chlorpyrifos mixture more harmful to earthworms than bulk ZnO? A multigeneration approach

Chemosphere 247 (2020) 125885

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Is nano ZnO/chlorpyrifos mixture more harmful to earthworms than bulk ZnO? A multigeneration approach
Lon
Z. cari c a, D.K. Hackenberger a, *, I. Jug b, B.K. Hackenberger a a b

Department of Biology, Josip Juraj Strossmayer University of Osijek Cara Hadrijana 8A, HR-31000, Osijek, Croatia Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek, Vladimira Preloga 1, HR-31000, Osijek, Croatia

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Effects of CHP/bZnO and CHP/nZnO mixtures differ depending on the soil type.
Tested mixtures had synergistic effect in artificial soil and additive effect in natural soil.
Different biomarker responses were obtained for the 1st and 2nd generation.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 July 2019 Received in revised form 6 January 2020 Accepted 9 January 2020 Available online 10 January 2020

As chlorpyrifos is one of the most widely used organophosphorus insecticides and ZnO-NPs are identified as NPs of the highest concern due to their negative effects on aquatic and soil organisms the objective of this study was to evaluate mixture toxicity of CHP and ZnO (bulk and nanoparticles (20 nm)) on two types of soil, artificial (AS) and natural (NS), and over two generations of earthworms. Primary endpoint measured was reproduction inhibition and biochemical biomarkers (acetylcholinesterase, catalase, glutathione-S transferase and malondialdehyde content). Results showed that mixture toxicity differs in respects to all tested factors: soil type, ZnO particle size and earthworm generation. CHP/ZnO mixtures had synergistic effects and significantly reduced a number of juveniles in both generations in AS, while the effects were additive or even antagonistic in NS. There was no difference in reproduction inhibition in respect to particle size of ZnO used in the mixtures. Negative effects could also be detected on growth dynamics of juvenile earthworms (2nd generation) as they had lower initial body mas, reduced growth rate and lower body mass as adults. Measured enzymes responded differently in respect to ZnO particle size used in the mixtures, with CHP/bZnO producing stronger effects. Measured concentrations of the bioavailable Zn in the soils showed no difference in the concentration of bioavailable Zn2þ between mixtures, but significantly more Zn2þ was retrieved from AS. General biomarker response indicated that 2nd generation of earthworms had lower capability to cope with oxidative stress. © 2020 Elsevier Ltd. All rights reserved.

Handling Editor: Tamara S. Galloway Keywords: Mixture toxicity Multigenerational effects Bayes nano(eco)toxicology Isobolograms

1. Introduction Earthworms are important soil organisms and may constitute

* Corresponding author. E-mail address: [email protected] (D.K. Hackenberger). https://doi.org/10.1016/j.chemosphere.2020.125885 0045-6535/© 2020 Elsevier Ltd. All rights reserved.

up to 80% of the total biomass. They live in close contact with the soil, have a permeable cuticle, and consume large amounts of soil. For those characteristics earthworms are considered as the key soil organism for ecological risk assessment. Chlorpyrifos is one of the most widely used organophosphorus insecticides in the world for both crop protection and pest control. Main mechanism of its acute toxicity is the inhibition of the enzyme

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acetylcholinesterase (AChE, EC 3.1.1.7). Its effects on non-targeted soil fauna are well investigated on both population and biochemical level. It is known that chlorpyrifos negatively affects biochemical processes/enzyme activities (Sanchez-Hernandez et al., 2014; Bednarska et al., 2017), growth and reproduction (Zhou et al. 2007, 2011) and can induce biomass loss and aestivation in earthworms (Reinecke and Reinecke, 2007). Engineered nanomaterials (ENMs) have been used for several decades and their production and usage is still increasing. Various studies showed that ENMs can increase the pollution level of air, water, and soil accumulating in the environment, and affecting the life-cycle of living systems present in the environment (Kabira et al., 2018). In particular, zinc-oxide nanoparticles (ZnO-NPs) have received a lot of attention as they are used in medicine, cosmetics, electronics and optics, coatings, paints, and pigment products. Estimated global production of ZnO-NPs is 30,000 tons per year, and it is estimated that 8700 tons per year ends up in the soil and 21,100 tons per year in the landfills (Keller et al., 2013). Predicted increase of concentration per year in the soil compartment for ZnO-NPs is 0.093 mg kg1y1 in Europe and 0.050 mg kg1y1 in the U.S (Gottschalk et al., 2009). Several authors identified ZnO-NPs as ENMs of the highest concern (Stone et al., 2009; Aschberger et al., 2011; Coll et al., 2016; Rajput et al., 2018). Negative effects of zinc (in bulk, nano and ionic form) on earthworm populations have been demonstrated. It is known that zinc can have adverse effects on biomarkers (Li et al., 2011), and can ~ as et al., induce oxidative stress and reproduction inhibition (Can mez et al., 2014). However, primary mechanisms 2011; García-Go of ZnO-NPs nanoparticles toxicity are still not clear. Even though some studies suggested that environmental toxicants such as pesticides can induce epigenetic alterations in soil organisms which can be inherited to future generations, multigenerational studies on the effects of pesticides and/or metals on soil organisms are scarce (Brunninger et al., 1994; Spurgeon and Hopkin, 2000; Langdon et al., 2009, Oliveira et al., 2018, Zhang and Qiao, 2018). Various pollutants are continuously being accumulated in environmental compartments in which they form complex mixtures. ENMs are highly reactive structures and as such they can interact with other pollutants to generate more or less toxic structures (Kabira et al., 2018). Uwizeyimana et al. (2017) showed that 60% of investigated mixtures of pesticides and heavy metals produced synergistic interactions, among which the mixtures of organophosphate insecticides and heavy metals are very likely to produce synergistic effects. The interaction between NPs and/or pesticides and the soil matrix can modify their availability due to several processes such as aggregation, release of the metal ion, oxidation, and sorption to soil components which in turn can modify their toxicity (Ahmad et al., 2001; Tourinho et al., 2012, Heggelund et al., 2014). Chemical mixtures in the environmental usually do not produce the same degree of toxicity towards organisms as individual toxicant of that mixture whose toxicity can be influenced by many factors. Although individual effects of ZnO and chlorpyrifos (CHP) have been investigated their joint toxic effects are still not known. Therefore, the main aim of this research was to investigate the toxicity of chlorpyrifos and ZnO on earthworms using equitoxic mixtures which are evaluated using different exposure media artificial soil and natural soil. Primary endpoint was reproduction inhibition e.g. a reduction in the number of juvenile earthworms. As we wanted to determine possible differences in mixture toxicity based on the particle size, two types of ZnO were tested e bulk and nano ZnO. Besides reproduction, biochemical biomarkers were measured to see if their responses can predict higher level

biomarkers such as reproduction success. And finally, to test a possible development of tolerance of earthworms to toxic effects of tested mixtures, all toxicity parameters were measured on two generations of earthworms. 2. Materials and methods 2.1. Organisms Earthworms (Dendrobaena veneta Rosa 1886) were obtained from a synchronized culture maintained in our laboratory. For all experiments adult earthworms with well-developed clitellum were used. 24 h before each experiment, earthworms were left on moist filter paper to void the gut content (OECD, 1984). 2.2. Chemicals All reagents used in the experiments were of analytical grade. For the preparation of the exposure mixtures, commercial preparation of insecticide chlorpyrifos was used e Nufos (480 g L1 chlorpyrifos), nanoparticles of zinc(II)-oxide were purchased form Iolitec, Germany (ZnO, 99.5%, 20 nm, NO-0011-HP, CAS Number 1314-13-2), and ZnO bulk (CAS Number 1314-13-2) was purchased from T.T.T. Ltd. For measurement of molecular biomarkers following chemicals were used: Sodium phosphate monobasic monohydrate (NaH2PO4x H2O, CAS Number 10049-21-5, 137.99 g mol1), Sodium phosphate dibasic anhydrous (Na2HPO4 CAS Number 7558-79-4), Hydrogen peroxide solution (H2O2 CAs Number 7722-84-1, 34.01 g mol 1), 5,50 dithiobis-2 nitrobenzoic acid (DTNB, CAS Number 69-78-3, molecular weight: 396.35 g mol1); acetylthiocholine iodide (AcSChI, CAS Number, 1866-15-5, molecular weight 289.18 g mol1); 1-chloro 2,4-dinitrobenzene (CDNB, CAS Number 97-00-7, molecular weight 202.55 g mol1); trichloroacetic acid (TCA, CAS Number 76-03-9, molecular weight 163.39 g mol1); reduced glutathione (GSH, CAS Number 70-18-8, molecular weight 307.32 g mol1); thiobarbituric acid (TBA, CAS Number 504-17-6, molecular weight 144.15 g mol1). For determination of concentration of bioavailable zinc following chemicals were used: Calcium chloride dihydrate (CaCl2x2H2O, CAS Number 10035-04-8, molecular weight 147.01 g mol1); Diethylenetriaminepentaacetic acid (DTPA, CAS Number 67-43-6, molecular weight 393.35 g mol1) and Triethanolamine (TEA, CAS Number 102-71-6, molecular weight 149.19 g mol1). 2.3. Experimental design Within this conducted.

research

three

separate

experiments

were

2.3.1. Preliminary experiment e reproduction test The main aim of the preliminary experiment was to determine concentrations of chlorpyrifos (CHP), bulk ZnO (bZnO) and ZnO nanoparticles (nZnO) that cause 50% reduction in number of juveniles after reproduction test (e.g. EC50). Reproduction test was performed according to the OECD (1984) guidelines. Artificial soil used in the experiments consisted of 70% fine quartz sand, 20% kaolinite clay and 10% organic matter (sphagnum peat), with pH adjusted to 6.0 ± 0.5 with CaCO3. 400 g of prepared soil was placed in glass jars and appropriate concentrations of chlorpyrifos were added to the test vessels. Five concentrations of CHP were used: 0.01 mg kg1DW. SOIL, 0.1 mg kg1DW. SOIL, 1 mg kg1DW. SOIL, 10 mg kg1DW. SOIL and 100 mg kg1DW. SOIL. Zinc (II) oxide (bZnO and


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nZnO) were dry mixed in artificial soil (Waalewijn-Kool et al., 2012) and after soil spiking water was added to achieve 50% of the water holding capacity. Five concentrations were used: 10 mg kg1DW. 1 1 1 SOIL, 100 mg kg DW. SOIL, 500 mg kg DW. SOIL, 1000 mg kg DW. SOIL and 5000 mg kg1DW. SOIL. For the controls only distilled water was added. After preparation of the test vessels 10 adult earthworms were placed in each vessel, and each concentration was done in triplicate. Test vessels were then placed in environmental chamber at temperature 20 ± 1  C in constant dark. Earthworms were fed with horse manure once a week. After 28 days all adult earthworms were removed and soil was left in environmental chamber for additional 28 days for cocoons to hatch. On the 56th day test vessels were immersed in a warm water bath (60 ± 5  C) for 1 h, and emerged juveniles were collected, counted and weighted (Supplementary material, S1a). 2.3.2. 1st generation mixture exposure experiment After preliminary experiment, EC50 (reproduction) values for single compounds were calculated and those concentrations were used for testing of the effects of mixtures. EC50 for CHP was 7 mg kg1DW. SOIL. bZnO and nZnO had the same EC50 of 900 mg kg1DW. SOIL. Components CHP/bZnO and CHP/nZnO are mixed in the ratio of their individual EC50 values with 5 equitoxic mixtures in five different ratios: 100%CHP:0%ZnO (M1), 75%CHP:25% ZnO (M2), 50% CHP:50%ZnO (M3), 25%CHP:75ZnO (M4), 0%CHP:100% ZnO (M5). Toxic effects of mixtures were evaluated in artificial soil (AS) as well as in natural soil (NS). Artificial soil was prepared as previously described. Natural soil was collected from an uncontaminated site in Baranja (Osijek-Baranja County) situated in the north-eastern part of the Republic of Croatia. Natural soil had following characteristics: Clay soil (30.2% sand, 27.2% silt and 42.6% clay), pH ¼ 5.77 (in water), organic matter content 4.3%. Prior to the experiment natural soil was air dried, and sieved to <2 mm. Reproduction tests (AS and NS) were conducted according to the standard protocols (OECD, 1984). 400 g of the prepared soil was put in the glass jars and appropriate mixtures of CHP/bZnO and CHP/ nZnO were mixed in the soil; for the controls only distilled water was added. After application of the mixtures soil was left to equilibrate for 6 h. For each mixture triplicates were prepared, with 10 adult earthworms in each test vessel. Prepared test vessels were left in environmental chamber at temperature 20 ± 1  C in constant dark. Earthworms were fed with horse manure once a week. After 28 days adult earthworms were collected from the test vessels, left on Petri dish on moist filter paper to void their gut content, weighed, homogenized and stored at 80  C until the measurement. Test vessels were then returned to the environmental chamber for additional 28 days. On the 56th day the number of juvenile earthworms was counted (Supplementary material S1b). After reproduction test has finished, juvenile earthworms were transferred to the clean artificial soil and left in environmental chamber until they were fully grown with well-developed clitellum. Biomass of juvenile earthworms in each vessel was measured every two weeks. After earthworms were fully grown, they were exposed to the same mixtures as the adult earthworms (2nd generation experiment). 2.3.3. Reproduction test - 2nd generation experiment Reproduction test on the second generation of earthworms was performed in the same manner and with the same concentrations as the 1st generation experiment, only in the artificial soil (Supplementary material, S2a). 2.4. Sample preparation and measurement of molecular biomarkers Earthworms were weighted and homogenized with addition of

3

ice cold sodium phosphate buffer (0.1 M, pH ¼ 7.4) in ratio 1:5 (w:v). Homogenization was performed using Omni Tissue Homogenizer. After homogenization part of the homogenate (200 mL) was used for measurement of TBARS and the rest of the homogenate was centrifuged at 9000g for 30 min at 4  C to obtain postmitochondrial fraction (S9). Aliquots of the S9 fraction were stored at 80  C until use. Acetylcholinesterase (AChE) activity was measured according to the method described by Ellman et al. (1961), catalase (CAT) activity was determined by method of Claiborne (1985) and glutathione-S transferase (GST) activity was measured by method of Habig et al. (1974). All three were measured spectrophotometrically. Lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances, following method described by Gagne (2014) fluorometrically. All enzyme activities as well as MDA content are express as relative activities compared to its respective control. 2.5. Concentration of bioavailable zinc and chlorpyrifos The concentration of bioavailable Zn in artificial and natural soil was determined by extraction method with a DTPA solution, according to the ISO 14870: 2001 - Soil quality – Extraction of trace elements by buffered DTPA solution. Zinc content in the soil was determined with the AAS methods using the atomic absorption spectrophotometer (Shimadzu Scientific Instruments/AA-7000). Chlorpyrifos (CHP) concentrations in soil were measured with a gas chromatography. Total CHP concentrations were measured only at the beginning of the experiment and were in agreement with the nominal ones. 2.6. Statistical analysis and data presentation Data analysis was performed using the statistical software R version 3.5.0 (R Core Team, 2018) and R Studio Team (2016). All data were first tested for normality using ShapiroeWilk test, and homogeneity of variance was confirmed with Bartlett’s test. As no significant differences from normality were detected all data were analyzed using three-way ANOVA. Dataset was separated in two groups. In the first group of data the differences in biomarker responses between two ZnO present particle sizes used in the mixtures (bZnO and nZnO) and between first and second generation of earthworms were tested. And in the second group of data, the differences in biomarker responses between artificial soil (AS) and natural soil (NS) and two particle sizes of the ZnO used in the mixtures were tested. If significant differences were detected after three-way ANOVA, pairwise contrasts were evaluated to detect which groups are statistically significant (poct-hoc test) with package emmeans (Lenth, 2018). Linear regression was used to compare the relationship between ZnO concentration and soil DTPA-extractable Zn (e.g. bioavailable Zn). For analysis of differences in DTPA-Zn concentration between different soil types and ZnO forms package lsmeans was used (Lenth, 2016). For curve fitting, calculation of EC50 values for single exposure experiments and isobologram construction package drc (Ritz et al., 2015) was used. Dose response curves for single toxicants (CHP and ZnO) were fitted to the three-parameter logistic model. To evaluate toxic interactions (additivity, synergy or antagonism) of binary equitoxic mixtures, isobolographic method was used. Mixture toxicity was evaluated based on the toxic unit (TU) concept (Warne, 2003; Brodeur et al., 2014). Here, one toxic unit (TU) is defined as a concentration of toxicant that cause 50% reduction in the number of juvenile earthworms after reproduction test (EC50). In the case of the mixtures, for example, value of 1 TU contains 0.5 TU of CHP (1/2

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of EC50) and 0.5 TU of ZnO (1/2 EC50). Line of additivity that connects dose pairs that cause EC50 effect was added to the isobologram, to enable visual contrast of additive from synergistic and antagonistic interactions. For every mixture ratio combination index (CI) was calculated as a standard measure of the combination effects. CI ¼ 1 implies additive effect, CI < 1 means synergism and CI > 1 means antagonistic effect. All the models during dose response fitting were chosen based on lack-of-fit test and according to AIC criteria. To compare the differences in growth dynamics of juvenile earthworms Bayes approach was used. Analysis of growth curves was done using package babar (Rickett et al., 2015). Data on earthworm mass (mass per earthworm) were first fitted to 4 parameter Baranyi model (Baranyi et al., 1993) available within babar package. Juvenile earthworm growth curve consists of a lag phase, then a steep exponential linear phase and when they reach maturity they usually go through a stationary phase (Mathieu, 2018). Similarly, 4 parameter model of Baranyi describes bacterial growth which consist of the same growth phases. Parameters of the model are: y0 - initial mass of juvenile earthworms, ymax maximum final weight of earthworms; stationary phase, mmax maximum specific growth rate and h0 ¼ l mmax, where l is the lag time. As the model gave good fit to the data, to compare differences in growth dynamics of juveniles, each fitted curve was compared to the normal growth curve (e.g. control treatment). For comparison of two growth curves function Bayescompare() was used. Within this function three different hypothesis can be tested: first hypothesis (H1) is that two curves are replicates and therefore the same set of parameters can be used to describe each curve; second

hypothesis (H2) is that the two curves have the same growth rate (mmax) but they differ in all other parameters and the third hypothesis (H3) is that the curves have no common parameters. Result of comparison of two hypothesis is Bayes factor which quantifies how well a hypothesis predicts the empirical data relative to a competing hypothesis. For example, BF10 (H1 over H0) of 2 means that empirical data is 2 times more probable if H1 were true than if H0 were true. For the analysis of differences in growth curves Bayes factor BF31 (H3 over H1) with uniform distribution as prior was calculated and interpreted according to the modified Jeffreys’ scale (Jeffreys, 1961 modified by Lee and Wagenmakers, 2014).

3. Results 3.1. Biomarker activities in response to mixture, ZnO particle size and soil type All treatments significantly inhibited AChE activity compared to the control treatment (Fig. 1A). Three-way ANOVA showed significant effects of all three tested factors; mixture (M, p < 0.001), ZnO particle size (Z, p < 0.001) and soil type (S, p < 0.001), as well as significant two-way interaction of mixture and ZnO particle size (M x Z, p < 0.01), and mixture and soil type (M x S, p < 0.001) (Table 1A). AChE inhibition was stronger in CHP/bZnO mixtures with significant differences at M3, M4 and M5 (p < 0.001) (S2Fig. 1). Significant differences in AChE activities were also detected between AS and NS at mixtures with higher ratio of CHP; M1 (p < 0.001) and M2 (p < 0.01) (S2-Fig. 2).

Fig. 1. Differences in biomarker responses of A) AChE, B) GST, C) CAT and D) TBARS in Dendrobaena veneta after exposure to the binary mixtures of CHP/bZnO and CHP/nZnO in artificial soil (AS) and natural soil (NS). All activities are expressed as relative values; dotted line represents the control value. Results are presented as mean ± standard deviation. Mixture of CHP/nZnO in NS ( ), CHP/bZnO in NS ( ), CHP/nZnO in AS ( ), CHP/bZnO in AS ( ). Detail graphical presentation of the results of three-way ANOVA are presented in S1.


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Table 1 Three-way ANOVA analysis of the effects of binary mixtures of CHP/ZnO on molecular biomarkers (AChE, GST, CAT and MDA) in earthworm Dendroabena veneta after reproduction test. A) F values for the three-way ANOVA of the effects of mixture (M), ZnO particle size (Z) and soil type (S). B) F values for the three-way ANOVA of the effects of mixture (M), ZnO particle size (Z) and earthworm generation (G). Statistically significant differences are marked with asterisk. A

Mixture (M) Particle size (Z) Soil type (S) MxZ MxS ZxS MxZxS

B

df

AChE

GST

CAT

MDA

5 1 1 5 5 1 5

75.28*** 42.46*** 18.1*** 4.03** 6.59*** 0.06 1.15

6.44*** 3.99* 32.99*** 4.25*** 7.48*** 1.62 1.45

5.75*** 4.21* 111.09*** 6.08*** 6.63*** 19.94*** 2.35*

6.4*** 12.21*** 33.57*** 4.11** 9.67*** 40.51*** 3.85**

The activity of CAT varied between treatments (Fig. 1C). A threeway ANOVA showed significant effects of all three main factors; mixture (M, p < 0.001), ZnO particle size (Z, p < 0.05) and soil type (S, p < 0.001), significant two-way interaction of mixture and ZnO particle size (M x Z, p < 0.001), mixture and soil type (M x S, p < 0.001), ZnO particle size and soil type (Z x S, p < 0.001) as well as a significant three-way interaction of all three tested factors (M x Z x S, p < 0.05) (Table 1A). The interaction of M x Z showed a significant difference in CAT activity between two ZnO particle sizes at mixture ratio M2 (S2-Fig. 5). Significantly different responses in CAT activity were detected between AS and NS at all tested mixtures (p < 0.001) (S2-Fig. 6). Additionally, CAT activities increased in AS at all mixtures except M4, while in NS, CAT activities decreased compared to the control at all mixtures. In AS different responses of CAT activities were detected between CHP/bZnO and CHP/nZnO mixtures (p < 0.001) and no such difference in CAT activities was observed in NS (S2-Fig. 7). Changes in GST activity in response to applied mixtures of CHP/ ZnO and soil type are presented in Fig. 1B. A three-way ANOVA showed significant effects of all three tested factors (M, p < 0.001; Z, p < 0.05; S, p < 0.001) and significant two-way interactions of applied mixture and soil type (M x S, p < 0.001), and mixture and ZnO particle size (M x Z, p < 0.001) (Table 1A). Different responses of GST activity were detected between CHP/bZnO and CHP/nZnO mixtures at M4 in which GST activity had stronger increase in CHP/ nZnO mixture (p < 0.001) (17.6%) (S2-Fig. 10). Two-way interaction of applied mixture and soil type shows significant differences in GST activities between AS and NS with a stronger increase of GST activity in NS (S2-Fig. 11). MDA concentrations in response to applied mixture and soil type are presented in Fig. 1D. A three-way ANOVA showed significant main effects of all three tested factors (M, p < 0.001; Z, p < 0.001; S, p < 0.001) and significant two-way interactions of mixture and soil type (M x S, p < 0.01); mixture and ZnO particle size (M x Z, p < 0.001) as well as significant two-way interaction of ZnO particle size and soil type (Z x S, p < 0.001). A three-way interaction was also significant (M x Z x S, p < 0.01) (Table 1A). Interestingly, mixtures with only CHP (M1) and ZnO (M5) did not cause any changes in MDA concentration levels compared to the control treatments (S2-Fig. 15) when tested in NS (S2-Fig. 17). MDA concentrations differed between CHP/bZnO and CHP/nZnO mixtures depending on the soil type used for the test. (S2-Fig. 16). 3.2. Biomarker activities in response to mixture, ZnO particle size and earthworm generation All applied treatments significantly inhibited AChE activity compared to the control treatment in both generations (Fig. 2A). Three-way ANOVA showed significant influence of the two main factors; applied mixture (M, p < 0.001) and ZnO particle size (Z,

Mixture (M) Particle size (Z) Generation (G) MxZ MxG ZxG MxZxG

df

AChE

GST

CAT

MDA

5 1 1 5 5 1 5

89.50*** 34.05*** 0.17 2.287* 8.61*** 0.86 0.67

2.31* 6.68* 27.77** 2.89* 6.16*** 44.41*** 5.87***

12.08*** 2.38 187.79*** 1.81 12.32*** 22.63*** 4.97***

8.33*** 8.85** 31.53*** 2.13 2.79* 0.45 0.19

p < 0.001). However, there was no significant difference for the generation (G) factor. Significant two-way interaction of applied mixture and ZnO particle size (M x Z, p < 0.05), and applied mixture and earthworm generation (M x G; p < 0.001) was also detected (Table 1B). Again, a stronger inhibition of AChE was detected when earthworms were exposed to CHP/bZnO mixtures (S2-Fig. 3). CHP caused a stronger inhibition of AChE activity in the 1st generation in M1 and M2 mixtures, There was no difference between 1st and 2nd generation when exposed to the pure bZnO or nZnO (M5) (S2Fig. 4). CAT activities in response to applied mixtures, ZnO particle size and earthworm generation are presented in Fig. 2C. Three-way ANOVA showed significant main effects of applied mixture (M, p < 0.001) and earthworm generation (G, p < 0.001); significant two-way interactions of mixture and earthworm generation (M x G, p < 0.001), and ZnO particle size and earthworm generation (Z x G, p < 0.001), as well as significant three-way interaction of all three tested factors (M x Z x G, p < 0.001) (Table 1B). Significant differences in CAT activities between 1st and 2nd generation were detected at all tested mixtures (p < 0.001) (S2-Fig. 8). In general CAT activities increased or remained at the control levels in 1st generation, while in the 2nd generation CAT activities decreased compared to the control. Different responses of CAT activity between 1st and 2nd generation were detected between CHP/bZnO and CHP/nZnO mixtures (both p < 0.001) (S2-Fig. 9). GST activities in response of applied mixtures, ZnO particle size and earthworm generation are presented in Fig. 2B. A three-way ANOVA showed significant effects of all three main factors (M, p < 0.05; Z, p < 0.05; G, p < 0.001), significant two-way interaction of mixture and ZnO particle size (M x Z, p < 0.05); mixture and earthworm generation (M x G, p < 0.001) and also significant threeway interaction of all three tested factors (M x Z x G, p < 0.001) (Table 1B). The GST activity increased in the 1st generation compared to the control and compared to the 2nd generation of earthworms at mixture ratios with a higher percentages of CHP (M1, p < 0.001; M2, p < 0.001 and M3, p < 0.001) (S2-Fig. 8A). Mixtures M4 and M5, in CHP/bZnO treatment in the 1st generation caused a GST activity to remainat control levels, and to increase in the 2nd generation. Contrary, in CHP/nZnO treatments in the 1st generation of earthworms GST increased, and in second generation GST activity decreased (S2-Fig. 8B). MDA concentrations in response to mixtures and earthworm generation are presented in Fig. 2D. A significantly different concentrations of MDA between mixtures were detected between 1st and 2nd generation of earthworms. A three-way ANOVA showed significant effects of all three main factors (M, p < 0.001; S, p < 0.01 and G, p < 0.001) and significant two way integration of mixture and earthworm generation (M x G, p < 0.05) (Table 1B). Significantly different MDA concentrations between earthworm generations were detected at all mixtures, except M5, with generally higher MDA concentrations observed in the 2nd generation (S2-

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Fig. 2. Differences in biomarker responses of A) AChE, B) GST, C) CAT and D) TBARS in Dendrobaena veneta after exposure to the binary mixtures of CHP/bZnO and CHP/nZnO in the first and second generation of earthworms. All activities are expressed as relative values; dotted line represents the control value. Results are presented as mean ± standard deviation. Mixture of CHP/nZnO, 1st generation ( ), CHP/bZnO, 1st generation ( ), CHP/nZnO 2nd generation ( ), CHP/bZnO 2nd generation. Detail graphical presentation of the results of three-way ANOVA are presented in S1.

Fig. 18). The highest difference in MDA concentration between 1st and 2nd generation was detected at M3 with 27.7% higher MDA concentration in the 1st generation.

3.3. Reproduction test The effects of binary mixtures of CHP/nZnO and CHP/bZnO on earthworm reproduction are presented in Fig. 3. Combination index (CI) is calculated for each mixture and is reported at EC50 value as CI ± SE with corresponding p value showing significant difference from additive effect (CIs1). The same mixture had different effects in AS and NS in the 1st generation, while the effects of mixtures were consistent between the 1st and the 2nd generation. In AS (1st generation), both equitoxic mixtures of CHP/bZnO and CHP/nZnO, had synergistic effect (Fig. 3A and B), while in NS effects were additive or antagonistic (Fig. 3C and D). Values of the CI for the 1st generation in AS for CHP/ bZnO mixtures were all significantly smaller than 1, indicating synergism: CI(M2) 0.70 ± 0.04 (p < 0.001), CI(M3) 0.85 ± 0.05 (p < 0.01), CI(M4) 0.83 ± 0.04 (p < 0.001). CI for CHP/nZnO mixtures were also significantly smaller than 1 also indicating synergism; CI(M2) 0.35 ± 0.02 (p < 0.001), CI(M3) 0.69 ± 0.03 (p < 0.01), CI(M4) 0.65 ± 0.31 (p < 0.001). In NS synergistic effects could not be detected in CHP/bZnO or CHP/nZnO mixtures. In CHP/bZnO mixtures CI at mixtures M3 and M4 was not significantly different from additive effect, but CI(M2) 1.1 ± 0.05 (p < 0.01) indicate a significant antagonistic effect (Fig. 3C). In CHP/nZnO mixtures, similar effects could be observed, with only CI(M3) 1.14 ± 0.05 (p < 0.05) showing significant antagonism, and M2 and M4 not significantly different

from additive effect (Fig. 3D). In the 2nd generation effects of CHP/ bZnO and CHP/nZnO mixtures were also synergistic, (Fig. 3E and F). In CHP/bZnO mixtures, CI was significantly smaller than 1 for all ratios indicating synergism; CI(M2) 0.96 ± 0.04 (p < 0.001), CI(M3) 0.77 ± 0.06 (p < 0.001). Exception was M4 where the effect was additive (CI 0.96 ± 0.04) (Fig. 3E). In CHP/nZnO mixtures effects were synergistic with CI(M4) 0.74 ± 0.05 (p < 0.001), CI(M3) 0.40 ± 0.03 (p < 0.001) and CI(M4) 0.90 ± 0.03 (p < 0.001) (Fig. 3F).

3.4. Growth dynamics of juvenile earthworms Results of Bayesian growth dynamics analysis of juvenile earthworms are presented in Table 2. After exposure of the adult earthworms (1st generation) to the mixture of CHP/bZnO at M3, M4 and M5, juvenile earthworms (2nd generation) had significantly lower initial body mass (y0), lower final mass (ymax) and reduced maximum specific growth rate (mmax) compared to the juvenile earthworms in the control treatment. The exposure of adult earthworms (1st generation) to the mixture of CHP/nZnO had significant effects on growth dynamics of juveniles (2nd generation) at M2, M3 and M4. Juvenile earthworms also had significantly lower initial body mass (y0), lower final mass (ymax) and reduced maximum specific growth rate (mmax) compared to the juvenile earthworms in the control treatment. Contrary to the expected, when significant result is obtained (difference in dynamics compared to the control) lag phase in juveniles was shortened compared to the control for 1e7 days, except at the M4 when lag phase was prolonged for 11 days. M1 corresponding to the EC50 of CHP did not have significant effect on the growth dynamics of

Fig. 3. Isobologram of the effects of CHP/bZnO and CHP/nZnO mixtures on reproduction success of Dendrobaena veneta in AS and NS, and 1st and 2nd earthworm generation. Dotted lines are showing the estimated ED50 values ± SE (solid line segments) for every mixture ratio. The theoretical additivity line (presented as a full line) connects the experimental EC50 points for single components of the mixtures. A) Isobologram showing reproduction success of D. veneta (1st generation of earthworms) after exposure to CHP/bZnO mixture in AS. B) Isobologram showing reproduction success of D. veneta (1st generation of earthworms) after exposure to CHP/nZnO mixture in AS. C) Isobologram showing reproduction success of D. veneta (1st generation of earthworms) after exposure to CHP/bZnO mixture in NS. D) Isobologram showing reproduction success of D. veneta (1st generation of earthworms) after exposure to CHP/nZnO mixture in NS. E) Isobologram showing reproduction success of D. veneta (2nd generation of earthworms) after exposure to CHP/bZnO mixture in AS. F) Isobologram showing reproduction success of D. veneta (2nd generation of earthworms) after exposure to CHP/nZnO mixture in AS. Significant differences from additive effect are marked with asterisk e *p < 0.05, **p < 0.01, ***p < 0.001.


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Table 2 Bayesian analysis of growth dynamics of juvenile earthworms e comparison of parameters of 4 parameter model of Baranyi: y0 - initial mass of juvenile earthworms, ymax emaximum final weight of earthworms (stationary phase), mmax e maximum specific growth rate and h0 ¼ l mmax, where l is the lag time. BF31 (H3 over H1) indicating evidence for H3 (curves have no common parameters). ZnO

Mixture

y0

mmax

ymax

0.063 ± 0.022

0.988 ± 0.091

l

h0

0.017 ± 0.003

2.513 ± 0.431

Jeffery’s scale

145.3 ± 7.3

BF31

Label

Bulk

100% CHP: 0% 75% CHP: 25% 50% CHP: 50% 25% CHP: 75% 0% CHP: 100%

ZnO ZnO ZnO ZnO ZnO

M1 M2 M3 M4 M5

0.056 0.056 0.031 0.047 0.040

± ± ± ± ±

0.016 0.019 0.011 0.011 0.012

0.832 0.951 0.539 0.723 0.634

± ± ± ± ±

0.069 0.082 0.042 0.054 0.050

0.015 0.016 0.015 0.015 0.013

± ± ± ± ±

0.002 0.002 0.002 0.002 0.002

2.179 2.145 2.066 2.192 1.795

± ± ± ± ±

0.329 0.372 0.302 0.308 0.264

144.7 136.7 134.2 145.5 141.8

± ± ± ± ±

5.6 6.8 5.5 5.4 5.6

<1 <1 >100 12.6 >100

No evidence No evidence Extreme evidence Strong evidence Extreme evidence

Nano

100% CHP: 0% 75% CHP: 25% 50% CHP: 50% 25% CHP: 75% 0% CHP: 100%

ZnO ZnO ZnO ZnO ZnO

M1 M2 M3 M4 M5

0.056 0.046 0.033 0.038 0.058

± ± ± ± ±

0.019 0.013 0.013 0.009 0.019

0.773 0.692 0.702 0.714 0.847

± ± ± ± ±

0.085 0.063 0.073 0.044 0.083

0.015 0.014 0.014 0.014 0.015

± ± ± ± ±

0.003 0.002 0.002 0.001 0.002

2.125 1.978 1.832 2.030 2.167

± ± ± ± ±

0.467 0.319 0.299 0.233 0.391

142.9 142.5 135.2 146.2 140.7

± ± ± ± ±

12.2 6.7 7.1 3.6 7.7

<1 33.2 4.14 16.16 <1

No evidence Very strong evidence Moderate evidence Strong evidence No evidence

Control

juvenile earthworms, as well as the mixture M5 corresponding to the EC50 value of nZnO. 3.5. Concentration of bioavailable zinc Concentrations of soil DTPA-extractable Zn (bioavailable Zn) increased linearly with increasing concentrations of the bZnO and nZnO in the soils (Fig. 4). There were no significant differences in the soil DTPA-extractable Zn concentrations between bZnO and nZnO, however there were significant differences in retrieved zinc concentrations between AS and NS (p < 0.001). In general, significantly more bioavailable Zn could be retrieved from the AS than from the NS. In the AS 60.4 ± 3.2% of the zinc from the nZnO was converted to into DTPA-extractable form (e.g. bioavailable form) and 55.3 ± 2.4% from the bZnO. In the NS 38.1 ± 5.2% of nZnO and 35.7 ± 5.9% of bZnO of Zn was converted into bioavailable form. 4. Discussion The most consistent response that remained same at all tested factors was inhibition of AChE. Even though, the mechanism for acute toxicity of OP insecticides such as chlorpyrifos is the inhibition of the enzyme acetylcholinesterase, AChE was also inhibited by M5 corresponding to EC50 (reproduction) of pure bZnO and nZnO, and also by all mixture ratios of CHP/ZnO. It is known that some metals can inhibit AChE activity (de Lima et al., 2013), yet mechanism of this inhibition is still unknown. Metals can bind to

Fig. 4. Soil DTPA-extractable Zn concentrations released from the bZnO and nZnO and extracted from the AS and NS. Different letters (a, b) indicate significant differences between the slopes (p < 0.05).

functional groups of the protein which can cause decrease in enzyme activity or loss of function (Najimi et al., 1997), or sometimes stimulate AChE activity at low metal concentrations (Sant’Anna et al., 2011). de Lima et al. (2013) showed in vitro that if high metal concentrations are present, and if metals are in direct contact with the enzyme, inhibitory effects of metals are more due to the denaturation of the enzyme than to the binding of metals to specific target site. However, in vivo inhibition of esterases by metals, is attributed to the indirect effects (physiological changes) and not direct mechanism of action of the metal in the enzyme. Responses of other enzymes used in this study should be commented in the contexts of the differences among tested factors obtained by statistical analysis. Different responses of measured biomarkers were observed depending on the soil type used in the experiments with, in general, stronger effects in AS at mixtures with higher ratio of CHP (M1 and M2). There are several possible explanations for this difference in toxicity among AS and NS. It is known that main soil properties affecting bioavailability and degradation of pesticides in the soil are organic matter (SOM), quantity and type of clay, moisture content and pH. Yu et al. (2006) demonstrated positive correlation between SOM content and soil adsorption capacity for chlorpyrifos, so it is expected that higher SOM content would reduce toxicity of CHP. In our experiments however, SOM content between AS were NS was similar, 6.9% and 4.3% respectively, but the amount of clay in the NS was twice the amount of clay in AS (20% in the AS and 42.6% in the NS). This indicates that clay content should be considered as the main factor explaining observed difference in toxicity of CHP. Chlorpyrifos has a high affinity for soil (Yu et al., 2006; Murray et al., 2001) with lower bioavailability and lower bioaccumulation factor in the soil with higher TOC and clay content, where higher SOM content reduced the availability of CHP and higher clay content contributed to faster  degradation of CHP due to clay-catalysed hydrolysis (Svobodova et al., 2018). Several authors demonstrated that increase in clay content increases chlorpyrifos hydrolysis (Getzin, 1981; Awasthi and Prakash, 1997; Svobodov a et al., 2018), and, therefore, reduces its bioavailability in the soil. For the mixtures M3, M4 and M5 differences in biochemical biomarkers responses between soil types were unambiguous. Namely, there was no statistically significant differences in the AChE activity between the soil types, induction of CAT was significantly higher in AS, but GST activities were more strongly induced in NS (M4 and M5). NPs, as well as pesticides, undergo various transformation processes when released into the environment. Those transformations are results of processes such as aggregation/agglomeration, dissolution, complexation and different types of reactions with biological macromolecules (Maurer-Jones et al., 2013). All those processes can


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affect the transport, fate and finally the toxic properties of NPs in the environment. Soil properties such as soil organic content (SOM) can decrease toxicity of ZnO NPs and reduce oxidative stress levels in earthworms (Mwaanga et al., 2017). In addition, soil pH, cation exchange capacity (CEC), soil moisture content can also affect toxicity of NPs, however SOM and clay content are considered as main soil properties responsible for modulating toxicity and bioavailability of NPs (Moghaddasi et al., 2017; Zhang et al., 2017). Differences in biomarker responses were obtained depending on ZnO particle size used in the mixtures. In general, when significantly different responses between biomarkers were detected, CHP/bZnO mixtures produced stronger effects. Different toxic effects of the tested mixtures with respect to the ZnO particle size indicate the complexity and number of confounding factors present in natural environments, which hinders the determination of the exact causes of toxicity, especially for nanoparticles. Toxic effects of metal oxides, particularly those that are present as nanoparticles, are usually attributed to dissolution e.g. the release of cations from the metal oxides (Franklin et al., 2007; Bondarenko et al., 2016). Our results from DTPA-extractable Zn (e.g. bioavailable Zn), show that both types of mixtures (CHP/bZnO and CHP/nZnO) in the same soil type released the same amounts of ionic zinc, so causes of observed differences in toxicity should be attributed to some other factors. Similar results were obtained by Xiong et al. (2011) who showed that metal ions released by bZnO and nZnO contributed to toxic effects on zebrafish but were not the main lethal mechanism. Various environmental factors can modify toxicity of nanoparticles. Dissolution of ZnO NPs is enhanced in soil extracts, but at the same time interaction of NPs with soil matrix changes their bioavailability usually by reducing its toxicity (Campbell, 1994). Also, intracellular distribution of particulate ZnO and dissolved zinc ion is different and the toxicity depends on the uptake route; dermal or ingestion (Li et al., 2011). Li et al. (2011) showed that ionic zinc is distributed mainly in cell membranes and tissues (75% of total accumulated zinc) and zinc from NPs is mainly stored in organelles and cytosol (76% of total accumulated zinc). Dominant uptake routes are also concentration dependent (Laycock et al., 2015), and different mechanisms are responsible for the regulation of Zn concentrations in animals exposed to different forms of metals  ˛ tek et al., 2017). Taking into account all of the above (Swia mentioned factors, does not fully explain the observed differences in toxicity of bZnO and nZnO, with, contrary to other research, bZnO containing mixtures producing stronger oxidative stress. Stronger toxic effects of CHP/bZnO mixtures could be, at least partially, explained by different dynamics of dissolution which would then lead to different biomarker responses, as it is known that biomarker responses are not only concentration but also time dependent (Tennekes and Sanchez-Bayo, 2011). Yang and Xie (2006) investigated dynamics of dissolution of different ZnO particles and showed that release of Zn2þ from nanoparticles is significantly higher during initial several days (approx. 8 days) while the release of Zn2þ from bulk ZnO is initially slower, and then more constant during longer periods of time. Higher initial release of Zn2þ could move the equilibrium towards complexation of Zn2þ cations with soil organic matter which would, in the end, produce weaker toxic effects in earthworms exposed to mixtures containing nZnO. On the other hand, there was no difference in the effect on the reproduction in respect to particle size of ZnO used in the mixtures. However, the response was different in AS and NS. Synergistic effects of the applied mixtures were observed in AS, while in NS response was no different from additive or was even antagonistic (M4 of CHP/bZnO and M3 of CHP/nZnO). In the AS, in both

9

generations of earthworms, CHP/bZnO and CHP/nZnO mixtures had synergistic effects and significantly reduced the number of juveniles. Furthermore, negative effects of some mixtures were persistent and negative effects could be detected by monitoring growth dynamics of juveniles (2nd gen.). They had lower initial body mass, lower growth rate and final weight compared to the control earthworms. Some research suggests adaptation mechanism in earthworms as a results of long term exposure to pesticide residues (Givaudan et al., 2014) and metals (Fisker et al., 2013). Research of Brunninger et al. (1994) showed that earthworms exposed to terbuthylazine significantly increased their biomass compared to the control, and second generation of earthworms had grown significantly faster as juveniles and consequently produced more cocoons than the controls after reaching maturity. Also, research of Spurgeon and Hopkin (2000) in which earthworms E. fetida were exposed to zinc LC99 for parent worms resulted in the highest survival for F2 worms with the lowest survival in the parent generation. Effects such as adaptation or stimulative effects of toxicants, could not be detected in our study. However, authors in the above mentioned studies used single toxicants while we used a mixture of CHP/ZnO, so it is possible to hypothesize that earthworms can more easily adapt when exposed to single chemicals, but are less likely to adapt when exposed to mixtures of different chemicals. This hypothesis should be further investigated. Biomarker responses between two generations of earthworms were significantly different. Comparison of AChE inhibition between 1st and 2nd generation shows that 2nd generation of earthworms became less susceptible to toxic effects of CHP, as AChE was less inhibited in M1 and M2. Oxidative stress biomarkers of the 2nd generation of earthworms, CAT, GST and MDA indicated failure of antioxidant defenses to cope with oxidative stress. Similar results were obtained by Oliveira et al. (2018) showing that although the first generation of Folsomia candida exposed to carbamazepine and fluoxetine was able to efficiently prevent oxidative damage, following generations gradually loose the capacity to successfully deal with the stressor. Results of the reproduction test were consistent between generations, with synergistic effects of the applied mixtures in both generation of earthworms, with stronger synergism in the 2nd generation at M3.

5. Conclusions As demonstrated in this study, the effects of mixture of CHP/ZnO are dependent on the soil type, ZnO particle size used in the mixtures and generation of earthworms. Contrary to the majority of studies, mixtures containing bZnO produced stronger effects compared to the mixtures containing nZnO. This indicates that number of different factors has to be taken into account when assessing toxicity of mixtures, as well as the need to properly characterize the soil that is used for toxicity testing, as the soil has its own “buffer” mechanisms that can alleviate the toxic effects of chemicals released in the environment. This study showed that effects observed in the first generation are not confirmed in the following generations in respect to all measured parameters. Comparison of the results of our study with available literature, provided some indications that it is possible for earthworms to more easily adapt on single toxicants but are less likely to adapt to mixtures. This hypothesis should be further tested. Detailed understanding of mixture toxicity itself as well as of environmental factors is of extreme importance during estimation of the risks for target and non-target organisms. Hence, further studies are necessary and emphasize the need for multigenerational tests when assessing the impacts of chemicals.

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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. CRediT authorship contribution statement
Lon
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