3.1. Survival pattern, synergistic and antagonistic action of metals (Zn, Fe) and herbicide (PQ)
The survival patterns of caterpillars that were single and co-exposed to Zn, Fe and PQ changed. Survival rates followed similar curves, but differed with respect to time of death and morphological abnormalities, showing that the cause of death was dose and compound dependent (Fig. 2A-F). Specifically, larval mortality began after 7 days with 10 mg Fe, but began after 4, 4 and 8 days for PQ, Zn/PQ, and Fe/PQ, respectively. At 10 days, total mortality was Fe (15%), PQ (29%), Zn/PQ (15%), and Fe/PQ (15%) (Fig. 2A, D). No mortality was recorded in the control, Zn, Fe, and Zn/Fe/PQ treatments. For 20 mg dose, mortality at 5–7 days was control (0%), Zn (17%), Fe (15%), and PQ (58%); at 2–10 days it was Zn/PQ (43%), Fe/PQ (43%), and Zn/PQ/PQ (29%) (Fig. 2B, E). All treatments exhibited mortality at 5–9 days with a dose of 40 mg, with the exception of Zn/Fe/PQ (72% mortality), while total mortality at 10 days remained 100%. (Fig. 2C, F). Zn/Fe clearly enhanced survival (10, 20 mg 100%, 40 mg 85%), with the Zn/Fe/PQ complex lowering individual compound toxicity and prolonging survival time of caterpillars (Fig. 2A-F).
Survival of caterpillars in the 10 mg dose of Zn, Fe, Zn/Fe, PQ, Zn/PQ, Fe/PQ, and Zn/ Fe/PQ was 83, 85, 100, 71, 85, 85, and 100%, respectively. At 20 mg, it was 100, 85, 100, 42, 57, 57, and 71%, respectively. At 40 mg, it was 66, 71, 85, 0, 0, 0 and 29%, respectively. Compared to the control, Kaplan-Meier data showed that the survival rate significantly differed for larvae exposed to 10 mg (log-rank 2 = 5.027; df = 7; p.65), 20 mg (log-rank 2 = 13.73; df = 7; p.05), and 40 mg (log-rank 2 = 44.8; df = 7; p 0.001) (Fig. 2).
3.2. Influence of single and co-exposure of Zn, Fe, and PQ on larval and pupal development
Single and mixed exposure of metals and the herbicide affected larval (Fig. 3A, B) and pupal weight (Fig. 4A, B). Single and mixed exposure to chemicals significantly differed, showing a dose-specific pattern (two-way ANOVA, P < 0.0001, F = 397.3; P < 0.0001, F = 396.3). Compared to the control, the bodyweight of caterpillars considerably increased when treated with Zn, Fe, and Zn/Fe (10, 20 mg; P < 0.001). In contrast, the larval weight of Zn, Fe and, Zn/Fe (40 mg) exposed caterpillars significantly declined (P < 0.001) (Fig. 3A). Compared to caterpillars treated with PQ, LW increased at 10, 20 mg of Zn/PQ, Fe/PQ, and Zn/Fe/PQ exposure (P < 0.001). LW was lower following exposure to 40 mg Zn/PQ and Fe/PQ (P < 0.001). Interestingly, 40 mg Zn/Fe/PQ exposure mitigated compound toxicity, with a significant increase in LW (P < 0.001) (Fig. 3B).
After 48 h of pre- and post-PQ exposure, the pupal weight (PW) changed (P < 0.001, F = 125.6; P < 0.001, F = 136). Zn, Fe, and Zn/Fe treatments considerably reduced PW overall; however, the Zn/Fe treatment distinctly declined for all applied doses (P < 0.001) (Fig. 4A). Compared to PQ treated caterpillars, PW significantly increased in Zn/PQ, Fe/PQ, and Zn/Fe/PQ treatments at 10 mg exposure (P < 0.001). Of note, 40 mg exposure resulted in zero pupal ecdysis, due to 100% mortality of caterpillars (Fig. 4B).
3.3. Effect of single and mixed Interaction of Zn, Fe, and PQ on diet consumption and frass production
Single and mixed interactive effects were recorded for diet consumption after 48 h (Fig. 5A). Compared to the control, pre-post PQ food consumption increased significantly at 10 mg and 20 mg for Zn, Fe, and Zn/Fe. In contrast, it decreased significantly at 40 mg for Zn, Fe, and Zn/Fe (P < 0.001). Food consumption significantly increased at 10 mg Zn/PQ, Zn/Fe/PQ, 20 mg Zn/PQ, Zn/Fe/PQ, and 40 mg Zn/Fe/PQ (P < 0.001). In contrast, significant decreases were recorded at 10 and 40 mg Fe/PQ (P < 0.001) (Fig. 5B).
Frass production during pre-post PQ exposure significantly varied with the interaction of doses and compounds (P < 0.0001, F = 76.3; P < 0.0001, F = 245). Compared to control caterpillars, frass production significantly increased with Zn/Fe (P < 0.001) at all exposed doses. In contrast, it decreased at 10, 20, and 40 mg of Zn and 20 mg Fe (P < 0.001) (Fig. 6A). Frass production significantly declined in Zn/PQ, Fe/PQ, and Zn/Fe/PQ at both 10 mg and 20 mg (P < 0.001). Co-exposure to Zn/PQ and Zn/Fe/PQ caused frass production to increase significantly at 40 mg (P < 0.001) (Fig. 6B).
3.4. Influence of single and mixed interactions of Zn, Fe, PQ on pupal death, pupal and imago ecdysis, and metamorphosis related deformity
Pupal mortality, pupal and imago ecdysis, and overall deformities clearly showed the effects of single and combined exposures (Table 1). Fe exposure caused 15% (10, 20 mg) and 29% (40mg) larval death. The toxicity of single and combined metals with PQ was generally dose dependent, with the lowest and highest larval mortality occurring at 10 mg and 40 mg exposure, respectively. PQ exposed larvae had the highest mortality, with 29% (10 mg), 58% (20 mg), and 100% (40 mg) mortality. Interaction of PQ with Zn and Fe reduced mortality at low exposure, with 15% (10 mg) and 43% (20 mg) mortality. In contrast, 40 mg exposure had 100% mortality. Interestingly, the interaction of Zn/Fe/PQ rescued larval mortality, with 0% (10 mg), 29% (20 mg), and 72% (40 mg) mortality. The length of larval mortality varied greatly, and was noticeably influenced by single and combined interactions and doses (Table 1).
Pupal ecdysis showed dose-dependent variation, with a minimal effect on pre-PQ exposure 100% (10 and 40 mg). Exposure to 20 mg Fe and Zn/Fe resulted in 94% and 89% larval ecdysis, respectively. PQ exposure significantly reduced larval ecdysis; however, its interaction with metals restored ecdysis in a dose-dependent way. Zn/Fe/PQ interaction had the highest pupal ecdysis, at 92% (10 mg) and 81% (20 mg). The length of pupal emergence was dose-dependent, with pre-post PQ exposure emergences of 6–10 days at 10 mg, 5–18 days at 20 mg, and 6–8 days at 40 mg. There was no pupal emergence with the post-PQ exposure (Table 1).
Significant dosage and treatment differences were recorded for ecdysis to imago. Pre-PQ treatments had almost no toxic effects at 10 and 20 mg of Zn and Fe (100% ecdysis). In comparison, 40 mg had the lowest (42%) emergence for Fe, Zn (59%), and Zn/Fe (65%). PQ had the maximum toxicity on imago emergence, with 42% (10 mg) and 22% (20 mg) emergence. Combined exposure with Zn and Fe recovered ecdysis, while Zn/Fe/PQ had maximum ecdysis at 76% (10 mg) and 60% (20 mg) (Table 1).
Table 1
Summary of single and co-exposure of Zn, Fe, and PQ on pupal death (% days− 1) and pupal and imago ecdysis (% days− 1) of S. littoralis (N = 360 individuals in total; 15 individuals per test). The experiments were completed in triplicate, with the results presenting the combined data. Note: a Pupal malformation. Days are counted from the day of the indicated treatment (start of the penultimate larval instar) until imago ecdysis.
Treatment | Pupal ecdysis (%)/Days | Pupal death (%)/Days | Ecdysis to Imago (%)/Days |
10 mg 20 mg 40 mg | 10 mg 20 mg 40 mg | 10 mg 20 mg 40 mg |
Control Zn Fe Zn / Fe PQ Zn / PQ Fe / PQ Zn /Fe / PQ | 100/7–9 100/6–9 100/6–8 100/6–8 76/8–10 81/8–9 67/8–9 92/9 − 6 | 100/6–7 100/6–7 94/7–8 89/5–9 50/5–9 64/7–10 49/6–11 81/6–18 | 100/6–8 100/7–8 100/7–8 100/7–8 0 0 0 0 | 0 0 0 0 10/6a 12/8a 11/7a 5/12 | 0 0 6/6a 11/5a 23/7a 15/6-7a 15/8-9a 7/9–10 | 0 0 0 0 100 100 100 100 | 100/5–8 100/6–9 100/7–9 100/9–10 42/10 57/10–12 61/10–12 76/10 | 100/4–6 100/5–7 100/3–7 88/7–10 22/8–12 35/7–12 39/7–12 60/10–12 | 100/6–7 59/8–9 42/7–9 65/10–12 0 0 0 0 |
Prominent defective molt block at different stages of the larval and pupal molting process; regionally restricted molting, molts to "intermediates" combining the regions of newly secreted larval and pupal cuticles and wing deformity in recently emerged imagoes were also observed, and were recorded as the outcome of single and mixed interaction of metals and PQ (Fig. 7).