Acute oral toxicity of zinc phosphide: an assessment for wild house mice (Mus musculus)

**Background:** Zinc phosphide (ZnP) is the only registered broadacre rodenticide for controlling house mouse plagues in Australian grain production, where outbreaks cause economic losses of AU$64–200 million. Farmers have reported increasingly variable efficacy of ZnP over the past 10–15 years, raising questions about whether mice have developed tolerance, whether bait aversion is occurring, or whether the presence of alternative food reduces uptake of ZnP-coated grains. The previously reported LD50 for wild house mice was 32.7 mg/kg body weight, and the registered bait loading is 25 g ZnP/kg grain (approximately 1 mg ZnP per grain, intended to deliver an LD90 dose to a 15 g mouse).

**Methods:** Three groups of mice were studied: (1) wild mice from a farm on Yorke Peninsula, South Australia, where ZnP baiting occurred regularly (at least once per year for 9 of the last 12 years; "exposed"); (2) wild mice from a horse stable in Murrumbateman, NSW, where ZnP had never been used ("naïve"); and (3) an outbred laboratory strain. In Experiment 1, oral gavage of ZnP in vegetable oil was administered at doses ranging from 30 to 149 mg ZnP/kg body weight, following the OECD Up/Down protocol. Group sizes per dose were n ≥ 4. Mice were observed for 48 hours post-gavage. In Experiment 2, wild mice were offered ZnP-coated wheat grains at either 25 g ZnP/kg grain (F25 group, with alternative food) or 50 g ZnP/kg grain (F50 group with alternative food; UF50 group without alternative food for 4–6 hours). Grains were presented for 3 consecutive days. ZnP concentrations were independently verified by ICP-OES analysis (recovery 95.4–101.3% for oil suspensions; grain coating averaged 1.06 ± 0.11 mg ZnP/grain for 25 g/kg batch and 2.4 ± 0.37 mg ZnP/grain for 50 g/kg batch). Statistical analysis used generalized linear models with binomial error structure in R.

**Key Results:** In Experiment 1, the estimated LD50 values were 79.18 ± 6.24 mg/kg for outbred laboratory mice, 72.11 ± 9.09 mg/kg for naïve wild mice, and 75.22 ± 4.39 mg/kg for exposed wild mice—all significantly higher than the previously reported 32.7 mg/kg. LD90 values were 114.58 ± 14.6, 119.50 ± 18.01, and 102.27 ± 9.34 mg/kg, respectively. No significant differences were found between mouse groups, sexes, or fed/unfed status. Time to death ranged from 2.5 to 48 hours post-gavage, with higher doses associated with more rapid onset of toxicity. In Experiment 2, 94.4% (17/18) of mice in the UF50 group (no alternative food, 50 g ZnP/kg grain) died after consuming ZnP-coated grains. In contrast, mortality was 46.7% (7/15) in the F50 group and 42.1% (8/19) in the F25 group where alternative food was available. Aversion (consuming a sublethal dose on day 1 and refusing ZnP-coated grains thereafter) occurred in 46.7% of F50 mice, 42.1% of F25 mice, but only 6% of UF50 mice. Most mice consumed 1–2 ZnP-coated grains on the first day of presentation.

**Clinical Implications:** The finding that Australian wild house mice have an LD50 more than double the previously reported value has direct implications for rodent control. The current registered bait loading of 25 g ZnP/kg grain (∼1 mg ZnP/grain) may deliver a sublethal dose to many mice, promoting bait aversion and reducing control efficacy. The authors recommend increasing the ZnP loading to approximately 50 g ZnP/kg grain (∼2 mg ZnP/grain) to ensure a single grain delivers a lethal dose. However, the presence of alternative food significantly reduces mortality even with the higher dose, suggesting that baiting strategies should consider food availability in the field. The study found no evidence of genetic resistance or tolerance development from repeated ZnP exposure, as LD50 values were similar between exposed and naïve wild mice. The authors note that a field assessment of the higher dose has been conducted (Ruscoe et al. 2022) and has validated these laboratory findings. They also call for reassessment of ZnP LD50 values for other rodent species internationally, as the current findings for house mice suggest that previously reported values for many species may need revision.