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Toxicological Effects of Combined Exposure of Cadmium and Enrofloxacin on Zebrafish

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Overview

Controlled-exposure zebrafish study (AB strain; 6,000 fish purchased, of which 3,900 were used in the chronic 20-d arm and an additional ~570 across the acute 96-h Cd-alone, ENR-alone, and combined arms) characterizing the joint toxicity of cadmium (Cd) and the fluoroquinolone antibiotic enrofloxacin (ENR). Co-exposure approximately doubled acute lethality of each compound (LC50 Cd 89.12 mg/L → 46.35 mg/L; LC50 ENR 190.11 mg/L → 99.39 mg/L) with the Concentration Addition model classifying the interaction as additive (MDR 0.96, 95% CI 0.58–1.04). Under chronic exposure, ENR enhanced Cd accumulation in zebrafish liver, intestine, and muscle by 1.11–2.33-fold over single-Cd controls and exacerbated hepatic oxidative stress (SOD, CAT, GSH-Px elevated 1.34–7.06-fold at day 8 then declining 9.9–48.98% by day 20; MDA rising to 4.06-fold above controls). 16S rRNA sequencing of gut microbiota showed Cd alone reduced α-diversity (Shannon −57 to −63%) with a 16–20% drop in beneficial Cetobacterium and a 44–114% increase in Aeromonas; co-exposure further amplified the dysbiosis. The paper measures no food-matrix contamination; its HMI relevance is mechanistic (Cd toxicokinetics, antibiotic-driven Cd bioavailability enhancement, and Cd-driven gut dysbiosis).

Key numbers

All Cd and ENR values below are exposure concentrations (mg/L water-borne) or tissue accumulations (ng/g wet weight) measured in zebrafish under controlled laboratory conditions, not food-matrix contamination. Page and figure references are to Toxics 2025, 13, 378 (21 pp.).

Acute 96-h LC50 (Section 3.1, Figure 1, p. 6):

  • Cd alone: 96-h LC50 = 89.12 mg/L (six exposure concentrations 31–122 mg/L, n = 30 fish per concentration in triplicate).
  • ENR alone: 96-h LC50 = 190.11 mg/L (six exposure concentrations 120–320 mg/L).
  • Combined Cd + ENR at fixed Cd:ENR ratio 1:2.1: 96-h LC50 for the Cd component = 46.35 mg/L; for the ENR component = 99.39 mg/L. Combined exposure was 1.92× more toxic than Cd alone and 1.91× more toxic than ENR alone.
  • Concentration Addition model predicted combined effect concentration = 139.91 mg/L (sum); Model Deviation Ratio (MDR) = 0.96 with 95% CI 0.58–1.04 — classifies the interaction as additive, not synergistic, by the CA criterion. (Note: the abstract’s phrase “synergistic effects” describes the empirical LC50 shift; the formal CA/MDR classification in Section 3.1 is additive.)

Chronic 20-d exposure design (Section 2.2, pp. 3–4):

  • Five groups: CK (control); L-Cd (Cd 0.046 mg/L); H-Cd (Cd 0.46 mg/L); L-CE (Cd 0.046 + ENR 0.099 mg/L); H-CE (Cd 0.46 + ENR 0.99 mg/L). Co-exposure ratios set at 1/100 and 1/1000 of the combined 96-h LC50.
  • 3,900 fish total; three replicates per group; 260 fish per tank; water replaced every 4 days; Cd by ICP-MS (Agilent 7800), ENR by UHPLC-MS/MS (Agilent 1290-6470A); experimental concentrations held within 80–120% of theoretical values; controls below instrument detection limits.

Cd tissue accumulation under chronic co-exposure (Section 3.2, Figure 2, p. 7):

  • Tissue accumulation rank order at day 20: intestine > liver > muscle.
  • Cd-only liver at day 20: rose with prolonged exposure; the H-Cd liver accumulated ≈1,700 ng/g at day 20 (Figure 2a bar chart).
  • Cd-only intestine at day 20: H-Cd intestine ≈50,000 ng/g (Figure 2b bar chart).
  • Cd-only muscle at day 20: H-Cd muscle ≈1,150 ng/g (Figure 2c bar chart).
  • Co-exposure vs single-Cd at day 20, low-concentration arm (L-CE vs L-Cd): liver +1.13-fold, intestine +1.20-fold, muscle +1.27-fold.
  • Co-exposure vs single-Cd at day 20, high-concentration arm (H-CE vs H-Cd): liver +2.33-fold, intestine +2.20-fold, muscle +2.26-fold.
  • Overall co-exposure increased Cd accumulation 1.11–2.33-fold across tissues and time points relative to single Cd; tissue concentrations of Cd in the intestine at day 20 were 9.54–31.60 times those in liver and 16.93–38.49 times those in muscle (Section 4.1, p. 14); liver Cd 1.22–1.77 times muscle Cd.

Hepatic oxidative-stress markers (Section 3.3, Figure 3, p. 7; days 1, 8, 20):

  • All exposure groups had significantly elevated SOD, CAT, GSH-Px activities and MDA content vs CK (p < 0.05); the three enzymes showed a biphasic time course (initial rise then decline); MDA accumulated monotonically.
  • Initial elevation across all exposure groups (paper-level summary, abstract and Section 3.3): SOD ×1.34–7.06, GSH-Px ×0.98–3.28, CAT ×1.53–3.65 at day 8.
  • Later decline at day 20: SOD, CAT, GSH-Px reduced 9.9–48.98% relative to day-8 peaks.
  • MDA at day 20: up to 4.06-fold above controls.
  • Low-concentration co-exposure increment (L-CE vs L-Cd) at days 1/8/20: SOD activity +20.36%, +36.62%, +28.82%; CAT +11.24% to +17.15%; GSH-Px +20.78% to +28.53%; MDA +12.37% to +14.40%.
  • High-concentration co-exposure increment (H-CE vs H-Cd) at the same time points: SOD +15.12% to +33.80%; CAT +29.87% to +31.06%; GSH-Px +18.80% to +25.11%; MDA +16.66% to +34.45%.
  • Abstract-level summary: co-exposure elevated oxidative stress 11.24–34.48% relative to single exposures.

Gut microbiota — α-diversity and composition (Section 3.4, Figures 4–7, pp. 9–11):

  • 15 samples sequenced; 263,591 optimized sequences, 55,328,742 bp; average read 1,049 bp; 1 domain, 1 kingdom, 14 phyla, 28 classes, 79 orders, 123 families, 258 genera, 407 species, 541 OTUs.
  • Shannon α-diversity index decreased in single-Cd groups (L-Cd, H-Cd) vs CK; increased in combined groups (L-CE, H-CE) vs single Cd. Simpson index dropped further in combined groups than in single Cd. Chao and ACE indices rose under co-exposure (H-CE highest).
  • Abstract-level summary: Cd exposure reduced α-diversity Shannon index by 57–63% relative to control.
  • PCA: significant separation between L-Cd vs L-CE and H-Cd vs H-CE (R² = 0.6588, p = 0.002); PC1 explained 39.41%.
  • PCoA / PERMANOVA between single and combined exposure groups: R² = 0.6692, p = 0.002; PCo1 60.84%, PCo2 28.10% (cumulative 88.94%).
  • Phylum-level dominants: Pseudomonadota (≡Proteobacteria), Fusobacteriota, Bacillota (≡Firmicutes), Actinomycetota (≡Actinobacteriota), Bacteroidota (≡Bacteroidota).
  • CK and L-Cd groups dominated by Fusobacteriota (49.80% and 41.72% respectively); H-Cd, L-CE, H-CE dominated by Pseudomonadota (40.64%, 49.53%, 40.44%).
  • Compared to CK, Fusobacteriota decreased and Bacillota increased in Cd-alone groups. L-CE vs L-Cd showed increases in Pseudomonadota and Actinomycetota and decreases in Fusobacteriota and Bacillota; H-CE vs H-Cd showed a significant decrease in Fusobacteriota and increases in Bacillota and Actinomycetota.
  • Top genera: Aeromonas, Cetobacterium, ZOR0006, Shewanella, Mycoplasma.
  • Abstract-level summary: 16–20% reduction in beneficial Cetobacterium; 44–114% increase in pathogenic Aeromonas under Cd exposure; co-exposure exacerbated this dysbiosis.
  • Genus-level comparisons: vs CK, Aeromonas and ZOR0006 increased and Cetobacterium decreased in Cd-alone groups. L-CE vs L-Cd: Aeromonas and ZOR0006 increased, Cetobacterium decreased. H-CE vs H-Cd: Aeromonas and Cetobacterium decreased significantly, ZOR0006 increased significantly.
  • LEfSe (LDA > 3.0): 40 indicator species identified — CK (10), L-Cd (5), H-Cd (7), L-CE (8), H-CE (10).
  • Co-occurrence network: CK 50 nodes/183 edges (modularity 0.59); L-Cd 43/197 (0.60); L-CE 41/119 (0.50); H-Cd 45/155 (0.48); H-CE 43/162 (0.51). Network degree and centrality decreased under most exposures vs CK (except low-Cd group); network complexity rose under combined exposure (Figure 8H).

Methods (brief)

Static-renewal aquatic-toxicology design under China’s “Chemicals — Acute Toxicity Test for Fish” GB/T 27861-2011 protocol (Approval No. BMY-ZY-202402, Guizhou Provincial Analytical Testing Research Institute). Zebrafish (AB strain, 3–4 months) acclimatized 15 days; rearing water aerated 48 h, quartz-filtered, UV-sterilized; 25 ± 1 °C; pH 7.2–7.5; 14 h light/10 h dark. Acute test: 96-h static exposure, six concentration gradients per compound and per mixture, 10 fish per replicate × 3 replicates per concentration, mortality scored at 24, 48, 72, 96 h, LC50 via probit-concentration regression and Fieller’s theorem in SPSS 2019. Chronic test: 20-d exposure at 1/100 and 1/1000 of combined LC50; water replaced every 4 days; Cd in tissues by Agilent 7800 ICP-MS after polytetrafluoroethylene-vessel microwave digestion (Mars 6, CEM) in 8 mL HNO₃ + 2 mL H₂O₂ for 4 h; ENR by Agilent 1290-6470A UHPLC-MS/MS. Antioxidant enzymes (SOD, CAT, GSH-Px), MDA, and total protein measured on 10% liver homogenates (Sorvall Legend Micro 21R, Thermo Fisher) using Nanjing Jiancheng Bioengineering Institute kits read on a Multiskan GO microplate reader (Thermo Fisher). 16S rRNA V3–V4 region (primers 338F/806R, Sangon Biotech) amplified, library-prepped, sequenced on Illumina NextSeq2000 (Majorbio); OTUs clustered at 97% UPARSE; taxonomy annotated against SILVA v138; analyses in R packages vegan (α-diversity), PCA/PCoA (β-diversity), LEfSe (LDA > 3.0, p < 0.05), and Hmisc + igraph for Spearman co-occurrence networks (|r| > 0.6, p < 0.05). Concentration Addition model and Model Deviation Ratio used to classify joint toxicity. Joint toxicity assessment formulas: ECx,mix = (Σ pᵢ / ECx,i)⁻¹; MDR = ECx,PRE / ECx,OBS; MDRUpper = ECx,Upper/ECx,OBS; MDRLower = ECx,Lower/ECx,OBS. Synergy if MDR > MDRUpper; antagonism if MDR < MDRLower; addition otherwise.

Limitations the authors acknowledge: exposure concentrations are higher than typical environmental levels (the cited environmental ENR range is 0.019–11.9 µg/L; Cd freshwater EU limit 0.25 µg/L); zebrafish responses extrapolate to other teleosts and humans only with caution; no molecular-mechanism work was performed.

Implications

  • Certification (HMTc): The paper measures no food-matrix Cd contamination and proposes no certification threshold. The relevance to HMTc is indirect — it documents an antibiotic-driven mechanism (Cd-ENR complexation enhancing Cd absorption 1.11–2.33-fold) that strengthens the case for considering co-pollutant context when interpreting Cd biomonitoring data from aquaculture-sourced ingredients. No threshold contribution.
  • Courses: Teachable as a case study in joint-toxicity classification (CA model vs empirical LC50 doubling) and in the distinction between “synergistic” as a colloquial descriptor and as a formal CA/MDR classification.
  • App: Does not contribute primary occurrence values to any ingredient’s contamination_profile. Belongs in the cadmium mechanism evidence layer, not the contamination layer.
  • Microbiome: Direct evidence for Cd-driven gut microbial dysbiosis in a teleost model (Shannon −57 to −63% under Cd; Cetobacterium −16 to −20%; Aeromonas +44 to +114%). Relevant to microbiome/cadmium-gut-axis crosswalk for WikiBiome federation and to the broader Cd-microbiome literature accumulating on this wiki.

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