Environment International 77 (2015) 95–105

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Environment International
journal homepage: www.elsevier.com/locate/envint

The synergistic toxicity of the multiple chemical mixtures: Implications

for risk assessment in the terrestrial environment
Chen Chen a,1, Yanhua Wang b,1, Yongzhong Qian a,⁎, Xueping Zhao b, Qiang Wang b
a
Key Laboratory of Agro-Product Quality and Safety of Ministry of Agriculture, Institute of Quality Standards and Testing Technology for Agro-Products,
Chinese Academy of Agricultural Sciences, Beijing 100081, China
b
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control/Key Laboratory for Pesticide Residue Detection of Ministry of Agriculture,
Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

a r t i c l e i n f o a b s t r a c t

Article history: The combined toxicity of five insecticides (chlorpyrifos, avermectin, imidacloprid, λ-cyhalothrin, and phoxim), two
Received 9 July 2014 herbicides (atrazine and butachlor) and a heavy metal (cadmium) has been examined with the earthworm acute
Received in revised form 11 November 2014 toxicity test. Toxicological interactions of these chemicals in four, five, six, seven, and eight-component mixtures
Accepted 5 January 2015
were studied using the combination-index (CI) equation method. In four-component and five-component mix-
Available online xxxx
tures, the synergistic effects predominated at lower effect levels, while the patterns of interactions found in six,
Keywords:
seven, and eight-component mixtures displayed synergism. The λ-CY + IMI + BUT + ATR + CPF + PHO combi-
Insecticides nation displayed the most strongly synergistic interaction, with CI values ranging from 0.09 to 0.15. The nature of
Herbicides the interaction changes with the effect level and the relevance of synergistic effects increase with the complexity of
Heavy metal the mixture. The CI method was compared with the classical models of concentration addition (CA) and indepen-
Eisenia fetida dent action (IA) and we found that the CI method could accurately predict the combined toxicity. The predicted
Acute mixture toxicity synergism resulted from co-existence of the pesticides and the heavy metal especially at low effect levels may
Combination index have important implications in risk assessment for the real terrestrial environment.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Pesticides and heavy metals are two types of pollutants that are
commonly present in the terrestrial environment (J.H. Wang et al.,
2012). Insecticides and herbicides are commonly used in agricultural
fields to protect crops and improve the quality and quantity of the
harvest. Their co-application is commonplace where herbicides are
used to control weeds and insecticides to control insect pests
(Choung et al., 2013). On the other hand, the release of heavy metals
into the environment, mainly as a consequence of anthropogenic ac-
tivities, also constitutes a potential risk for unintended adverse health
impacts to both humans and non-target wildlife (Laetz et al., 2009;
Soares and Soares, 2012). These pollutants have been detected simulta-
neously in agricultural lands, resulting in complex exposure scenarios
for soil-dwelling organisms such as earthworms.
Traditional effect and risk assessment have been routinely focused
on exposures to single chemicals and additive behaviors, which may
underestimate the risk associated with toxic action of mixtures (Barata
et al., 2006). Assessing the combined (additive, synergistic or antago-
nistic) toxicity of pollutant mixtures has therefore been an enduring
⁎ Corresponding author.
E-mail address: qyzcaas@aliyun.com (Y. Qian).
1
Chen Chen and Yanhua Wang contributed equally to this study.
challenge for environmental health and ecotoxicology for the past
several decades (Laetz et al., 2009). Recently, there are an increasing
number of studies dealing with mixtures of contaminants (Jin-Clark
et al., 2002; Munkegaard et al., 2008; Gomez-Eyles et al., 2009;
Rodea-Palomares et al., 2010; Mehler et al., 2011; Bjergager et al.,
2012; Harabawy and Ibrahim, 2014). However, only few studies have
been conducted on the toxicity of mixtures to soil invertebrates (e.g.
Anderson and Lydy, 2002; Amorim et al., 2012; Santos et al., 2010;
Schnug et al., 2014), despite soils being the primary target for pesticides
applied in agriculture. Besides, there are not many reports in the litera-
ture which have studied the nature of interactions of multi-component
chemical mixtures. Hence, in order to investigate the toxicity of multi-
component chemical mixtures on soil-dwelling invertebrates, more
studies are needed (Amorim et al., 2012).
In the present study, we investigated the ecotoxicity of the five
insecticides, two herbicides, and a heavy metal to the earthworm Eisenia
fetida which is an important component in improving the soil structure
of the terrestrial ecosystem (Fourie et al., 2007). Atrazine (ATR) is a
triazine-ring herbicide, known to act as an endocrine disruptor in
frogs and fish (Hecker et al., 2005). Chlorpyrifos (CPF) and phoxim
(PHO) are organophosphorus cholinesterase inhibitors after metab-
olism, allowing acetylcholine to accumulate at cholinergic synapses
(IPCS, 1989). Imidacloprid (IMI) is a relatively new systemic insecticide
that acts as an agonist at nicotinic acetylcholine receptors (nAChRs)

http://dx.doi.org/10.1016/j.envint.2015.01.014
0160-4120/© 2015 Elsevier Ltd. All rights reserved.
96 C. Chen et al. / Environment International 77 (2015) 95–105
(Anatra-Cordone and Durkin, 2005). The pyrethroid insecticide
λ-cyhalothrin (λ-CY) can interfere with sodium ion movement
through the nerve membrane causing hyperactivity of the nervous sys-
tem (Clark and Brooks, 1989). Butachlor (BUT) is a chloroacetanilide
herbicide for the control of grasses and weeds that acts by inhibiting
elongase responsible for the elongation of very long-chain fatty acids
and geranylgeranyl pyrophosphate cyclisation enzymes (Böger et al.,
2000). Avermectin (AVM) is used for insect control, acting as a partial
agonist at a GABA-binding site. Cadmium (Cd) could be accumulated
in the granules in the chloragogenous tissue surrounding the digestive
tract and in the nephridia of earthworms, causing demographic and
reproductive abnormalities (Fourie et al., 2007). The above herbicides
and insecticides are being commonly used in agriculture in China,
where herbicides are used to control weeds and insecticides are
co-applied to control insect pests. Moreover, Cds are widespread occu-
pational and environmental toxicants with high acute ecotoxicity and
recognized to be the most hazardous chemicals to various ecosystems.
These chemicals were selected due to their different modes of action,
usage frequency, or the content in terrestrial environment.
The main objective of the present study was to evaluate the acute
toxicity of the combined effects of the multi-component chemical mix-
tures on E. fetida in an artificial soil test. In order to identify and quantify
the nature of interactions among the chemicals, we tested four, five, six,
seven and eight-component mixtures of these chemicals by the method
of the combination index equation which has recently been used to
study the nature of chemical interactions (Rodea-Palomares et al.,
2010; Rosal et al., 2010; Boltes et al., 2012; Rodea-Palomares et al.,
2012; González-Pleiter et al., 2013).
2. Materials and methods
2.1. Test organisms
E. fetida is an invertebrate currently used for ecotoxicological assess-
ment of substances in soil and is the test species recommended by the
Organization for Economic Co-operation and Development (OECD)
and International Standardization Organization (ISO) (OECD, 1984,
2004; ISO, 1993). Adult earthworms weighing between 350 and
500 mg with well-developed clitella were purchased from the Animal
Sciences College, Zhejiang University, China, and cultured in the labora-
tory in artificial soil according to the OECD guidelines (OECD, 1984,
2004). Soils were mixed with decayed leaves and decomposed pig ma-
nure, and kept at room temperature (20 ± 1 °C). The water content of
the soil was measured weekly and the moisture was adjusted to 35%
of the maximum water-holding capacity by adding distilled water.
2.2. Test chemicals
Chlorpyrifos (CAS2921-88-2; 96% TC) was purchased from Yangnong
Agrochemical Group (Yangzhou, Jiangsu, China). Atrazine (CAS 1912-
24-9; 96% TC), imidacloprid (CAS 138261-41-3; 105827-78-9; 97% TC)
and avermectin (CAS 71751-41-2; 95.3%) were donated by Red Sun
Chemical Co. Ltd. (Nanjing, Jiangsu, China). Lambda-cyhalothrin (CAS
91465-08-6; 97% TC) was obtained from Changlong Chemical Industrial
Group (Changzhou, Jiangsu, China). CdCl2·2.5H2O (CAS10108-64-2;
99% active ingredient) was purchased from Jinshanting New Chemical
Industrial Group (Shanghai, China). Butachlor (CAS 23184-66-9; 95%
TC) was provided by Qingfeng Chemical Industrial Group (Hangzhou,
Zhejiang, China). Phoxim (CAS 14816-18-3; 95% TC) was gifted from
Liben Chemical Co. Ltd. (Lianyungang, Jiangsu, China).
Stock and working stock solutions of each chemical were prepared
insolvent (insecticides and herbicides were dissolved in analytical-
grade acetone, 99% purity; CdCl2·2.5H2O was dissolved in distilled
water) and stored in a refrigerator at 4 °C. The stock solutions were
stored for up to 1 month. All working stock solutions were made imme-
diately before use.
2.3. Acute toxicity test in artificial soil
Mortality rate was chosen as the endpoint, which is the most com-
mon acute toxicity test used to evaluate chemical effects on the tested
organisms in laboratories (Y. Wang et al., 2012). Artificial soil used for
the toxicity test consisted of 10% ground sphagnum peat (b 0.5 mm),
20% kaolinite clay (N 45% kaolinite) and 70% fine sand (OECD, 1984).
In the toxicity tests, a small amount of calcium carbonate was added
to adjust the pH to 6.0 ± 0.5 and the water content was adjusted to
35% of the maximum water-holding capacity. For each tested concen-
tration, the desired amount of the chemical was dissolved in 10 mL sol-
vent and mixed into a small quantity of fine quartz sand. The sand was
mixed for at least 1 h to evaporate the acetone and then mixed thor-
oughly with the premoistened artificial soil in a household mixer. The
final moisture contents of artificial soil were adjusted to the descried
level by the addition of distilled water. A total of 0.65 kg soil (including
0.5 kg dry artificial soil and 150 mL distilled water) was placed in a
500 mL glass jar (surface area = 63.6 cm2). Ten adult earthworms
were added to each jar. Controls were prepared similarly but only
with 10 mL solvent and no chemical. The jars were loosely covered
with polypropylene lids, allowing exchange of air, and stored at 20 ±
1 °C with 80–85% relative humidity under 400–800 lx of constant
light. Survival was assessed 14 d after the treatment.
Preliminary studies were conducted to determine the range of con-
centrations that caused 0–100% mortality for each chemical. Six test
concentrations in a geometric series and a control were used to obtain
the LC50 value. Toxicity tests were conducted in triplicate for each con-
centration. The earthworms were preconditioned for 24 h under the
same conditions described above in the untreated soil before the
dose–response test. Survival in the controls was 100% in all earthworm
tests.
2.4. Experimental design of chemical mixtures
Solutions of chlorpyrifos (CPF), atrazine (ATR), butachlor (BUT),
λ-cyhalothrin (λ-CY), imidacloprid (IMI), avermectin (AVM), phoxim
(PHO) and CdCl2·2.5H2O (Cd) prepared as described above were used
in fifteen four-component mixtures (including combinations of
λ-CY + PHO + BUT + IMI; ATR + PHO + BUT + λ-CY; λ-
CY + PHO + BUT + CPF; λ-CY + PHO + BUT + AVM;
λ-CY + PHO + BUT + Cd; λ-CY + IMI + ATR + CPF;
ATR + AVM + λ-CY + IMI; IMI + Cd + λ-CY + ATR;
BUT + λ-CY + IMI + ATR; BUT + λ-CY + IMI + CPF;
BUT + IMI + λ-CY + AVM; λ-CY + IMI + Cd + BUT;
AVM + IMI + ATR + CPF; λ-CY + Cd + ATR + CPF;
ATR + AVM + CPF + Cd), ten five-component mixtures (includ-
ing combinations of λ-CY + IMI + BUT + ATR + PHO, λ-
CY + IMI + BUT + AVM + PHO, λ-CY + IMI + BUT + CPF + PHO,
λ-CY + IMI + Cd + BUT + PHO, λ-CY + IMI + BUT + ATR + CPF, λ-
CY + IMI + BUT + ATR + AVM, λ-CY + IMI + Cd + BUT + ATR, λ-
CY + IMI + ATR + CPF + AVM, λ-CY + IMI + Cd + ATR + CPF,
IMI + Cd + ATR + CPF + AVM), six six-component mixtures (includ-
ing combinations of λ-CY + IMI + BUT + ATR + CPF + PHO,
λ-CY + IMI + BUT + ATR + AVM + PHO, λ-CY +
IMI + Cd + BUT + ATR + PHO, λ-CY + IMI + Cd +
BUT + ATR + CPF, λ-CY + IMI + BUT + ATR + CPF + AVM,
λ-CY + IMI + Cd + ATR + CPF + AVM), three seven-component
mixtures (including combinations of λ-CY + IMI + BUT +
ATR + CPF + AVM + PHO, λ-CY + IMI + Cd + BUT +
ATR + CPF + PHO, λ-CY + IMI + Cd + BUT + ATR + CPF + AVM),
one eight-component mixture (the combination of λ-CY + IMI +
Cd + BUT + ATR + CPF + AVM + PHO). Earthworms were treated
with serial dilutions of each chemical with a fixed equi-toxic constant
mixture ratio (the same effect came from each individual chemical),
based on the measured individual LC50 values. Five to seven dilutions
(with a serial dilution factor of 2) of each chemical with their four,
 C. Chen et al. / Environment International 77 (2015) 95–105 97

five, six, seven, and eight-component combinations were tested.
Besides, a control was tested in three independent experiments
with replicate samples.
2.5. Chemical measurements in soil samples
2.5.1. Chlorpyrifos and butachlor
Soil sample (approximately 20 g) was taken from each test container
both before the organisms were added and after the organism samples
were removed at the end of the test. A total of 5 g moist soil was trans-
ferred to a 50 mL polytetrafluoroethylene tube, and 5 g anhydrous
MgSO4 was then added to dry the sample. Acetonitrile (10 mL) was
used for extraction, and triphenyl phosphate (1 μg mL−1) was added
as internal standard. The samples were shaken for 16 h and then centri-
fuged at 8000 g for 8 min. Subsequently, an aliquot was analyzed by gas
chromatography (GC, Varian CP-3800). The limit of quantification was
0.02 μg mL− 1, and the average recovery for the spike levels ranged
from 80% to 120%. In addition, 15 g of soil of each replicate was dried
at 105 °C for 12 h to determine the water content of the samples. All
concentrations were normalized by dry soil weight.
2.5.2. Atrazine, avermectin, lambda-cyhalothrin, imidacloprid and phoxim
Approximately 10 g of soil sample was collected and placed in coni-
cal flasks with glass stoppers, and the obtained samples were extracted
using a 30 mL solution containing methylene chloride and acetone (6:1,
v:v) for three times under mechanical vibration for 120 min. The pooled
soil extracts (approximately 80 mL) were then centrifuged at 10,000 g
for 5 min at 4 °C. The extract was concentrated to near dryness by rotary
vacuum evaporator and recovered in acetonitrile, and its final volume
was adjusted to exactly 2 mL. The extract was stored in amber glass
vials at − 20 °C prior to further analysis. High-performance liquid
chromatography (HPLC, Agilent 1200) coupled with a reversed-phase
column was used to analyze the soil extracts. Pesticides were isocratically
eluted with acetonitrile and then quantified by a diode array detector at a
wavelength of 200–300 nm. The recovery rate was estimated by adding
1 mL methanol containing pure pesticide (1 μg mL−1) to soil. The

Table 1
Dose–effect relationship parameters and mean combination index (CI) values of multi-component mixtures on Eisenia fetida acute toxicity test.

Multi-component mixtures Dose–effect parameters CI values

−1
Dm (mg kg ) m r LC10 LC50 LC90

Four-component mixtures
λ-CY + IMI + BUT + PHO 52.3 2.65 ± 0.53 0.945 0.06 Syn 0.09 Syn 0.14 Syn
λ-CY + BUT + ATR + PHO 80.8 3.50 ± 1.17 0.904 0.11 Syn 0.12 Syn 0.14 Syn
λ-CY + BUT + CPF + PHO 67.3 2.86 ± 0.55 0.950 0.07 Syn 0.09 Syn 0.13 Syn
λ-CY + BUT + AVM + PHO 70.0 3.44 ± 0.46 0.974 0.09 Syn 0.11 Syn 0.13 Syn
λ-CY + Cd + BUT + PHO 105 3.38 ± 0.48 0.971 0.08 Syn 0.11 Syn 0.13 Syn
λ-CY + IMI + ATR + CPF 32.9 3.92 ± 0.81 0.960 0.11 Syn 0.13 Syn 0.16 Syn
λ-CY + IMI + ATR + AVM 113 2.46 ± 0.40 0.951 0.39 Syn 0.65 Syn 1.10 Add
λ-CY + IMI + Cd + ATR 422 3.54 ± 0.65 0.968 1.87 Ant 2.56 Ant 3.53 Ant
λ-CY + IMI + BUT + ATR 172 3.13 ± 0.24 0.991 0.33 Syn 0.44 Syn 0.58 Syn
λ-CY + IMI + BUT + CPF 146 3.56 ± 0.28 0.991 0.25 Syn 0.31 Syn 0.40 Syn
λ-CY + IMI + BUT + AVM 233 2.41 ± 0.31 0.968 0.34 Syn 0.58 Syn 0.99 Add
λ-CY + IMI + Cd + BUT 494 3.22 ± 0.33 0.984 0.48 Syn 0.64 Syn 0.88 Syn
IMI + ATR + CPF + AVM 98.2 2.92 ± 0.47 0.963 0.44 Syn 0.68 Syn 1.06 Add
IMI + Cd + ATR + CPF 633 4.17 ± 0.24 0.997 1.05 Add 1.30 Ant 1.62 Ant
Cd + ATR + CPF + AVM 396 3.28 ± 0.57 0.958 0.56 Syn 0.80 Syn 1.16 Ant
% of synergistic mixtures 87% 87% 60%

Five-component mixtures
λ-CY + IMI + BUT + ATR + PHO 91.4 4.19 ± 0.96 0.951 0.17 Syn 0.19 Syn 0.21 Syn
λ-CY + IMI + BUT + AVM + PHO 56.9 2.68 ± 0.61 0.930 0.08 Syn 0.12 Syn 0.18 Syn
λ-CY + IMI + BUT + CPF + PHO 81.9 3.39 ± 0.54 0.963 0.12 Syn 0.16 Syn 0.20 Syn
λ-CY + IMI + Cd + BUT + PHO 107 3.65 ± 0.76 0.960 0.12 Syn 0.15 Syn 0.18 Syn
λ-CY + IMI + BUT + ATR + CPF 92.0 3.15 ± 0.48 0.967 0.17 Syn 0.22 Syn 0.31 Syn
λ-CY + IMI + BUT + ATR + AVM 342 3.16 ± 0.38 0.979 0.69 Syn 0.91 Add 1.23 Ant
λ-CY + IMI + Cd + BUT + ATR 430 3.77 ± 0.77 0.961 0.56 Syn 0.67 Syn 0.81 Syn
λ-CY + IMI + ATR + CPF + AVM 44.9 2.79 ± 0.26 0.991 0.14 Syn 0.23 Syn 0.35 Syn
λ-CY + IMI + Cd + ATR + CPF 78.2 3.88 ± 1.04 0.935 0.14 Syn 0.18 Syn 0.22 Syn
IMI + Cd + ATR + CPF + AVM 396 3.56 ± 0.35 0.985 0.80 Syn 1.10 Add 1.52 Ant
% of synergistic mixtures 100% 87% 87%

Six-component mixtures
λ-CY + IMI + BUT + ATR + CPF + PHO 55.2 4.03 ± 0.56 0.981 0.10 Syn 0.12 Syn 0.14 Syn
λ-CY + IMI + BUT + ATR + AVM + PHO 51.3 4.30 ± 0.47 0.988 0.11 Syn 0.13 Syn 0.14 Syn
λ-CY + IMI + Cd + BUT + ATR + PHO 116 3.39 ± 0.57 0.961 0.15 Syn 0.19 Syn 0.24 Syn
λ-CY + IMI + Cd + BUT + ATR + CPF 150 3.46 ± 0.33 0.986 0.21 Syn 0.27 Syn 0.35 Syn
λ-CY + IMI + BUT + ATR + CPF + AVM 119 4.38 ± 0.52 0.986 0.31 Syn 0.35 Syn 0.40 Syn
λ-CY + IMI + Cd + ATR + CPF + AVM 95.5 3.74 ± 0.22 0.997 0.20 Syn 0.25 Syn 0.33 Syn
% of synergistic mixtures 100% 100% 100%

Seven-component mixtures
λ-CY + IMI + BUT + ATR + CPF + AVM + PHO 58.2 4.30 ± 0.47 0.988 0.13 Syn 0.15 Syn 0.17 Syn
λ-CY + IMI + Cd + BUT + ATR + CPF + PHO 81.0 2.28 ± 0.19 0.993 0.08 Syn 0.14 Syn 0.25 Syn
λ-CY + IMI + Cd + BUT + ATR + CPF + PHO 130 4.67 ± 0.83 0.970 0.25 Syn 0.28 Syn 0.31 Syn
% of synergistic mixtures 100% 100% 100%

Eight-component mixtures
λ-CY + IMI + Cd + BUT + ATR + CPF + AVM + PHO 73.4 3.81 ± 0.44 0.987 0.12 Syn 0.14 Syn 0.18 Syn
% of synergistic mixtures 100% 100% 100%

The parameters m, Dm and r are the slope and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose–effect curve, the potency (LC50), and con-
formity of the data to the mass-action law, respectively (Chou and Talalay, 1984; Chou, 2006). CI b 1, CI = 1, and CI N 1 indicate synergism (Syn), additive effect (Add), and antagonism
(Ant), respectively. LC10, LC50 and LC90, are the doses required to cause 10%, 50% and 90% mortalities of the earthworms, respectively.
98 C. Chen et al. / Environment International 77 (2015) 95–105

extractions were carried out using the abovementioned procedures, and effect curve: m = 1, m N 1, and m b 1 indicate hyperbolic, sigmoidal,
the average recovery for the spike levels ranged from 80% to 120%. and negative sigmoidal dose–effect curves, respectively.
The quantification of synergism or antagonism for a combination of
2.5.3. Cadmium a series of chemicals is given by combination index (CI) values at 10%,
Soil sample was homogenized and dried at 105 °C. Briefly, 1.0 g of 50% and 90% mortality rate:
soil sample was collected, and 3 mL solution containing HNO3 and
( )
H2O2 (2:1, v:v) was added. This mixture was gently shaken and then ½D
ðDx Þ1−n Xn j

∑ ∑
dried on a hot plate. After cooling, 2 mL nitric acid (0.75 M) was n n
ðDÞ j ½D
added, and then the sample was centrifuged. The clear digests were an- ðCIÞx ¼ ¼  1
1=m j ð2Þ
alyzed using direct slotted tube atom trap flame atomic absorption ðDx Þ j ð f ax Þ j
ðDm Þ j
spectrometry (STAT-FAAS, AFS-9230) for cadmium. Blank analysis was j¼1 j¼1 ½1−ð f ax Þ j 
carried out using the same procedure.
Average measured concentrations of the tested substances in soil where n(CI)x is the combination index for n chemicals at x% mortality
both before the test start and after the completion of the test generally rate; (Dx)1 − n is the sum of the concentrations of n chemicals causing
varied less than 20% from the nominal start concentrations of each indi- x% mortality rate in mixture, [D]j/∑n1[D] is the proportionality of the
vidual chemical. Therefore, all calculations were based on nominal dose of each of n chemicals causing x% mortality rate in combination;
concentrations. and (Dm)j{(fax)j/1 − (fax)j}1/mj is the concentration of each individual
chemical causing x% mortality rate. CI b 1, CI = 1 and CI N 1 indicates
2.6. Median-effect and combination index (CI)-isobologram equations for synergism, concentration addition and antagonism, respectively.
determining individual and combined toxicities
2.7. Analysis of results
The response to toxic exposure of E. fetida in artificial soil tests was
estimated using the median-effect equation, as described by Chou and Computer program CompuSyn (Chou and Martin, 2005) was used
Talalay (1984): for the calculation of dose–effect curve parameters and CI values. The
fa–CI plot represents CI versus fa which is the fraction affected by a par-

m ticular dose.
fa D
¼ ð1Þ
fu LC 50
2.8. Mixture toxicity predictions based on CA, IA and CI equations

where D represents the dose affecting a fraction fa, LC50 is the dose Experimental toxicities of the chemical mixtures were computed
at which 50% mortality rate is reached, fu is the unaffected fraction based on the predictive equations of the two most widely used defini-
(fa = 1 − fu), and m identifies the coefficient of the shape of the dose– tions of additivity, concentration addition (CA) (Eq. (3)) and independent

a λ-CY+IMI+BUT+PHO λ-CY+BUT+ATR+PHO b λ-CY+IMI+ATR+CPF λ-CY+IMI+ATR+AVM

λ-CY+BUT+CPF+PHO λ-CY+BUT+AVM+PHO λ-CY+IMI+Cd+ATR λ-CY+IMI+BUT+ATR
0.19 4.5
4
0.17
Combination Index, CI
Combination Index, CI

3.5
0.15
3
0.13 2.5
0.11 2
1.5
0.09
1
0.07 0.5
0.05 0
0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1
Fraction affected, fa Fraction affected, fa
c IMI+ATR+CPF+AVM λ-CY+IMI+BUT+AVM d λ-CY+Cd+BUT+PHO λ-CY+IMI+BUT+CPF
λ-CY+IMI+Cd+BUT IMI+Cd+ATR+CPF Cd+ATR+CPF+AVM
2 1.6
1.8
1.4
Combination Index, CI
Combination Index, CI

1.6
1.2
1.4
1
1.2
0.8
1
0.8 0.6

0.6 0.4
0.4 0.2
0.2 0
0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1
Fraction affected, fa Fraction affected, fa

Fig. 1. Combination index plot (fa–CI plot) for four-component mixtures of the eight chemicals for the earthworm acute toxicity test. CI values are plotted as a function of the mortality rate
(fa) of the earthworms by computer simulation (CompuSyn). CI b 1, =1 and N1 indicates synergism, additive effect and antagonism, respectively. CPF = chlorpyrifos, ATR = atrazine,
BUT = butachlor, λ-CY = λ-cyhalothrin, IMI = imidacloprid, AVM = avermectin, PHO = phoxim, Cd = cadmium.
 C. Chen et al. / Environment International 77 (2015) 95–105 99

action (IA) (Eq. (4)) (Faust et al., 2001; Altenburger et al., 2004). CA is 3. Results
based on the assumption that the mixture components have the same
sites and similar mode of action (MOA), and is computed by equation: 3.1. Toxicological interactions of multi-component chemical mixtures

!−1 Table 1 summarizes the dose–effect curve parameters (Dm, m and

Xn
pi
EC x;mix ¼ ð3Þ r) of the eight chemicals in four, five, six, seven and eight-component
i¼1
ECxi mixtures using the 14-d acute toxicity test towards E. fetida; 95% confi-
dence intervals are indicated for the m parameter.
Figs. 1–2 display the fa–CI plots for the four, five, six, seven and eight-
where ECx,mix is the effect concentration of the mixture eliciting x% effect,
component mixtures of the E. fetida acute toxicity tests. The fa–CI plot
ECx,i denotes the concentration of the ith component when it exists indi-
depicts the CI value versus fa (the effect level or the mortality rate of
vidually and elicits the same effect (x%) as the mixture, pi is the relative
the earthworms with respect to the control) and shows the types of
mass proportions of the ith component in the mixture. In the present
the interaction (synergism, antagonism or additive effect) as a function
study, for survival data, simply exchange ECx with LC50 (lethal concentra-
of the level of the effect of a particular mixture on the tested organism
tion 50).
(Rodea-Palomares et al., 2010). Average CI values for three representa-
IA is based on the assumption that the components in the mixture
tive effect levels (LC10, LC50 and LC90) are also shown in Table 1. The
have dissimilar MOA. The following equation applies for IA.
individual concentrations of eight chemicals in all multi-component
mixtures can be found in the Supplemental data.
n
Eðcmix Þ ¼ 1−∏ ð1−Eðci ÞÞ ð4Þ Regarding four-component mixtures, nine out of fifteen combina-
i¼1 tions showed synergistic effects in practically the whole range of fa
values. The λ-CY + BUT + CPF + PHO combination was the most
strongly synergistic (with CI values ranging from 0.06 to 0.16 as effect
where cmix and E(cmix) are the total concentration and total effect of the
level increased) in the whole range. Four combinations resulted in
mixture, respectively. E(ci) denotes the effect of the ith component with
dual synergistic/antagonistic on the fa–CI behaviors: at low and mean
the concentration of ci in the mixture.
effect levels, the mixtures were synergistic, but the synergism signifi-
The predictive equation based on the CI values was computed as fol-
cantly decreased with increasing fa levels until it approached an additive
lows:
effect at fa levels between 0.85 and 0.9 and turned into slight antago-
!−1 nism at fa values N0.95 (e.g., λ-CY + IMI + ATR + AVM was synergistic
X
n
pi at fa levels below 0.8, additive at fa values between 0.8 and 0.9 and
EC x;mix ¼ : ð5Þ dominated by antagonism above 0.9). Two combinations exhibited in-
i¼1
EC xi  CI xcomp
creasingly antagonistic effects along with the fa range, in which the
λ-CY + IMI + Cd + ATR combination showed the most strongly antag-
CIxcomp is the computed combination index value for the mixture onistic interaction, with the CI values ranging from 1.69 to 4.26.
at the x level of effect (x%) from the experimental toxicity curve of the In the ten five-component mixtures, eight combinations showed
mixture (Chou, 2006). synergistic effects in practically the whole range of fa values. The

a λ-CY+IMI+BUT+CPF+PHO λ-CY+IMI+BUT+ATR+PHO b λ-CY+IMI+BUT+ATR+AVM λ-CY+IMI+ATR+CPF+AVM

λ-CY+IMI+BUT+AVM+PHO λ-CY+IMI+Cd+BUT+PHO IMI+Cd+ATR+CPF+AVM λ-CY+IMI+Cd+BUT+ATR
λ-CY+IMI+BUT+ATR+CPF λ-CY+IMI+Cd+ATR+CPF
0.4 2
1.8
0.35
Combination Index, CI

1.6
Combination Index, CI

0.3 1.4

0.25 1.2
1
0.2 0.8
0.15 0.6
0.4
0.1
0.2
0.05 0
0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1
Fraction affected, fa Fraction affected, fa
c λ-CY+IMI+BUT+ATR+CPF+PHO λ-CY+IMI+BUT+ATR+AVE+PHO d λ-CY+IMI+BUT+ATR+CPF+AVM+PHO
λ-CY+IMI+Cd+BUT+ATR+PHO λ-CY+IMI+Cd+BUT+ATR+CPF λ-CY+IMI+Cd+BUT+ATR+CPF+PHO
λ-CY+IMI+BUT+ATR+CPF+AVM λ-CY+IMI+Cd+ATR+CPF+AVM λ-CY+IMI+Cd+BUT+ATR+CPF+AVM
0.43 λ-CY+IMI+Cd+BUT+ATR+CPF+AVM+PHO
0.35
0.38
Combination Index, CI

0.3
Combination Index, CI

0.33
0.25
0.28
0.2
0.23
0.15
0.18

0.13 0.1

0.08 0.05
0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1
Fraction affected, fa Fraction affected, fa

Fig. 2. Combination index plot (fa–CI plot) for five-component (a, b), six-component (c), seven, and eight-component (d) mixtures of the eight chemicals for the earthworm acute toxicity
test. CI values are plotted as a function of the mortality rate (fa) of the earthworms by computer simulation (CompuSyn). CI b 1, =1 and N1 indicates synergism, additive effect and
antagonism, respectively.
100 C. Chen et al. / Environment International 77 (2015) 95–105

Fig. 3. Experimental toxicity values (Exp) and predicted dose–response curves of four-component mixtures based on concentration addition (CA), independent action (IA) and combina-
tion index (CI) models for the earthworm acute toxicity test.
 C. Chen et al. / Environment International 77 (2015) 95–105 101

Fig. 4. Experimental toxicity values (Exp) and predicted dose–response curves of five-component mixtures based on CA, IA and CI models for the earthworm acute toxicity test.
102 C. Chen et al. / Environment International 77 (2015) 95–105
λ-CY + IMI + BUT + AVM + PHO combination was the most strongly
synergistic (with the CI values ranging from 0.07 to 0.24 as the effect
level increased) in the whole range. Two combinations led to dual syn-
ergistic/antagonistic behaviors (e.g., λ-CY + IMI + BUT + ATR + AVM
mixture was synergistic at effect levels below 0.5, additive between
0.5 and 0.8 and turned into antagonism above 0.8).
All of the six six-component mixtures presented strong synergistic
effects along with the fa range, in which the λ-CY + IMI +BUT +
ATR + CPF + PHO combination displayed the most strongly synergistic
interaction, with CI values ranging from 0.09 to 0.15 as the effect level
increased. Similarly, the synergism of the three seven-component and
one eight-component mixtures was evident throughout the entire
effect level ranges but decreased with increasing effect levels. The
results suggest that the co-existence of the pesticides and the heavy
metal represents an enhanced risk to terrestrial ecosystems.
3.2. Experimental and predicted toxicity of the mixtures of chemicals under
CA, IA and CI methods
To validate the toxicological interactions of the multi-component
mixtures predicted by the combination index (CI) equation, the predict-
ed dose–response curves of the mixtures were generated based on the
Fig. 5. Experimental toxicity values (Exp) and predicted dose–response curves of six-component mixtures based on CA, IA and CI models for the earthworm acute toxicity test.
classical CA and IA models. The predicted dose–response curves of the
mixtures based on the CI values were also generated at the different fa
levels of the mixtures. Figs. 3–6 demonstrate the predicted dose–
response curves under CA, IA and CI models together with the experi-
mental values for four, five, six, seven and eight-component mixtures
in earthworm acute toxicity tests. Generally, when comparing the
experimental and predicted toxicities of all the multi-component mix-
tures based on the three models (CA, IA and CI), the predicted toxicity
of the mixtures according to CI method is closer to the experimental
toxicity values than that using CA or IA in nearly all of the multi-
component mixtures. For example, when analyzing the toxicity of the
λ-CY + IM I + BUT + PHO mixture, neither the CA nor the IA model
predicted the observed synergistic interaction, while it was accu-
rately predicted by the CI method (Fig. 3a). The CI method improved
the predictive power of the classical CA and IA models, allowing us to
achieve more accurate predictions of combined toxicity in synergis-
tic mixtures.
4. Discussion
The CA and IA are two traditional concepts that have been widely
utilized in predicting mixture toxicities (Altenburger et al., 2003;
 C. Chen et al. / Environment International 77 (2015) 95–105
Backhaus et al., 2003; Jonker et al., 2004, 2005; Loureiro et al., 2009).
These two concepts provide a reference structure to which the experi-
mental mixture toxicity could be compared (Backhaus et al., 2004).
However, the classic models for the prediction of the mixture toxicity
are based on simple assumptions on the mode of toxic action (Rodea-
Palomares et al., 2012). Chemicals with known mechanisms of action,
once released in the environment, might have diverse dose–response
relationships. CA had severe limitations when the dose–response curves
of the individual chemicals were not identical (Cleuvers, 2003). In an
extensive statistical evaluation on the accuracy of the predictive models,
only a half of the experimental toxicities of the mixtures could be cor-
rectly predicted by either CA or IA models (Cedergreen et al., 2007). Re-
cently, Rosal et al. (2010) reported an environmental application of the
median-effect/combination index (CI)-isobologram equation originally
developed by Chou (2006), which allows quantitative determinations
of chemical interactions at different concentrations and effect levels. Be-
sides, knowledge on the component–component type of interaction is
not required to assess the overall interaction of the mixture (Chou,
2006). In the present study, we determined the interaction types for a
series of effect levels of eight chemicals in multi-component mixtures
in earthworm acute toxicity test by applying this method, which im-
proved the predictive power of the CA and IA models.
An important feature of the observed multi-component mixture
toxicity is that the nature of the interaction changes with the effect
level. The percentage of mixtures representing synergism at three rep-
resentative effect levels (LC10, LC50 and LC90) was 87%, 87%, 60%, respec-
tively, in four-component mixtures, while this figure reached 100%, 87%,
and 87%, respectively, in five-component mixtures. The synergistic
effects predominated at the lower effect levels. González-Pleiter et al.
reported an antagonistic effect of the multi-component (four and
five-antibiotics) mixtures at very low to low effect levels, with syner-
gism clearly predominating at higher effect levels (González-Pleiter
et al., 2013).
Fig. 6. Experimental toxicity values (Exp) and predicted dose–response curves of seven, and eight-component mixtures based on CA, IA and CI models for the earthworm acute
toxicity test.
103
The nature of the toxicological interaction was partially influenced
by the toxicological mode of action of each component (Rodea-
Palomares et al., 2010). Atrazine contains five electron-donor atoms
that can potentially complex with Cd to form atrazine–Cd complexes
that might change the toxicity characteristics of the individual com-
pound (Meng and Carper, 2000), which might reduce the amount of
exposed atrazine and Cd to the earthworms. In the present study, two
mixtures containing atrazine and Cd showed additive or antagonistic
effects. However, the patterns of interactions found in the rest of the
multi-component (six, seven, and eight-component) mixtures cannot
be easily explained in terms of the known mechanisms of action.
These mixtures exhibited clearly synergistic toxicities in the whole
range of the effect levels. The results point out that the complexity of
the mixture tends to increase the relevance of synergistic effects.
Koutsaftis and Aoyama (2007) examined the toxicity of the mixtures
of antifouling biocides on the brine shrimp Artemia salina and synergism
was observed in the quaternary combination. Rodea-Palomares et al.
(2010) investigated a complex mixture including pharmaceuticals and
a real wastewater sample consisting of thirty micropollutants on the
cyanobacterium Anabaena CPB4337. They concluded that synergism
was the predominant interaction in a wide range of the effect level.
Petersen et al. (2014) noted that the combined effects of pharmaceuti-
cals, personal care products, biocides and organic contaminant multi-
compound mixtures on the marine algae Skeletonema pseudocostatum
conformed to the concentration addition concept. Taking into account
that CA may underestimate the actual risk, synergistic effect could still
be expected. Yang et al. (2008) studied the combined effects of 12 anti-
bacterial agents on the green alga Pseudokirchneriella subcapitata
finding that the synergistic effects predominated at low antibiotic
concentrations.
Different insecticides, herbicides, and heavy metals may co-occur in
the same terrestrial environment. The predicted synergism in the
majority of the mixtures especially at the low effect levels indicated a
104 C. Chen et al. / Environment International 77 (2015) 95–105
potential ecotoxicological risk associated with the co-occurrence of
these chemicals at environmentally relevant concentrations. This may
have important implications in the terrestrial environment.
5. Conclusions
The combination index (CI)-isobologram equation was applied to
study the nature of the multi-component interactions of five insecti-
cides, two herbicides, and a heavy metal on the earthworm E. fetida
with acute toxicity test. In the four-component and five-component
mixtures, the synergistic effects predominated at the lower effect levels.
The patterns of interactions found in the rest of the multi-component
displayed strong synergism. The relevance of synergistic effects increases
with the complexity of the mixture. The CI method could improve the
predictive power of the mixtures when compared with the classical CA
and IA models. The predicted synergism resulted from co-existence of
the pesticides and the heavy metal represents an enhanced risk to the
terrestrial ecosystem, which may have important implications in risk
assessment for the real terrestrial environment.
Acknowledgments
The research was supported by the National Natural Science
Foundation of China (Grant No. 31401767), Science and Technology
Innovation Program of the Chinese Academy of Agricultural Sciences,
and the Innovation Project of the Zhejiang Academy of Agricultural
Sciences (2014CX010).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.envint.2015.01.014.
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