Individual and combined contamination of the toxic metals in commercial cat and dog food

Sample collection and preparation

The samples examined in this paper were pet food imports produced between 2021 and 2022, sourced from various locations in supermarket and store chains across Beijing, Shenzhen, and Wuhan. A total of 93 pet food products were sampled, including 32 from the United States, 29 from Spain, 17 from Thailand, 10 from Germany and 5 from New Zealand, with 45 types of cat food and 48 types of dog food. In terms of food form, the samples consisted of 79 dry pet food formulations and 14 canned pet food formulations. After collection, the samples were stored according to the product storage instructions, that is the room with RH 40–60% and temperature 20–23℃. To avoid sample contamination and degradation, the sample determination should be completed as soon as possible. Before analysis, dry food samples were ground using a grinding mill (TAISITE INSTRUMENT, FW100) to ensure that 95% of the ground samples passed a 20 mesh sieve. Canned samples were homogenised using a homogeniser (Precision Labs, HM-7300).

Sample preparation for heavy metal determination

According to the national standard of China, samples for the determination of Pb (GB/T 13,080–2018), Cd (GB/T 13,082–2021), Cr (GB/T 13,088–2006) and As (GB/T 13,079–2006) were digested by the dry ashing method. A 5 g sample (0.0001 g) was charred in a porcelain crucible on a DK-98-II adjustable electric furnace, followed by ashing in a muffle furnace (Nabertherm, L9/11/B170). For Pb and Cd dettermination, 5 mL 6 mol/L HCl (Sinopharm Chemical Reagent Co.Ltd) and 5 mL 6 mol/L HNO₃ (Sinopharm Chemical Reagent Co.Ltd) were added to the ashed samples. For Cr, 5 mL 20% HNO₃ (Sinopharm Chemical Reagent Co.Ltd) was added to the ashed samples. Then the resulting solution was diluted to 50 mL with deionized water, subjected to being mixed and filtered. At the same time, the reagent blank solution was prepared. Samples for As determination were digested with 5 mL 150 g/L Mg(NO₃)₂ (Sinopharm Chemical Reagent Co., Ltd), followed by being charried and ashed in a muffle furnace (Nabertherm, L9/11/B170). Then the ashed sample were cooled and dissolved in 10 mL 3 mol/L HCl (Sinopharm Chemical Reagent Co., Ltd), followed by being transferred to a 50 mL volumetric flask, then 2.5 mL 50 g/L thiourea solution (Sinopharm Chemical Reagent Co., Ltd) was added to it, and the above solution was diluted to 50 mL by deionized water, meanwhile the reagent blank solution was prepared.

According to the national standard of China, samples for the determination of Hg (GB/T 13,081–2006) were digested using the microwave digestion method. A sample weighing 0.20–1.0 g (accurate to 0.0001 g) was placed in a digestion vessel along with 2–10 mL of 69%−70% HNO₃ (J.T.BaKer) and 2–4 mL of 30% H₂O₂ (Sinopharm Chemical Reagent Co., Ltd) in a SINEO MASTER 40 microwave digestion system (Shanghai Sineo Microwave Chemistry Technology Co., Ltd) in three heating stages. First, the samples were heated for 10 min until reaching 150℃ at 2100 W. In the second stage, the temperature was maintained at 170℃ for 5 min at 2100 W. In the last stage, the temperature was increased for 35 min to 190℃ at 2100 W. After digestion, the vessel was washed with a nitric acid solution (50 + 450) (J.T.BaKer), and the rinse was collected in a 50 mL volumetric flask and diluted to volume. Simultaneously, a reagent blank solution was prepared.

Sample analysis method

The levels of Pb, Cd and Cr were detected by flame atomic absorption spectrometry (ZEEnit 700 P, Analytik Jena AG) and the wavelengths were determined: Pb: 283.3 nm; Cd: 228.8 nm; Cr: 359.3 nm. The levels of Hg and As were analyzed by atomic fluorescence spectrometry (AFS-8530, Beijing Haiguang Instrument Co., Ltd). Quantification of toxic metals based on standard calibration in samples. Standard solutions of Pb, Cd, Cr, Hg and As (1000 μg/mL) (Guobiao (Beijing) Testing & Certification Co., Ltd) were used to configure standard series solutions, and the calibration curves for Pb, Cd, Cr, Hg and As were plotted. The standard solution has a national certification and accurate content.

The contamination rate of Pb and Cd indicates the proportion of samples where the toxic metal concentrations exceed the corresponding limit of quantification (LOQ). The contamination rate of Cr, Hg, and As indicates the proportion of samples where the toxic metal concentrations exceed the corresponding limit of detection (LOD). The exceedance rate is the proportion of samples where toxic metal concentrations exceed the Hygienical Standard for Pet Feed of China (Announcement No. 20 of 2018). The LOD and LOQ for detection method are presented in Table 1.

Quality control

Each batch of samples consists of 21 samples, each with parallel samples. Every batch also includes blank samples and certified reference materials to ensure the accuracy and stability of the testing. The reliability of the results is evaluated by calculating the recovery rate and precision. The recovery was calculated by spiking the toxic metal standard, the blank sample was not spiked. The recovery rates for Pb, Cr, Cd, Hg, and As were 105.28%, 117.93%, 102.52%, 89.9% and 107.6%, respectively. Standard solutions (Pb (5 mg/kg), Cr (1 mg/kg), Cd (0.5 mg/kg), Hg (8 µg/kg), and As (100 µg/kg)) were detected 4 times, and the relative standard deviation (RSD) was calculated to be in the range of 2.88–4.16%.

Health risk assessment

To assess the health risk of toxic metals in the samples, we calculated the daily intake of food for cats and dogs based on the metabolizable energy (ME) listed on the food labels. For foods that did not provide this information, the ME was estimated using the method described by the NRC²⁶. The short-term intake (ESTI; μg/kg/day) and the acute hazard index (aHI) were used to represent the health risk. A simulation was therefore carried out with dogs of different sizes (5 kg, 10 kg and 30 kg) and cats weighing between 2 and 5 kg. The ME for dogs was calculated using the Eq. 95 kcal × BW^0.75, while for cats, it was calculated using the Eq. 100 kcal × BW^0.6727.. The ESTI was calculated as follows:

where C represents the highest concentration of each toxic metal in all samples, and K is the calculated daily intake of the sample.

The acute hazard index (aHI) is the ratio between the exposure to a single dose of a toxic substance and the acute reference dose of toxicity for it, was calculated as follows²⁸:

where ARfD is the acute reference dose, but it should be emphasised that there are no toxicological reference values for dogs and cats for the heavy metals investigated in the present study. Therefore, in the absence of appropriate reference values for pets, we used the same reference dose (RfD; µg/kg/day) as for mammals.

Data statistics

Microsoft Excel 2016 and PowerPoint 2016 for Windows version 10 were used for statistical analysis. Data were expressed as contamination rate, maximum, minimum and mean.