Combined toxic effects of polyethylene microplastics (PE-MPs) and 4-nonylphenol on the common carp (Cyprinus carpio) juvenile: multi-biomarker approach

There is inadequate knowledge of the cumulative impact of exposure to 4-nonylphenol (4-NP) and polyethylene microplastics (PE-MPs), and in particular of whether their joint action departs from additivity. The objective of this study was to determine and formally compare the single and combined effects of 4-NP and PE-MPs on multiple biomarkers in juvenile Cyprinus carpio. Fish were assigned to a fully crossed 2 × 2 factorial design comprising a control group and three treated groups receiving 10 mg/L PE-MPs, 200 µg/L 4-NP, or their binary mixture, for a two-week exposure period followed by a two-week depuration period. Because the four groups constitute a factorial arrangement, data were analysed by two-way ANOVA with a PE-MP × 4-NP interaction term, and departures from additivity were quantified against both effect-addition and independent-action null models.Both contaminants significantly suppressed antioxidant enzyme activity (SOD, CAT, GPx), suppressed neurological markers (AChE, MAO, NO), and elevated pro-inflammatory cytokines (IL-1β, IL-6), DNA strand breakage and erythrocyte apoptosis (all P < 0.05). The PE-MP × 4-NP interaction was significant at nine of ten endpoints, but its direction depended on the biological process examined. Suppression of CAT, SOD and GPx and induction of apoptosis were greater than additive, whereas inhibition of AChE, MAO and NO and elevation of IL-1β and IL-6 were less than additive; DNA strand breakage showed no significant interaction. All departures from additivity were below the two-fold threshold conventionally required to denote toxicologically meaningful synergism, so the joint action is best described as approximately additive with endpoint-specific modulation rather than as broadly synergistic.A two-week depuration period abolished the antagonistic interactions at cholinergic and monoaminergic endpoints and returned DNA strand breakage in the single-contaminant groups to control values, but did not restore antioxidant enzyme activity, resolve cytokine elevation, or reverse apoptosis, which remained more than five-fold above control in the mixture group. Serum protein electrophoretic profiles varied in fraction number and molecular weight distribution across treated groups; these profiles derive from a single pooled sample per group and are reported descriptively. A three-way analysis showed that the interaction itself changed significantly between exposure and depuration at six of ten endpoints, indicating that mixture interaction is a time-dependent rather than a fixed property. These outcomes indicate that interaction assessments based on a single biomarker or a single time point may misrepresent the joint hazard of microplastic-associated contaminants in aquatic ecosystems.