Metals are widely applied in homes, industries, agriculture and medicine, resulting in their omnipresence in the environment [1]. Children absorb these metals through the respiratory and digestive systems or skin from contaminated air, food, drinking water or toys. Typically, the sources of exposure of children aged 2–6 years in closed environments to heavy metals are mainly by biomagnification, for example when heavy metals are enriched in vegetables, fruits and cereals, or when they are secondarily enriched in fish, shrimps, livestock, etc., or when they are enriched as additives in tableware, food packaging, toys, stationery, which are taken up by the child's body through normal diet or body contact. Some metals, such as lead (Pb) and arsenic (As, despite its classification as a non-metal, arsenic is frequently present in heavy metal arsenic compounds and sulphur-arsenides, which possess toxic properties similar to other heavy metals. As a result, conversations surrounding heavy metal poisoning frequently include As), are toxic even at a low exposure level [2], [3]. Metals accumulate in the body leading to adverse health effects, such as nickel (Ni) can enter the brain tissue through the blood-brain barrier and cause damage to the central nervous system, inhalation of cadmium (Cd) -containing nanoparticles can cause lung damage, As exposure associated with a higher risk of cardiovascular disease and cancer [4]. Children are especially vulnerable to the toxic effects of metal pollutants [5]. Studies on metal exposure have been increasing, involving blood, urine, hair and other samples as biological matrices [6], [7]. However, many of these studies focus on populations engaged in specific risk behaviours, such as smoking [8], or on specific locations with high potential for environmental contamination, such as areas located close to recycling sites [9], smelting complexes [7] or mines [10]. What's more, due to the diversity and co-existence of metals in our living environment, we can hardly escape from exposure to a single metal, although studies on metal exposure in preschoolers are numerous, they mostly examined a limited number of metal categories, and we explored as many as 23 metals, which more truly reflects the status of metal exposures in preschoolers.

Haemoglobin (Hb) is a protein responsible for transporting oxygen in higher organisms, varying with age, sex, and race. Malnutrition and infection are the most frequent factors associated with abnormal Hb in children, leading to growth impairment, cognitive deficiencies, impaired immunity [11], [12]. In addition, the toxic effects of environmental pollutants may inhibit the synthesis of Hb in children and result in anaemia [13], [14], [15]. Animal and cellular experiments have found that heavy metal exposure leads to organismal damage mainly through oxidative stress or inflammatory pathways[16], [17], also causes anaemia by interfering with the haematopoietic system through inhibition of key enzymes[18]. Many epidemiological studies have shown an association between Pb exposure and anaemia or iron (Fe) deficiency. A cross-sectional study in the United States found that higher dietary Fe intake was associated with lower blood Pb levels in 299 urban children aged 9 months to 5 years [19]. Similarly, Fe deficient children were found to have higher blood Pb levels in 319 children in California and 279 children in Korea, with a relative reduction in blood Pb concentrations after Fe supplementation [20], [21]. A survey of 1078 children under 3 years of age in India in 2005 showed that blood Pb levels were associated with an increased risk of anaemia [22], and this association was also found in a cross-sectional study of 136 children living in a rural village near a Pb smelter in rural Brazil [23]. Blood Pb levels were negatively correlated with blood calcium (Ca) and Fe levels, but not with Hb levels in Chinese children aged 0–7 years [15]. The association of elevated blood Cd and blood manganese (Mn) with Fe deficiency has also been reported in children [24], [25]. Rare metals such as argentum (Ag), As, barium (Ba), bismuth (Bi), cerium (Ce), europium (Eu), erbium (Er), gallium (Ga), lanthanum (La), niobium (Nb), neodymium (Nd); Pb; praseodymium (Pr); samarium (Sm), stannum (Sn), tantalum (Ta), thorium (Th), thallium (Tl), uranium (U) and vanadium (V) were found to be negatively associated with Hb levels in a 2017 case-control study in the African adult population [26]. Mixed metal [Pb, Mn, Cd, and selenium (Se)] exposure in adolescents has been found to be associated with Hb, and their interaction relationships have also been found [27]. Another study in adolescents and women of childbearing age, elevated Cd level was associated with the prevalence of iron deficiency anaemia [28]. Additionally, the uptake of Cd could lead to anaemia when some essential metals are deficient, including Fe, Cu, Zn and calcium (Ca) [29].Due to competition between environmental metals and Fe for cellular uptake, we cannot ignore metal-to-metal interactions and cumulative effects) [30]. However, most former studies have estimated the association of single metal exposure with Hb by adjusting for other metals simultaneously using the traditional regression model ignoring non-linearity or interactions within multiply metals [31], [32], [33].

Therefore, we conducted this cross-sectional study among Chinese preschoolers and applied the joint effect models, including Quantile g-computation [34] and Bayesian kernel machine regression (BKMR) [35], [36], to estimate the single and multiple metal effects of urinary metals mixtures as well as their interactions with the Hb.