxicities of organosilicone surfactants and indoxacarb to B. rerio
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The B. rerio juvenile fish were also used as a model to assess the toxicities of
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organosilicone surfactants and indoxacarb at a higher trophic level for comprehensive
258
consideration. The acute toxicity of potassium dichromate to B. rerio was initially
259
investigated as a reference. As displayed in Table 1, the EC50 value of potassium
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dichromate against B. rerio was 283.5 (259.4-309.2) mg/L, which indicated the
261
favorable physiological status of B. rerio and suitability for the toxicity test. As 12
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displayed in Figure 3a, Silwet-DRS-60 was slightly toxic to B. rerio (LC50 values of
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60.61-96.51 mg/L), whereas the other tested organosilicone surfactants were
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moderately toxic to B. rerio, with LC50 values of 5.61-6.93, 3.89-4.45 and 6.47-7.20
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mg/L for Silwet-408, Silwet-806 and Silwet-618, respectively, when the
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measurements were conducted 24, 48, 72 and 96 h after chemical exposure.
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Apparently, the single toxicities of all tested organosilicone surfactants calculated at
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different time nodes had no significant differences. In addition, the single toxicities of
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the chemicals did not change greatly, even for doses of LC20 and LC10 (Figures 3b,
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c). However, indoxacarb was highly toxic to B. rerio, regardless of the test or doses
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(LC10, LC20 or LC50) used.
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3.5 Joint toxicities of organosilicone surfactants and indoxacarb to B. rerio
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The joint toxicities of organosilicone surfactants and indoxacarb to B. rerio were
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largely different those to D. magna. As depicted in Figure 4, all of the combined
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effects of organosilicone surfactants and indoxacarb against B. rerio were determined
276
as exhibiting antagonism when using the additive index method (Figure 4a),
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concentration addition method (Figure 4b), and toxicity unit method (Figure 4d).
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The evaluation methods with confidence intervals were much different in identifying
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antagonism. In terms of the V-value method, Silwet-408+indoxacarb and Silwet-
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618+indoxacarb displayed additive effects against B. rerio, whereas Silwet-
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806+indoxacarb and Silwet-DRS-60+indoxacarb still exhibited antagonism (Figure
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4c). However, only the combined effect of Silwet-618 and indoxacarb against B. rerio
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was determined as an additive effect (Figure 4i) via the equilibrium curve method, 13
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while others were identified as exhibiting antagonism (Figures 4g, h, j). In addition,
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evaluation methods with larger confidence intervals, such as Smyth’s method (Figure
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4e) and the mixture toxicity index method (Figure 4f), were much more conservative
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in identifying antagonism, where all of the combined effects of organosilicone
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surfactants and indoxacarb against B. rerio were identified as the additive effect.
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Overall, Silwet-806 and Silwet-DRS-60 were more likely to cause antagonism than
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Silwet-618 and Silwet-408 when combined with indoxacarb.
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4. Discussion
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In recent years, the development of synergistic techniques and synergists has paid
293
extensive attention to yielding the high use efficiency of pesticides and other
294
agrochemicals (Chang et al., 2015). The key approaches to increasing the use
295
efficiency of pesticides lie in the promotion of pesticide deposition on the interfaces
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of targets, the improvement in the uniform distribution of pesticide dilutions, the
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regulation of the release profiles of pesticide formulations, and the enhancement of
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pesticide transport into the inner parts of targets (Yuan et al., 2000; Cao et al., 2014).
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Surfactants with favorable wettability, spreadability, and penetrability are always
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added as premixed or tank-mixed agents to assist in depositing pesticides onto targets,
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adhering to biological interfaces, and retaining and penetrating compact cell
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membrane barriers (Baur et al., 1997; Baur et al., 1999; Burton and Bhushan, 2006).
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With the help of surfactants, the use efficiency and control efficacy of pesticides have
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become highly encouraged in pest management (Antonious and Saito, 1981;
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Querejeta et al., 2012). 14
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Among them, organosilicone surfactants are one of the most highly consumed
307
synergists in agricultural production due to their synergistic effects on pesticides in
308
the management of agricultural pests. In the current study, we found that
309
organosilicone surfactants of the same class had apparently different synergistic
310
effects when controlling Lepidopteran pests (e.g., S. exigua and A. ipsilon), among
311
which Silwet-618 and Silwet-408 had much higher synergistic effects, and Silwet-
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DRS-60 had very small synergistic effects (via the contact toxicity test; Figure 1a, c).
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However, there were certain antagonistic effects for all of the mixtures against S.
314
exigua and partial mixtures against A. ipsilon in terms of the stomach & contact
315
toxicities, which were measured by dipping cabbage leaves into the toxicant solutions
316
and then inoculating them with pests. Indeed, the surface tension of the organosilicone
317
solutions significantly decreased compared with that of the water treatment, as
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displayed in Figure 5a. The retention of dilutions containing indoxacarb on the
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cabbage leaves also increased for all organosilicone treatments (Figure 5b). This
320
indicated that there were more toxicants on the cabbage leaves. However, why were
321
there certain antagonistic effects for all of the mixtures against S. exigua and partial
322
mixtures against A. ipsilon? We hypothesized that the decreased mobility of pests and
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the reduced consumption of the poisonous cabbage leaves could be key factors. We
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observed that the pests were less likely to move on the leaves after toxication,
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especially for the organosilicone+indoxacarb group; thus, their exposure to pesticides
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on the surfaces of the cabbage leaves was reduced. In addition, the average poisonous
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leaf consumption of S. exigua and A. ipsilon in the organosilicone+indoxacarb group 15
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was much smaller than that in the single indoxacarb groups. We have used the
329
average leaf consumption of S. exigua and A. ipsilon for a dose of LC50 for each
330
mixture as verification. As shown in Figure 5c, the average poisonous leaf
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consumptions of S. exigua and A. ipsilon in the organosilicone+indoxacarb groups
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were both significantly lower than those of the single indoxacarb treatments. This has
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great significance for pest management. For instance, imagine that we use the mixture
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of Silwet-618 and indoxacarb to control A. ipsilon; we would use less indoxacarb to
335
achieve the same efficacy. In addition, damage on the crops would be reduced, as A.
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ipsilon pests in the binary mixture group consume fewer leaves.
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Organosilicone surfactants exhibited much different toxicity to typical aquatic
338
organisms at different trophic levels. All of the tested chemicals were highly toxic to
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D. magna, whereas these chemicals were less toxic to B. rerio. Silwet-DRS-60 had a
340
low toxicity to B. rerio, while Silwet-408, Silwet-806 and Silwet-618 were
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moderately toxic to B. rerio. The integument of D. magna was very thin and it could
342
hardly delay the penetration of organosilicone surfactants into the body whereas B.
343
rerio had protective fish scales and thicker epidermis. This may be the main reason
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accounting for the fact that these chemicals were much toxic to D. magna than B.
345
rerio. It was also consistent with a previous review which reported that acute toxicity
346
tests with D. magna respond to a larger variety of chemicals with a higher sensitivity
347
than the acute toxicity tests with Danio rerio (Martins et al., 2007).
348
In this study, seven statistical methods were integrated to obtain comprehensive
349
evaluations on the combined effects of binary mixtures of organosilicone surfactants 16
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and pesticides. Overall, these methods showed similar trends in evaluating the joint
351
toxicity of binary mixtures, excluding the differences in the identification of
352
synergism, additivity or antagonism. Until now, many quantitative methods have been
353
proposed to evaluate the joint toxicity of chemicals (Lydy and Austin, 2004; Belden et
354
al., 2007; Li et al., 2018b); however, extensively applicable models and high
355
prediction accuracies were always scarce. In the current study, the additive index
356
method, concentration addition method and toxicity unit method were robust in
357
identifying synergism or antagonism, as they had no buffers (except for a critical
358
decision point), whereas the other methods, which had large buffers or confidence
359
intervals, were more conservative because quite a large proportion of the joint toxicity
360
was identified as additivity. Several previous studies have proposed the use of
361
endpoints equal to 0.5 and 2.0 to classify the toxicity unit and concentration addition
362
methods (Deneer, 2000; Boillot and Perrodin, 2008). However, we suggested the use
363
of a toxicity unit of 0.5-1.5 based on the additivity results in this study, which was
364
consistent with the recommendation of the European Inland Fisheries Advisory
365
Commission (EIFAC, 1980). In terms of the additive index method, we proposed a
366
cut-off of 0±0.2 to define the additive effect and reduce deviation or bias. In
367
summary, the V-value method and equilibrium curve method exhibited high
368
robustness and viability in evaluating the combined effects of binary mixtures.
369
Therefore, they deserve more attention in the assessment of joint toxicity.
370
In addition, the synergism of chemical mixtures against pests tends to be
371
consistent with their ecotoxicological risks to environmental organisms. In other 17
ACCEPTED MANUSCRIPT 372
words, surfactants with high wettability, spreadability and penetrability are more
373
likely to synergize pesticides in terms of pest management, and they may also cause
374
high toxicological risks or toxicity to other creatures. Thus, we should carefully select
375
organosilicone or other surfactants for premixed or tank-mixed pesticides in
376
agricultural production. Setting isolation belts and implementing other strategies are
377
imperative to reduce the discharge of various agrochemicals to nontarget organisms.
378
5. Conclusion
379
All of the tested organosilicone surfactants had obvious synergistic effects on the
380
contact toxicity of indoxacarb to S. exigua and A. ipsilon. However, all of the
381
organosilicone surfactants exhibited certain antagonistic effects for indoxacarb against
382
S. exigua in terms of stomach & contact toxicity; both Silwet-408 and Silwet-806
383
exhibited additive effects against A. ipsilon, whereas Silwet-618 and Silwet-DRS-60
384
exhibited synergism and slight antagonism, respectively. All of the tested chemicals
385
were highly toxic to D. magna, among which Silwet-DRS-60 had the lowest acute
386
toxicity (EC50 value of 94.91 µg/L). Silwet-DRS-60 was slightly toxic to B. rerio
387
(LC50 values of 60.61-96.51 mg/L), whereas other tested organosilicone surfactants
388
were moderately toxic to B. rerio, with LC50 values of 5.61-6.93, 3.89-4.45 and 6.47-
389
7.20 mg/L for Silwet-408, Silwet-806 and Silwet-618, respectively. The additive
390
index method, concentration addition method and toxicity unit method were robust in
391
identifying the synergism or antagonism of joint toxicity organosilicone surfactants
392
and indoxacarb to D. magna and B. rerio, whereas other methods were more
393
conservative. The V-value method and equilibrium curve method exhibited high 18
ACCEPTED MANUSCRIPT 394
robustness and viability in evaluating the combined effects of binary mixtures.
395
Overall, the current study would deepen the concept to assess the introduction of
396
chemicals into agriculture and the environment from all angles. And we should
397
carefully select organosilicone or other surfactants for premixed or tank-mixed
398
pesticides in agricultural production to obtain a balance between synergistic effects on
399
pests and environmental risks.
400
Acknowledgments
401
This work was supported by grants from the National Key R&D Program of China
402
(2017YFD0200307) and the National Natural Science Foundation of China
403
(31772203).
404
Declaration of interest statement
405
The authors declare no competing financial interests.
406
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Table 1 The acute toxicity of potassium dichromate to Daphnia magna and
574
Brachydanio rerio EC50 and 95% confidence Organism
Time (h)
Regression
R2
interval (mg/L) D. magna
24
1.282 (0.943-1.874)
Y=2.832X-0.306
0.955
B. rerio
24
283.5 (259.4-309.2)
Y=10.32X-25.32
0.936
575 576 577 578
Table 2 Single toxicity of indoxacarb or organosilicone surfactants to Daphnia
579
magna EC50 and 95% confidence Chemicals
Regression
R2
Toxicity
interval (µg/L) Indoxacarb
23.72 (12.53-38.68)
Y=1.105X+1.791
0.941
Highly toxic
Silwet-408
31.84 (12.19-100.7)
Y=0.537X+0.804
0.854
Highly toxic
Silwet-806
15.04 (5.888-41.16)
Y=0.561X+1.022
0.907
Highly toxic
Silwet-618
3.598 (1.312-8.874)
Y=0.586X+1.426
0.948
Highly toxic
Silwet-DRS-60
94.91 (27.56-603.1)
Y=0.681X+0.697
0.961
Highly toxic
28
ACCEPTED MANUSCRIPT 581
Figure captions
582
Figure 1 Contact toxicity (a) and stomach+contact toxicity (b) of indoxacarb and
583
organosilicone surfactants to Spodoptera exigua. Contact toxicity (c) and
584
stomach+contact toxicity (d) of indoxacarb and organosilicone surfactants to Agrotis
585
ipsilon. The synergistic ratios below the lower dotted lines (synergistic ratio<0.80),
586
above the upper dotted lines (synergistic ratio>1.20) and between the dotted lines
587
(0.80
588
effect, respectively.
589 590
Figure 2 Joint toxicity (at the EC50 level) of indoxacarb and organosilicone
591
surfactants to Daphnia magna evaluated by the (a) additive index method, (b)
592
concentration addition method, (c) V-value method, (d) toxicity unit method, (e)
593
Smyth’s method, (f) mixture toxicity index method, and (g-i) equilibrium curve
594
method. The term Ind represents indoxacarb, while S408, S806, S618 and DRS-60
595
represent Silwet-408, Silwet-806, Silwet-618, and Silwet-DRS-60, respectively.
596 597
Figure 3 Single toxicity of indoxacarb or organosilicone surfactants to Brachydanio
598
rerio. (a) LC50 values, (b) LC20 values and (c) LC10 values.
599 600
Figure 4 Joint toxicity (at the LC50 level) of indoxacarb and organosilicone
601
surfactants to Brachydanio rerio evaluated by the (a) additive index method, (b)
602
concentration addition method, (c) V-value method, (d) toxicity unit method, (e) 29
ACCEPTED MANUSCRIPT 603
Smyth’s method, (f) mixture toxicity index method, and (g-i) equilibrium curve
604
method. The term Ind represents indoxacarb, while S408, S806, S618 and DRS-60
605
represent Silwet-408, Silwet-806, Silwet-618, and Silwet-DRS-60, respectively.
606 607
Figure 5 (a) Surface tension of the organosilicone solutions. (b) Retention of the
608
dilutions containing indoxacarb on cabbage leaves. (c) The average leaf consumption
609
of Spodoptera exigua and Agrotis ipsilon at a dose of LC50 for each mixture. The data
610
are displayed as the mean ± standard deviation, and those with different lowercase
611
letters are significantly different at the p < 0.05 level by Tukey’s test.
30
ACCEPTED MANUSCRIPT Highlights
Organosilicone were synergistic on the contact toxicity of indoxacarb.
Organosilicone were highly toxic to D. magna but less toxic to B. rerio.
Additive index, concentration addition and toxicity unit were robust.
V-value and equilibrium curve were conservative in the joint toxicity evaluation.