<p>Two reference materials (RMs) were developed at the National Institute for Environmental Studies (NIES) for biomonitoring atmospheric mercury (Hg). First-year pine needles (<i>Pinus thunbergii</i>), reflecting atmospheric conditions over approximately one year were collected from Nara and Ibaraki prefectures and designated as NIES RM No. 1001 Pine Needle I (PN I) and NIES RM No. 1002 Pine Needle II (PN II), respectively. After removing surface dust, the needles were oven-dried at 70&#xa0;℃, ground, homogenized, bottled, and sterilized using ⁶⁰Co irradiation. Homogeneity and long-term stability tests were conducted for total Hg (THg) using cold-vapor atomic absorption spectrometry, confirming the material's suitability as RMs. The THg concentrations of PN I and PN II were 5.4 ± 0.4&#xa0;ng/g and 22 ± 2&#xa0;ng/g, respectively, lower than the NIST SRM 1575a Pine Needles (39.9 ± 0.7&#xa0;ng/g). Given decreasing background levels of atmospheric Hg, precise measurement of low-concentration samples is increasingly important. Hg isotopic analysis was performed using a two-stage furnace or microwave-assisted digestion. Isotopic values for PN I were δ²⁰²Hg = –2.63 ± 0.24‰, Δ¹⁹⁹Hg = –0.22 ± 0.17‰, Δ²⁰⁰Hg = –0.01 ± 0.10‰, and Δ²⁰¹Hg = –0.19 ± 0.17‰ (2SD, n = 8), while those for PN II were δ²⁰²Hg = –1.64 ± 0.23‰, Δ¹⁹⁹Hg = –0.51 ± 0.10‰, Δ²⁰⁰Hg = 0.00 ± 0.06‰, and Δ²⁰¹Hg = –0.52 ± 0.13‰ (2SD, n = 12). Various trace element concentrations (Al, Ba, Ca, Cd, Cu, Fe, K, P, Rb, and Zn) were also measured. These candidate RMs are suitable for quality control in heavy metal and Hg isotope studies of foliage and similar matrices.</p> Graphical abstract <p></p>