Ren et al. 2025 - cadmium and enrofloxacin co-exposure in zebrafish

Ren and colleagues tested acute and chronic co-exposure of cadmium (Cd) and the fluoroquinolone antibiotic enrofloxacin (ENR) in zebrafish. The paper reports Cd and ENR exposure concentrations, 96 h LC50 values, tissue-specific Cd accumulation in liver, intestine, and muscle, liver oxidative-stress biomarkers, and gut-microbiome changes. This is primary aquatic ecotoxicology and microbiome evidence, not food, ingredient, or consumer-product occurrence evidence.

Key numbers

Acute toxicity design and LC50s

The acute test followed the Chinese fish acute-toxicity test method GB/T 27861-2011. Cd chloride and ENR were tested singly and in combination for 96 h, with triplicate groups and 10 zebrafish per replicate.

Cd-only exposure concentrations were 31, 49, 68, 86, 104, and 122 mg/L. The text lists ENR-only concentrations as 120, 160, 240, 280, and 320 mg/L; Figure 1 also shows a 200 mg/L ENR point. The combined-exposure concentration pairs were:

The reported 96 h LC50 values were:

The authors state that the combined-exposure toxicity was 1.92 times that of Cd alone and 1.91 times that of ENR alone. Using the concentration-addition model, the predicted combined-effect concentration was 139.91 mg/L. The model-deviation ratio (MDR) was 0.96, within the reported confidence interval of 0.58 to 1.04, so the Results section classifies the Cd-ENR combined effect as additive.

Chronic exposure design

The 20 d chronic experiment used five groups:

The chronic experiment used 3900 zebrafish, randomly allocated to experimental tanks with three replicates per group and 260 fish per tank. Rearing water was changed every four days. Cd was measured by ICP-MS and ENR by UHPLC-MS/MS in water samples before and after water changes; the authors report that treatment concentrations remained within 80% to 120% of theoretical values, and both Cd and ENR were below detection limits in the control group.

Samples were collected on days 0, 1, 2, 4, 6, 8, 12, 16, and 20 for Cd in zebrafish tissues; on days 1, 8, and 20 for liver antioxidant biomarkers; and on day 20 for gut-microbiome sequencing.

Tissue Cd accumulation

Figure 2 reports Cd concentrations in zebrafish liver, intestine, and muscle. The point values are plotted graphically rather than tabulated, so this page records only the exact text-reported comparisons.

The tissue-order pattern was intestine > liver > muscle. Cd content increased with exposure time and with exposure concentration. For the same exposure duration, tissue Cd concentrations in the combined Cd+ENR groups were higher than in the Cd-only groups.

At 20 d in the low-concentration groups, adding ENR increased Cd accumulation relative to Cd-only exposure by:

At 20 d in the high-concentration groups, adding ENR increased Cd accumulation relative to Cd-only exposure by:

In the discussion, the authors add that after 20 d, intestinal Cd accumulation was 9.54-31.60 times liver Cd and 16.93-38.49 times muscle Cd, while liver Cd was 1.22-1.77 times muscle Cd.

Oxidative-stress biomarkers

The liver oxidative-stress biomarkers were SOD, CAT, GSH-Px, MDA, and total protein. Compared with controls, all exposure groups had significantly elevated SOD, CAT, GSH-Px, and MDA (p < 0.05). The authors describe a biphasic enzyme pattern: SOD, GSH-Px, and CAT rose early and then declined with longer exposure, while MDA accumulated over time.

The abstract reports that at day 8, SOD, GSH-Px, and CAT activities initially increased by 1.34-7.06-fold, 0.98-3.28-fold, and 1.53-3.65-fold, respectively, followed by 9.9-48.98% reductions after 20 d exposure. MDA reached up to 4.06-fold higher than controls.

Relative to Cd-only exposure, co-exposure elevated oxidative-stress indicators by 11.24-34.48%. In the low-concentration comparison (L-Cd vs L-CE), SOD activity increased by 20.36%, 36.62%, and 28.82% after 1, 8, and 20 d, respectively; CAT increased by 11.24-17.15%; GSH-Px increased by 20.78-28.53%; and MDA increased by 12.37-14.40%. In the high-concentration comparison (H-Cd vs H-CE), SOD increased by 15.12-33.80%, CAT by 29.87-31.06%, GSH-Px by 18.80-25.11%, and MDA by 16.66-34.45%.

Gut microbiome

The 16S rRNA analysis used 15 intestinal samples and produced 263,591 optimized sequences, 55,328,742 base pairs, an average sequence length of 1049 bp, and 541 OTUs. The annotation summary included 14 phyla, 28 classes, 79 orders, 123 families, 258 genera, and 407 species.

The abstract states that Cd exposure reduced the Shannon index by 57-63%, reduced beneficial Cetobacterium by 16-20%, and increased pathogenic Aeromonas abundance by 44-114%. In the Results section, combined Cd+ENR exposure increased Chao, ACE, and Shannon relative to single Cd exposure while decreasing Simpson, which the authors interpret as dysbiosis rather than a protective diversity gain.

At the phylum level, Fusobacteriota dominated the CK and L-Cd groups at 49.80% and 41.72%, respectively. Pseudomonadota dominated H-Cd, L-CE, and H-CE at 40.64%, 49.53%, and 40.44%, respectively. The dominant genera were Aeromonas, Cetobacterium, and ZOR0006.

Beta-diversity analysis found separation between L-Cd and L-CE and between H-Cd and H-CE. PCA reported R2 = 0.6588 and p = 0.002, with PC1 accounting for 39.41% of variance. PCoA reported PERMANOVA R2 = 0.6692 and p = 0.002, with PCo1 explaining 60.84% and PCo2 explaining 28.10%.

Network analysis found 50 nodes and 183 edges in the CK group. Exposure-group network counts were L-Cd 43 nodes and 197 edges, L-CE 41 nodes and 119 edges, H-Cd 45 nodes and 155 edges, and H-CE 43 nodes and 162 edges.

Methods (brief)

AB-strain zebrafish aged 3-4 months were acclimatized for 15 d at 25 +/- 1 degrees C, pH 7.2-7.5, and a 14 h light:10 h dark cycle. Acute exposure used static glass-tank tests with mortality recorded at 24, 48, 72, and 96 h. Chronic exposure used waterborne Cd and Cd+ENR groups for 20 d, with periodic water replacement and analytical verification of water concentrations.

Tissues were dissected on ice after euthanasia. Liver, intestine, and muscle samples were microwave-digested with nitric acid and hydrogen peroxide, then Cd was measured by ICP-MS. Antioxidant biomarkers were measured in liver homogenates using commercial SOD, CAT, GSH-Px, MDA, and total-protein assay kits. Gut microbiota was characterized by V3-V4 16S rRNA amplicon sequencing on an Illumina NextSeq2000 platform, OTU clustering at 97% similarity, SILVA v138 annotation, alpha- and beta-diversity analysis, LEfSe biomarker screening, and Spearman co-occurrence networks.

Implications

Certification: Do not use this source in HMTc food, ingredient, or product occurrence pools. The measured matrix is experimentally exposed zebrafish tissue from a laboratory Cd/antibiotic co-exposure study, not market fish, feed, water, or consumer products.

App: Context for combined-pollutant risk. The source supports the principle that antibiotic co-contamination can increase Cd bioaccumulation and toxicity in aquatic organisms relative to Cd-only exposure, so single-contaminant risk models may understate ecological risk in polluted waters.

Courses: Useful for teaching mixture-toxicology adjudication, especially the distinction between an exposure concentration, an LC50, tissue accumulation, oxidative-stress endpoints, and microbiome dysbiosis.

Wiki pages this source may touch

• cadmium
• index
• icp-ms

Verification notes

This page was built from the full PDF, including the abstract, Methods sections 2.1-2.3, acute and chronic exposure descriptions, Figures 1-8, Results sections 3.1-3.4, discussion, conclusions, and data-availability statement. Products and ingredients are intentionally empty because the source is a laboratory zebrafish ecotoxicology experiment. The abstract describes the mixture as having “significant synergistic effects,” but the Results section reports MDR = 0.96 within a 0.58-1.04 confidence interval and classifies the mixture effect as additive; the Conclusions section also says additive. This page follows the Results/Conclusion classification and records the abstract wording as an internal inconsistency. The ENR-only acute concentration list in the text omits the 200 mg/L point shown in Figure 1b, so the page preserves both observations without inferring missing mortality values.

Page history

The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.