*3.3. PTEs and PAHs in Milk*

Table 4 provides a comparison between the concentrations of inorganic elements and PAH found in the milk produced in farms close to and farther from industries. In addition, similarly to the soil and the forage, the variation in the mean (and median) concentration between both locations was calculated for each pollutant to assess its enrichment in the milk, depending on industrial proximity.


**Table 4.** Comparison of metals and polycyclic aromatic hydrocarbons in milk from farms < 5 or > 5 km from industrial areas.

Max: maximum; min: minimum; V: close-far variation; ML: Maximum Level according to EU regulations; *n*: number of farms. a: (CE 1881/2006).

The concentrations of PTEs in the milk were low, regardless of whether the farms were near to or farther from industrial areas. Hg and Pb showed substantial enrichment (50%) in farms closer to industries, while Cr, As, and Cd showed weaker enrichment (35%). These results are consistent with previous studies showing that the milk of cows on farms near industrial areas contained elevated contents of Cd [46] and Pb [47]. Nevertheless, the levels of Cd and Pb in milk were considerably higher in those studies than in the present work. Indeed, the levels of Cd in 11% of our milk samples and the level of Hg in 63% of our milk samples were below the limit of detection of our methodology (see Table 4). None of the milk samples exceeded the maximum recommended limit of 20 μg kg−<sup>1</sup> for Pb [48] (The European Union has not established limits in milk or dairy products for the other metals that we analyzed). None of our samples exceeded the maximum level of 2.6 μg kg−<sup>1</sup> for Cd, as recommended by the International Dairy Federation [49]. Perhaps these PTEs could be accumulated in the liver, kidney, or lung bovine organs, as previously stated [50].

The presence of PAHs in the milk was addressed to a lesser extent than the presence of PTEs; however, some works have reported PAH concentrations in milk from cows raised in industrial or in rural areas [4,18,19]. The presence of PAHs in milk can occur not only after ingestion of soil when livestock graze in fields, but also via feed (pasture or silage) when livestock is confined indoors [4].

We detected only three PAHs in the milk, and all three had low molecular weight: phenanthrene, fluoranthene, and pyrene. Their concentrations were similar to those reported in rural areas of France [18,19]. In contrast to the enrichment that we observed in the soil and the forage, we did not observe such enrichment in the milk, which was similar to a report comparing PAH levels in milk from rural or urban areas in France [19]. These results suggest that PAHs are not efficiently transferred into milk at such levels of pollution, as previously reported in a controlled experiment with goats, where C<sup>14</sup> PAHs were added to the diet [51].

#### *3.4. Transfer Factors between Soil and Forage and between Forage and Milk*

Table 5 provides the soil–forage transfer factor (TFsf) and forage–milk transfer factor (TFfm) for the inorganic elements.

In general, TFsf ratios were higher than TFfm ratios. Previous studies also reported very low forage–milk transfer of heavy metals, with values as low as 1:500 [52], implying that mammary glands act as barriers to prevent the entry of PTEs [53]. Na, K, Ca, and Mg had higher TFsf and TFfm, probably reflecting that they are major essential elements. TFsf values were above 1 for these elements, indicating a higher concentration of these minerals in the forage than in the soil. TFs varied across studies (Table 5), probably reflecting the complex influences on these factors, including plant species, soil properties, and dry matter intake by the animals [11]. Moreover, these TFsf could be affected by the sampling procedure, so it is necessary to remark that these data were obtained by collecting three subsamples in each location.

**Table 5.** Soil–forage (TFsf) and forage–milk (TFfm) transfer factors for potentially toxic metals.


SD: standard deviation.

The TFsf values in our work followed the trend Zn ≈Cu > Cd >> Pb, showing some discrepancies with previous work in the transfer of Cu and Zn [11,54], but consistent with a report that Zn and Cu accumulate to a much greater extent than Cd in edible plant parts [55]. Our trend is also consistent with the lower transfer of Pb from soil to plants observed in previous work, which led investigators to propose that this metal enters the human food chain via an alternative water–forage–milk pathway [9]. The TFfm values in our work followed the trend Zn > Cu > Pb > Cd, consistent with previous studies in Romania [11]. In contrast to that work, however, PTE concentrations in the present study were orders of magnitude larger in the forage than in the milk.

#### **4. Conclusions**

Our results suggest that PTEs and high-molecular-weight PAHs are enriched in soils near industrial areas, and that this enrichment led to somewhat elevated levels in the forage but not dangerously high levels in the milk (lower than the EU legislation maximum permitted level) from cows feeding on that forage. These results suggest that there is no risk for humans consuming cow's milk from these areas. Principal component analysis suggested that the sources of soil pollutants may be related to anthropogenic factors linked to industrial activity, as well as to natural soil mineralogy, as found in principal component 1 for As, Pb, Fe, and Se, emitted because of coal combustion of power plants or the steel industry. The calculated forage–milk transfer factors proved to be minimal for the most toxic elements (Cd, Hg, and Pb), with values lower than 10<sup>−</sup>3. Further, the content of PAHs and PTEs decreased along the soil–forage–milk food chain and only low molecular weight PAHs were detected in the milk. Future work should examine the fate of PTEs and PAHs in soils and farm-produced forage, as well as meat production and the health implications for cattle.

**Author Contributions:** Conceptualization, C.B., J.L.R.G., L.J.R., J.M.C.-F. and A.S. methodology, A.S., M.M.-M. and S.F.; validation, S.F., M.M.-M., A.S. and L.J.R.; formal analysis, S.F., C.B. and J.L.R.G.; investigation, A.S. and S.F.; resources, J.M.C.-F., L.J.R., S.F. and A.S.; data curation, S.F. and C.B.; writing—original draft preparation, S.F.; writing—review and editing, M.M.-M., C.B., J.L.R.G., L.J.R., J.M.C.-F. and A.S.; visualization, A.S. and S.F.; supervision, A.S. and J.L.R.G.; funding acquisition, L.J.R., J.M.C.-F. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Science and Innovation (PID2020- 117282RB-I00, MCI-20-PID2019-000081, PID2019-109698GB-I00, PID2021-126010OR-I00), and by Principado de Asturias Regional Government co-financed by the European Union through the European Regional Development Fund (grants IDI/2021/000081 and IDI/2021/000102). S.F. received an FPI fellowship (grant BES-2017-081314) supported by MCIN/AEI/10.13039/501100011033 and the Investments for the Future program of the European Social Fund, "El FSE invierte en tu futuro".

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
