Background

In agricultural production, pesticides are often applied to control pests and diseases in various plants [1, 2]. However, most pesticides do not remain attached to crops and are eventually released into aquatic environments through runoff, spray drift, and other pathways, resulting in extensive residue accumulation [3]. Additionally, pesticides that contaminate surface water usually contain a mixture of substances rather than an individual compound, and many types of crops are generally intermingled in agricultural areas [4]. This results in detrimental effects on ecosystems and threatens environmental organisms [5]. Present regulatory procedures for prioritization and environmental quality standards mainly focus on individual compounds [6, 7]. However, a mixture of pesticides may usually pose a greater risk than each constituent alone [8]. Consequently, compliance with environmental quality standards for individual pesticides may not sufficiently protect aquatic organisms against toxic impacts from exposure to pesticide mixtures.

The pollution of water ecosystems by pesticides may adversely affect non-target organisms, such as fish [9]. As a valuable model in aquatic ecotoxicology, zebrafish (Danio rerio) has been used in different research fields, including aquatic ecotoxicology [10, 11]. Among its several practical merits, including relatively easy maintenance and culturing, its embryos are transparent, and most organ systems can be fully developed within 96 h post-fertilization (hpf) [12]. The experiment on zebrafish embryos following the 3R (Replacement, Reduction, and Refinement) principles of animal research under acute exposure has replaced the mandatory adult fish acute test for the toxicity assessment of wastewater in some European countries [13, 22]. Additionally, exposure to low concentrations of pesticides during the early life stages of D. rerio can negatively affect their biology and the characteristics formed during later development stages [12]. However, previous studies on pesticides have mainly focused on their individual toxicity to zebrafish embryos, whereas the possible toxicities caused by pesticide mixtures have seldom been investigated [14,15,16].

A mixed formulation of organophosphate insecticide phoxim (PHO) and pyrethroid insecticide deltamethrin (DEL) is commonly used all over the world to reduce insect resistance and improve control efficiency and/or economic concerns [17]. Therefore, their residues are likely to coexist in the same aquatic environment [18]. Despite the coexistence of PHO and DEL in water samples, their ecological and health risk evaluations have been primarily carried out based on the toxicological impacts of single compounds [19,20,21]. The interaction between PHO and DEL has not yet been examined, and their combined toxicity at low doses and the underlying toxicity pathways remain largely unknown. This study investigated the acute toxicity, enzymatic activities, and gene expressions in embryonic zebrafish co-exposed to PHO and DEL. A comprehensive evaluation of PHO and DEL at various endpoints offered noteworthy insights into the overall toxicity triggered by their combinations in zebrafish and the potential mechanism.

Materials and methods

Ethical note

All rearing and exposure procedures were conducted according to the Experimental Animal Management Law of China and authorized by the Independent Animal Ethics Committee of the Zhejiang Academy of Agricultural Sciences.

Pesticides and reagents

PHO (purity of 97%) was obtained from Lianyungang Liben Agrochemical Group (Lianyungang, China). DEL (purity of 98%) was donated by Nanjing Red Sun Chemical Co., Ltd. (Nanjing, China). To prepare the stock solutions, pesticides were dissolved in N, N-dimethylformamide (DMF) and 10% Tween-80 (Wt: Vol) and then preserved at 4 ℃. These stock solutions were then further diluted to the required concentrations using reconstituted water [22]. Reagents of analytical grade were used in this study.

Experimental animals

Adult zebrafish (AB strain) were used as breeding stocks and purchased from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The organisms were reared in a flow-through system using dechlorinated and aerated water (27 ± 1 ℃, 14-h light:10-h dark) and fed twice with a commercial fish diet (Tetramin) ad libitum daily [7, 25]. For propagation, sexually mature fish at a female/male ratio of 1:2 were separated by isolation boards in spawning containers with a net bottom (Esen Corp, Beijing, China) overnight. Spawning was induced by the light stimulation at seven o’clock in the following morning, and the isolation boards were removed. Embryos were collected within 30 min of light exposure and rinsed with reconstituted water. Viable embryos with normal developmental blastula were transferred into crystallizing dishes (bottom diameter of 10 cm) incorporating Hank’s solution.

Detections of single and combined toxicity

Toxicity detection of single pesticides

The acute toxicities of single chemicals to zebrafish at various life stages (embryonic, larval, juvenile, and adult stages) were examined according to OECD guidelines [23, 24]. Embryos at 3 hpf and larvae at 72 h post-hatching were selected for embryo and larva experiments, respectively. Juvenile zebrafish of 1 month old and adult zebrafish of 3 months old were subjected to food deprivation for 24 h before the toxicity test. At least 4 concentrations with a geometrical ratio that caused 10–90% mortality according to the results from pre-trails were assayed for every pesticide. Each concentration and control were tested in triplicate. The optimal dose of chemicals and water quality were kept by exchanging the exposure solutions every 12 h. The temperature and light cycle remained the same as those in the culture environment during exposure. Mortality was assessed after exposure for 96 h. Detailed information on the test procedure during the multiple life stages is provided in the Additional file 1.

Combined toxicity detection

The combined toxicity of PHO and DEL was evaluated using zebrafish embryos. The toxicities of the single chemicals were linearly compared with their combined toxicity. To investigate the combined impacts of PHO and DEL, the zebrafish were subjected to serial dilutions of each chemical with a fixed equitoxic ratio according to the obtained single LC50 values. The sum doses of the mixture were systematically changed, while all of the above-mentioned factors were kept constant to construct the dose–response correlation. All tests were conducted three times for each dose. The other test programs of the equitoxic test were similar to the acute embryo toxicity tests for single chemicals.

Detections of biochemical and molecular parameters

Sample preparation

The doses of 1/400, 1/100, and 1/25 of 96-h LC50 value for each chemical to the embryos were deemed as the low, moderate, and high doses, respectively. Accordingly, low, moderate, and high doses in the mixture exposure of PHO + DEL (MIT) were the combinations of both PHO and DEL at the corresponding doses. Approximately 300 embryos at 3 hpf were placed into an 800 mL beaker containing 800 mL pesticide test solution as a replicate. Each experiment was repeated three times for each dose. After exposure for 96 h, the treated fish were collected. The collected specimens were preserved at – 80 ℃ for further experimentation.

Tests of biochemical parameters

About 200 fish from each beaker were placed in 50 mM potassium phosphate buffer (pH 7.0) consisting of 0.5 mM EDTA and then subjected to homogenization (1: 20, w/v) adopting an electric homogenizer. The tissue homogenate was centrifuged at 12,000 rpm for 30 min at 4 ℃, and the supernatant was applied to analyze biochemical indexes.

The parameters, including oxidative stress, apoptotic enzymes, detoxification enzymes, vitellogenin, and thyroid hormones, were detected using the available commercial kits (Nanjing Jiancheng Bioengineering Institute, China) according to previously established protocols [25]. Protein concentrations were examined using the Bradford method [26], and bovine serum albumin (BSA) was adopted to construct the standard curve. Details on the biochemical indices tested are found in the Additional file 1: Table S1.

Gene expression analysis

About 30 fish from each beaker were harvested and homogenized. Total RNA was extracted from the tissue homogenates using TransZol Up Plus RNA Kit (TransGen Biotech Ltd., China). Gene expressions were examined by quantitative real-time PCR (qRT-PCR) using previously established protocols [25]. Briefly, total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China). In addition, β-actin was taken as the internal reference gene. The primer sequences are listed in Additional file 1: Table S2. The relative expressions of the target genes were calculated using the 2−△△Ct method [27].

Chemical analysis

In order to determine the actual concentrations of PHO, DEL, and their mixtures, the water samples were monitored at the start (0 h) and before water renewal (12 h) during the experimental period. The water samples were filtered using a 13 mm filter (0.25 μm). The samples containing PHO and/or DEL were analyzed using liquid chromatography-tandem mass spectrometry (SHIMADZU, LCMS-8050) performed on an ACE C18 column (2.1 mm × 100 mm, 1.7 μm) with a mobile phase composed of an aqueous solution (5 mmol/L ammonium formate, A) and methanol (B). The separation was conducted using a gradient elution program as follows: 0–0.5 min, 95% A; 0.5–2.0 min, 5% A; 2.0–5.0 min, 5% A; 5.0–5.1 min, 95% A; 5.1–7.0 min, 95% A. Mass spectrometric detection was conducted under positive electrospray ionization (ESI) with a multiple reaction monitoring (MRM) mode. For PHO, the precursor ion was m/z 299.0, and the product ions were m/z 77.1 for quantization and m/z 129.1 for confirmation, with collision energies of 26 and 10 eV, respectively. For DEL, the precursor ion was m/z 523.0, and the product ions were m/z 281.0 for quantization and m/z 506.0 for confirmation, with collision energies of 16 and 11 eV, respectively. The analysis results revealed that the deviations of the nominal and the actual concentrations of PHO, DEL, and their mixtures were less than 20%. Accordingly, the nominal concentration was used as the actual concentration in this study.

Statistical analysis

The acute toxicities of pesticides to D. rerio were analyzed by adopting a probit analysis described by Chi [28]. The interplay pattern of the chemical combinations was assessed according to an additive index (AI) method [29, 30]. Details about the AI method are provided in the Additional file 1. The one-way ANOVA statistical divergence was determined using a Dunnett’s post-hoc test conducted with SPSS software (SPSS version 18.0, USA).

Results

Single pesticide toxicity determinations

Results from the single pesticide toxicity tests showed that the toxic selectivity of various chemicals to zebrafish varied greatly, and each chemical showed conspicuous toxicities to various life periods of D. rerio. PHO showed a narrower toxic range in different developmental periods of zebrafish, with the 96-h LC50 values ranging from 0.24 (0.12–0.33) to 3.39 (2.58–4.86) µM. In contrast, DEL elicited a broader toxic scope to the tested fish, with the 96-h LC50 values ranging from 0.0041 (0.0031–0.0060) to 2.97 (1.56–4.69) µM (Table 1). Overall, the embryonic stage was the most tolerant, while the adult period was the most sensitive to the 2 pesticides.

Table 1 Acute toxicity of phoxim and deltamethrin to various life stages of zebrafish
Full size table

Mixture toxicity determinations

The combination of PHO and DEL resulted in synergistic effects, and the corresponding AI values after exposure for 24, 48, 72, and 96 h were 2.29, 2.50, 3.41, and 4.24, respectively. Additionally, the AI values for the combination were increased with the increase in exposure time, indicating that the combined toxicity was positively associated with the exposure period (Table 2).

Table 2 Mixture toxicities of phoxim and deltamethrin to zebrafish embryos
Full size table

Biochemical index determinations

Evaluation of oxidative stress

The MDA level was noticeably enhanced under all the administrations (apart from the high dose of PHO and MIT administrations) relative to the baseline value. In contrast, a conspicuous decrease was found under the low and moderate doses of MIT administration relative to the corresponding PHO administration. The MDA level was also conspicuously reduced under the moderate dose of MIT administration relative to the corresponding DEL administration (Fig. 1A). The CAT activity was conspicuously elevated under all PHO and DEL treatments (apart from the moderate PHO and low DEL treatments) relative to the baseline value. Additionally, a dramatic enhancement was also observed under the low dose of MIT treatment relative to the baseline value and corresponding DEL treatment (Fig. 1B). T-SOD activity was dramatically raised under all the treatments relative to the baseline value. Conversely, an obvious decrease was recorded under the moderate dose of MIT treatment relative to the corresponding PHO treatment (Fig. 1C). Similar to the MDA level, the Cu/Zn-SOD activity was distinctly induced under all PHO and DEL treatments (apart from the high dose of PHO treatment) relative to the baseline value. Additionally, a distinct up-regulation was also observed under the low dose of MIT treatment relative to the baseline value. Contrastingly, its activity was conspicuously inhibited under the moderate dose of MIT treatment relative to the baseline value, as well as corresponding PHO or DEL treatments (Fig. 1D).

Fig. 1
figure 1

The oxidative responses in zebrafish treated with PHO, DEL, and PHO + DEL combinations. A MDA; B CAT; C T-SOD; D Cu/Zn-SOD; E T-GSH; F GSSG; G POD; H ROS. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

Similar to the T-SOD activity, the level of T-GSH was apparently increased under all the treatments relative to the baseline value. Additionally, a conspicuous rise was also observed under the moderate dose of MIT treatment relative to the corresponding PHO or DEL treatment. However, its level was obviously decreased under the high dose of MIT treatment relative to the corresponding PHO treatment (Fig. 1E). The GSSG activity was obviously enhanced under all PHO and MIT treatments (apart from the high dose of MIT treatment) relative to the baseline value. In addition, a clear increase was also found under the moderate dose of DEL treatment relative to the baseline value. Nevertheless, its level was distinctly reduced under the high dose of MIT treatment relative to the corresponding PHO treatment (Fig. 1F). The POD activity was considerably rised under all the treatments (except for the high dose of PHO treatment) relative to the baseline value. Moreover, a conspicuous elevation was also seen under the high dose of MIT treatment relative to the corresponding PHO treatment. Conversely, its activity was conspicuously reduced under the low and moderate doses of MIT treatment relative to the corresponding DEL treatment (Fig. 1G). The ROS level was apparently elevated under all the treatments (except for their treatments at the high dose) relative to the baseline value. An apparent up-regulation was also seen under the moderate dose of MIT treatment relative to the corresponding DEL treatment. Conversely, its level was apparently suppressed under the high dose of MIT treatment relative to the baseline value and corresponding DEL treatment (Fig. 1H).

The activities of apoptotic and detoxification enzymes

The activity of Caspase 3 was pronouncedly increased under the moderate dose of PHO treatment and the low dose of DEL treatment relative to the baseline value. In contrast, a pronounced reduction was monitored under the high doses of PHO and DEL treatments and the low dose of PHO treatment relative to the baseline value. Its activity was pronouncedly decreased under the low dose of MIT treatment relative to the corresponding DEL treatment (Fig. 2A). The activity of Caspase 9 slightly changed under all the treatments, apart from the low and moderate doses of DEL treatment, in which its activity was markedly weakened relative to the baseline value. No significant change was detected under all MIT treatments relative to the corresponding PHO or DEL treatments (Fig. 2B).

Fig. 2
figure 2

The apoptotic and detoxification enzyme activities in zebrafish treated with PHO, DEL, and PHO + DEL combinations. A Caspase 3; B Caspase 9; C CYP450; D CarE; E GST. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

The activity of CYP450 was significantly reduced under all the treatments at the high dose relative to the baseline value. A noticeable reduction was also seen under the low dose of PHO treatment relative to the baseline value. Contrastingly, its activity was conspicuously elevated under the low dose of MIT treatment relative to the corresponding PHO treatment (Fig. 2C). The activity of CarE was markedly elevated under all the treatments (apart from the high doses of PHO and MIT treatments) relative to the baseline value. Conversely, significant inhibition was observed under the high dose of PHO and MIT treatments relative to the baseline value. Its activity was also dramatically inhibited under the high dose of MIT exposure relative to the corresponding DEL exposure (Fig. 2D). The activity of GST was conspicuously increased under the low dose of DEL exposure relative to the baseline value. Contrarily, its activity was remarkably decreased under the low dose of MIT exposure relative to the corresponding DEL exposure (Fig. 2E).

Contents of THs and VTG

The content of T3 was conspicuously rised under the moderate dose of PHO treatment relative to the baseline value. A distinct enhancement was also found under all MIT treatments (apart from the moderate dose of MIT treatment) relative to the baseline value, as well as corresponding PHO and DEL treatments. Nevertheless, its activity was obviously reduced under the high dose of DEL treatment relative to the baseline value (Fig. 3A). The content of VTG was remarkably reduced under all PHO treatments relative to the baseline value. A marked reduction was also seen under the low dose of MIT treatment relative to the baseline value and corresponding DEL exposure. In contrast, its content was conspicuously rised under the moderate and high doses of MIT exposure relative to the corresponding PHO exposure (Fig. 3B).

Fig. 3
figure 3

A T3 and B VTG levels in zebrafish treated with PHO, DEL, and PHO + DEL combinations. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

Gene expression analysis

Expressions of antioxidation- and cell apoptosis-related genes

The expressions of antioxidation-related genes (Mn-sod and Cu/Zn-sod) were analyzed in our study. The expression level of Mn-sod was positively correlated with the dose of PHO, and there was a significant induction under all PHO treatments relative to the baseline value. Moreover, a dramatic elevation was found under the moderate dose of DEL and MIT treatments relative to the baseline value. Under the MIT treatment, a distinct elevation was also detected in all doses (apart from the moderate dose of MIT) relative to the baseline value, as well as corresponding PHO and DEL treatments (Fig. 4A). The expression level of Cu/Zn-sod was distinctly enhanced under the moderate and high doses of PHO treatment and the moderate dose of DEL treatment relative to the baseline value. Additionally, a marked up-regulation was also detected under all doses of MIT treatment relative to the baseline value. Meanwhile, it was also up-regulated under the low and high doses of MIT treatment relative to the corresponding PHO and DEL treatments (Fig. 4B).

Fig. 4
figure 4

Impacts on expressions of genes involved in oxidative stress and cellular apoptosis in zebrafish treated with PHO, DEL, and PHO + DEL combinations. A Mn-sod; B Cu/Zn-sod; C p53; D cas3; E cas9. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

The expressions of apoptotic genes (p53, cas3, and cas9) were analyzed in the present study. Relative to the baseline value, the expression of p53 was dramatically rised under the moderate dose of PHO treatment and low dose of DEL treatment. Nevertheless, a noticeable reduction was seen under the high dose of MIT treatment relative to the baseline value and corresponding individual treatments (Fig. 4C). The expression of cas3 was apparently rised under all treatments (except for the low dose of PHO and high dose of DEL) relative to the baseline value. Meanwhile, a marked elevation was also seen under the low dose of MIT treatment relative to the corresponding PHO and DEL treatments. A noticeable reduction was seen under the moderate dose of MIT treatment relative to the corresponding DEL treatment (Fig. 4D). The expression level of cas9 was markedly induced under all DEL and MIT treatments (apart from the moderate dose of DEL treatment) relative to the baseline value. A noticeable rise was also observed under the high dose of PHO treatment relative to the baseline value. Additionally, the expression of the cas9 gene was also markedly induced under the low dose of MIT treatment relative to the corresponding PHO treatment (Fig. 4E).

Expressions of the genes related to HPT and HPG axes in the endocrine system

The mRNA expressions of genes in the HPT axis (TRα, dio1, and tsh) were analyzed (Fig. 5). The expression of TRα was remarkably reduced under the moderate dose of DEL treatment relative to the baseline value. In contrast, a significant increase was seen under the low and high doses of DEL treatment relative to the baseline value. Its expression was also conspicuously rised under the moderate dose of MIT treatment relative to the corresponding DEL administration (Fig. 5A). The expression of dio1 was up-regulated under all administrations (apart from the low and high doses of DEL treatment) relative to the baseline value. Besides, a positive correlation was found between the expression of dio1 and the dose of the PHO. A large increase was seen under the low and high doses of MIT treatment relative to the corresponding DEL administration. Contrastingly, a conspicuous reduction was observed under the moderate dose of MIT treatment relative to the corresponding PHO and DEL administrations (Fig. 5B). The expression of tsh was increased dramatically under the PHO administration in a dose-dependent manner relative to the baseline value. A significant increase was seen under the moderate dose of DEL administration relative to the baseline value, and the same change was observed under the MIT administration. The expression was markedly rised under the low and high doses of MIT administration relative to the corresponding PHO and DEL administrations, respectively. Nevertheless, a pronounced inhibition was seen under the moderate dose of MIT administration relative to the corresponding DEL administration (Fig. 5C).

Fig. 5
figure 5

Impacts on expressions of genes involved in the HPT and HPG axes of zebrafish treated with PHO, DEL, and PHO + DEL combinations. A TRα; B dio1; C tsh; D ERα; E ERβ1; F vtg1; G cyp17; H cyp19a. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

The expressions of HPG axis genes (ERα, ERβ1, vtg1, cyp17, and cyp19a) were analyzed in our study. In the PHO administration, the expression level of ERα was remarkably rised relative to the baseline value, which was positively correlated with the administration dose. An apparent increase was also observed under the moderate dose of DEL treatment and all doses of MIT treatment relative to the baseline value. The expression was markedly increased under the low and high doses of MIT treatment, while it was conspicuously decreased under the moderate dose of MIT administration relative to the corresponding PHO and DEL administrations (Fig. 5D). The expression level of ERβ1 was noticeably induced under all doses of PHO treatment, the moderate dose of DEL treatment, and all doses of MIT treatment relative to the baseline value. Moreover, a conspicuous increase was observed under the low and high doses of MIT treatment relative to the corresponding PHO and DEL administrations, while a distinct down-regulation was seen under the moderate dose of MIT treatment relative to the corresponding DEL administration (Fig. 5E).

The expression of vtg1 was significantly increased under all administrations (apart from the low dose of DEL treatment) relative to the baseline value. Additionally, its expression under the high dose of PHO treatment was over 12 times higher than the baseline value. A significant induction under the low and high doses of MIT treatment and a significant reduction under the moderate dose of MIT treatment were detected relative to the corresponding PHO and DEL administrations (Fig. 5F). The expression level of cyp17 was noticeably induced under all PHO administrations relative to the baseline value. Additionally, a distinct increase was monitored under the moderate dose of DEL and MIT treatments relative to the baseline value. The expression of cyp17 was also markedly increased under the low and high doses of MIT treatment relative to the baseline value, as well as corresponding PHO and DEL administrations (Fig. 5G). The expression of cyp19a was conspicuously suppressed under all doses of DEL and MIT treatments relative to the baseline value, and its expression was negatively correlated with the dose of MIT treatment. Meanwhile, a conspicuous decrease was observed under the low dose of MIT treatment relative to the corresponding PHO administration (Fig. 5H).

Expressions of the genes related to HPA axis and immunity system

The expressions of the HPA axis genes (crh and gr) were analyzed (Fig. 6). The expression of crh was conspicuously enhanced under all administrations (apart from the low and high doses of DEL treatment) relative to the baseline value. A conspicuous increase was also detected under the low and high doses of MIT treatment relative to the corresponding PHO and DEL administrations, while a conspicuous reduction was seen under the moderate dose of MIT treatment relative to the corresponding DEL administration (Fig. 6A). The expression of gr gene was conspicuously rised under the high dose of DEL and MIT treatments relative to the baseline value. A dramatic increase was also observed under the moderate dose of DEL treatment relative to the baseline value (Fig. 6B).

Fig. 6
figure 6

Impacts on expressions of genes related to HPA axis and immune system of zebrafish treated with PHO, DEL, and PHO + DEL combinations. A crh; B gr; C CXCL-CIC; D IL-8. The results are presented as the means ± SD. *p < 0.05, significant difference relative to the control; #p < 0.05, significant difference relative to the corresponding PHO exposure; ★p < 0.05, significant difference relative to the corresponding DEL exposure. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose

Full size image

The expressions of the genes related to immunity response (CXCL-CIC and IL-8) were analyzed in the present study. The CXCL-CIC gene expression level was remarkably increased under the low dose of DEL treatment and high dose of PHO treatment relative to the baseline value. On the contrary, a conspicuous down-regulation was seen under the high dose of MIT treatment relative to the baseline value as well as corresponding PHO and DEL treatments (Fig. 6C). The expression of IL-8 was positively correlated with the dose of PHO. Moreover, its expression under the high dose of PHO treatment was approximately five times greater than the baseline value. Under the MIT administration, the expression of IL-8 was noticeably increased under the low dose relative to the baseline value. Nevertheless, a conspicuous decrease was seen under the moderate and high doses of MIT treatment relative to the corresponding PHO administration (Fig. 6D).

Integrated biomarker response (IBR) analysis

In order to assess the toxic impacts of PHO, DEL, and their mixtures at various doses, we integrated all determined biochemical and molecular indicators using an IBR analysis. The IBR index was calculated based on the data of zebrafish embryos treated with pesticides under different doses. The toxicity of single pesticides and their combinations are depicted in the bar graphs and radar charts, as shown in Fig. 7. Among all the tested biochemical and molecular indices, the most sensitive indices for PHO, DEL, and their mixtures were apoptotic biochemical biomarkers, anti-oxidant biochemical biomarkers, and antioxidant gene markers, respectively.

Fig. 7
figure 7

Star plots of integrated biomarker responses of different parameters. A Shows the PHO-L, PHO-M, and PHO-H treatment groups successively; B shows the DEL-L, DEL-M, and DEL-H treatment groups successively; C shows the PHO + DEL-L, PHO + DEL-M, PHO + DEL-H treatment groups successively. PHO phoxim, DEL deltamethrin, L low dose, M moderate dose, H high dose, AO-B Anti-oxidant biochemical biomarkers, AP-B Apoptotic biochemical biomarkers, DE-B Detoxification biochemical biomarkers, EN-B Endocrine system biochemical biomarkers, AO-G Anti-oxidant gene markers, AP-G Apoptotic gene markers, IM-G Immune system markers, EN-G Endocrine system gene markers

Full size image

Discussion

Acute toxicity results provide a first approximation of the impacts of pesticides and may be helpful in the detection of dose thresholds for the sequent investigation of sublethal impacts [31]. Our results showed that both PHO and DEL possessed relatively high toxicities to zebrafish during multiple developmental stages, with the 96-h LC50 values ranging from 0.24 to 1.44 µM and from 0.0041 to 2.97 µM, respectively. Fish are highly sensitive to DEL since this pesticide can be absorbed into gills at high rates, degraded slowly through hydrolysis, and hypersensitive to the piscine nervous system [32]. It has been reported that PHO exerts acute toxicity to most fish, such as Tilapia (Oreochromis niloticus), Black seabream (Acanthopagrus schlegelii), and Loach (Paramisgurnus dabryanus), which is consistent with our current findings [33,34,35]. Furthermore, its high toxicity to fish indicates that fish have a lower ability to metabolize PHO [36]. In the natural ecosystem, organisms are more often exposed to chemical mixtures rather than to an individual compound [3, 37]. Therefore, it is crucial to determine the mixture toxicity when evaluating the environmental quality [17, 38]. Mixtures of PHO and DEL have a synergistic effect on zebrafish embryos, implying that ecological risk assessments according to single chemicals cannot sufficiently protect aquatic environments from exposure to pesticide mixtures [39,40,41]. Therefore, PHO and DEL should be carefully mixed to avoid adverse impacts on the water ecosystem.

Antioxidation is a crucial topic in the research of environmental toxicology [42]. Oxidation stress is regarded as a cellular ROS imbalance that can lead to cell damage via alteration of DNA, proteins, and lipids [43]. Lipid peroxidation is a type of oxidative stress related to the instability of biofilms and can be measured by MDA quantification [7]. To resist oxidative stress from environmental contaminations, zebrafish always stimulate their antioxidation systems by increasing the expressions of enzymes, such as SOD, CAT, and POD [42]. In this study, the conspicuous increases of ROS and MDA contents in most exposures suggested that zebrafish embryos suffered from severe oxidative stress when exposed to PHO, DEL, and their mixture.

It is well known that SOD can catalyze superoxide radicals into hydrogen peroxide (H2O2), which is further broken down by CAT or POD into nontoxic H2O and molecular oxygen (O2) [44]. The levels of T-SOD and Cu/Zn-SOD under nearly all treatments were markedly enhanced relative to the baseline value, indicating that SOD was produced to transform the superoxide radicals into H2O2 in zebrafish embryos. It was noteworthy that the content of Cu/Zn-SOD was noticeably down-regulated under moderate and high doses of MIT treatment relative to the corresponding PHO and DEL administrations. The decrease suggested that the oxidative system was destroyed by the higher dose of combined pesticides, which might be one reason for the synergistic impact produced by the MIT administration [45]. In addition, the levels of CAT and POD were also induced under most treatments, suggesting that large amounts of CAT and POD enzymes were required to convert excess H2O2 into H2O, thereby relieving the toxicity from pesticide exposures [47]. Glutathione can occur in cells with reduced form (GSH) or oxidative form (GSSG) [48], and it is a major non-enzymatic antioxidant that contributes to removing some residual free radicals and cannot be decomposed by antioxidant enzymes [49]. In our present research, the levels of T-GSH and GSSG were clearly enhanced under most exposures relative to the baseline value. These results implied that two forms of glutathione (GSH and GSSG) played important roles in preserving the zebrafish embryos from oxidative stress.

Analyzing the expressions of antioxidation-related genes at the mRNA level will be helpful in assessing the ability of antioxidants in the organism [44]. Meanwhile, we found that the expressions of Mn-sod and Cu/Zn-sod genes were markedly rised under most administrations, which was consistent with the up-regulated activities of T-SOD and Cu/Zn-SOD. These results indicated that the up-regulation of antioxidative ability was achieved by enhancing the quality of mRNA transcription in zebrafish embryos when exposed to these pesticides [46]. The mRNA can be translated into more SOD protein in order to eliminate the negative effect of ROS, keeping the zebrafish away from oxidative stress [40]. The activity of Cu/Zn-SOD was significantly decreased in the moderate and high doses of MIT treatment relative to the baseline value, as well as corresponding PHO and DEL treatments. In contrast, significant increases in Mn-sod and Cu/Zn-sod expressions were discovered under the moderate and high doses of MIT treatment. The discrepancy between the mRNA level and enzyme activity of SOD could be explained as follows: (1) the mRNA level only represented a snapshot of antioxidant enzyme activity, and a time lag impact existed between transcription and translation; and (2) post-translation might regulate the enzymatic activity [52, 56].

Detoxification enzymes play crucial roles in excluding the exogenous contaminations to zebrafish [50]. Among these enzymes, CYP450 is one of the most important detoxification enzymes and is deemed an effective biomarker for environmental pollution detection [51]. CarE enzymes can hydrolyze ester compounds into alcohol and acid, resulting in a decline in the toxicity of ester contaminations [52]. The GST enzyme is the most critical phase II detoxification enzyme and can catalyze the conjugation of reduced glutathione (GSH) to the compounds [53]. The results of reduced activity of CYP450 in the present study implied that CYP450 would be the detoxification mechanism of zebrafish to PHO and DEL. Additionally, the activity of CarE was induced by the ester property of DEL, indicating that this enzyme was involved in the detoxification process. Nevertheless, the level of CarE declined under the high dose of MIT treatment relative to the corresponding DEL administration. We concluded that the inhibited enzymatic activity might be attributed to the synergistic impact of the combined exposure. No apparent change in GST activity was observed, indicating that the GST enzyme was not responsible for detoxification metabolism in the present study.

Apoptosis removes excess cells in the early developmental stage of zebrafish, contributing to normal tissue remodeling and morphogenesis [54]. The activities of Caspase 3 and Caspase 9 were somewhat altered under all PHO and MIT administrations, except under the high dose of PHO administration, in which the activity of Caspase 3 was apparently inhibited relative to the baseline value. An apparent inhibition of Caspase 3 activity was also observed under the moderate dose of DEL treatment relative to the baseline value. The activity of Caspase 9 was apparently increased under the low dose of DEL treatment relative to the baseline value. However, the expressions of p53, cas3, and cas9 were markedly up-regulated under most administrations relative to the baseline value, suggesting that PHO, DEL, and their combinations would probably induce apoptosis in the cell [55]. Two reasons might cause the disparity between gene expression and enzyme activity. Firstly, there was a delayed influence between transcription and translation, causing the transcriptome not to always reflect in the proteome. Secondly, modulation of enzyme activity might occur via post-translational events, such as post-translational modification and regulation of enzyme activity [56]. Therefore, PHO, DEL, and their combinations could induce cell apoptosis, which might be responsible for the increase in the number of developmental defects in zebrafish embryos.

The immune system of fish has many chemokines that maintain a stable environment in their bodies and are important components of the immune system [48]. Environmental substances can affect the gene expressions of relevant chemokines [57]. Chemokines (CXCL-CIC and IL-8) are secreted proteins that can regulate the nature of immune responses [25]. Stress factors greatly affect the immune system during zebrafish development [58]. Therefore, the immune system is an essential indicator when assessing the toxicity of chemicals in fish [59]. The innate immune system is the only defense system against infections caused by chemicals during the early life stages of zebrafish [60]. In the current work, the expression levels of CXCL-CIC and IL-8 genes were increased significantly under most of the administrations relative to the baseline value, indicating that the PHO, DEL, and MIT administrations triggered inflammatory responses, which might be explained by their intoxication-induced adaptation. The expressions of these inflammatory factors are essential for homeostasis in the body, and excessive amounts of them can lead to cell or tissue damage and may induce serious diseases, such as cancer and metabolic diseases [48]. Besides, the expressions of CXCL-CIC and IL-8 were considerably inhibited in the moderate and high doses of MIT treatment, indicating that the mixture of PHO and DEL could lead to immunity depression against various exogenous contaminant stimulations.

The endocrine systems of vertebrates are modulated by the hypothalamic-pituitary-thyroid/gonadal/adrenal (HPT/HPG/HPA) axes, which coordinate hormone synthesis, secretion, and metabolism to regulate kinetic processes [63]. Chemicals can disrupt the endocrine system, leading to impaired growth, development, and reproduction of aquatic vertebrates [61]. The current study demonstrated that the level of T3 was substantially altered after administration of PHO, DEL, and their combinations, indicating that the endocrine system of zebrafish was disturbed by exposure to these pesticides. THs regulated by the HPT axis have an important influence on the development and growth of fish [57]. We found that the expression levels of TRα, dio1, and tsh were obviously induced under most of the exposures relative to the baseline value. Additionally, apparent alterations of dio1 and tsh were observed under most MIT exposures relative to the baseline value and the corresponding single exposures. The variation of the mRNA expression in the HPT axis indicated PHO and DEL had the possibility to induce thyroid disruption, while their mixture elicited more serious injuries relative to the single pesticides. The HPA axis is responsible for regulating the adaptive stress response in organisms [18]. The current study showed that the expression levels of crh and gr in the HPA axis were distinctly enhanced under most administrations relative to the baseline value, suggesting that PHO, DEL, and their combinations could impact the growth, metabolism, and reproduction of zebrafish.

In oviparous vertebrates, the female-specific yolk protein precursors VTGs act to transport nutrients into oocytes during the maturation of oocytes [25]. By detecting the VTG content, the effects of pesticides on hormone endocrine disruptors can be detected [16]. Our analysis showed that the VTG content was increased dramatically after DEL exposure at the high dose relative to the baseline value, indicating that DEL had an estrogen-disrupting capacity [62]. Conversely, a dramatic decrease was observed under all PHO exposures relative to the baseline value, showing that PHO had antioestrogen-disrupting capacity in D. rerio. However, there was no significant regularity of VTG changes under MIT treatment, implying that PHO and DEL had opposite effects on VTG levels when these two pesticides were mixed.

The HPG axis can regulate sex hormones, which are closely related to fish reproduction [64]. VTG is modulated by the activation of estrogen receptors (ERs), which provide the nutrients for the development of zebrafish embryos [65]. ERs have an essential function in the synthesis of vtg [66]. The expressions of ERα, ERβ1, and vtg were clearly elevated under most administrations relative to the baseline value, along with a clear elevation of VTG level under individual treatments, indicating that PHO, DEL, and their combinations had potential oestrogenic impacts on D. rerio. Apparent induction was also observed under most MIT exposures relative to the corresponding PHO and DEL administrations, revealing that the mixture of PHO and DEL enhanced the oestrogenic impact mainly by revising genes in D. rerio. We deduced that the treatment of embryonic fish with PHO, DEL, and their mixture changed the expressions of ERs, which influenced the production of VTG, leading to significant absorption of nutrients. Additionally, the expression levels of cyp17 and cyp19a genes were conspicuously altered under most administrations relative to the baseline value, implying that PHO, DEL, and their combinations could affect the reproduction of D. rerio [67].

Our study showed that the concurrent pesticides in the aquatic environment caused greater toxicological impacts on biota than those reported from individual compounds. Therefore, evaluating an individual pesticide does not provide a realistic estimate of the impacts on aquatic ecosystems [68]. Our investigation also supported the hypothesis that the environmental interaction between various chemicals within the maximum permitted limits might harm aquatic life forms [69]. Therefore, future studies need to run an assessment of the environmental level of pollutants and test them in mixtures under laboratory conditions to clarify what is taking place in the environment. Ultimately, our findings might facilitate the selection of pesticide mixtures that are likely to exert synergistic interactions at environmentally-realistic doses.

Conclusions

Mixtures of PHO and DEL induced synergistic effects on zebrafish embryos. The activities of T-SOD, Cu/Zn-SOD, POD, and CarE were substantially altered under most treatments of PHO, DEL, and their combinations relative to the baseline value. The expressions of nine genes, including Mn-sod, Cu/Zn-sod, cas3, dio1, tsh, ERα, vtg1, cyp17, and crh, related to oxidative stress, apoptosis, immune response, and endocrine system were significantly altered under the mixture treatment relative to the individual pesticides. Overall, our results suggested that PHO, DEL, and their combinations caused different levels of toxic impacts on the early developmental stage of zebrafish. The enlarged adverse impacts of these chemicals in a mixture indicated that joint toxicity assessment played a vital role in developing more realistic water quality standards and monitoring guidelines.