*3.3. NP Monitoring in Diverse Food Samples*

The established analysis method was applied to 1185 food samples to determine NP concentrations. Results for each food group are listed in Table 4. NP contents appeared to be higher in aquatic products than in other food groups, such as agricultural and livestock products. Due to their lipophilic and bioaccumulative properties, alkylphenols remain in the fat tissue of animals throughout the food chain [8]. The NP levels of foods from animal sources were found to be higher than those from vegetables and fruits in Taiwan [8].

NP concentrations varied between species (Tables S1–S3). The concentration ranges for all aquatic products were wide, and the standard deviations were large even within the same food type. Many factors are expected to affect this heterogeneity of the NPs, including feeding patterns and metabolism, degrees of pollution levels in specific habitats, biotransformation, and excretion capabilities. Among these various factors, the habitat environments of each aquatic organism seemed to be a major cause of NP contamination. For example, eels collected from three distinct areas of lagoons and lakes in Poland had varying NP levels, demonstrating that NP concentrations in fish were highly dependent on the sampling area [18]. The alkylphenol concentrations in fish intestines were also closely related to how close sampling sites were to sewage treatment plants. The first sampling site downstream of a treatment plant had the highest NP levels, which declined as the distance from the plant increased. The number of samples was limited, and they were pretreated as pooled samples rather than individual liver samples, making specific correlations with environmental data unclear. However, NP concentrations appeared to follow a similar pattern as the ambient data [19]. Furthermore, different species of wild freshwater and marine fish from the same river or harbor had varying NP concentrations, implying that the potential of fish species for NP bioaccumulation may depend on habitat conditions, the metabolic activity of each aquatic organism, and their feeding strategy [20].


**Table 4.** Concentration of NPs in food groups.

\* N.D.: lower than the limit of detection.

NP concentrations in blue crabs, kelps, and seaweeds among marine algae were found to be relatively high, with mean levels around or higher than 100 μg/kg. Blue crabs, in particular, are known to be inhabitants of benthic environments and sediment dwellers, making them vulnerable to estrogenic chemicals that are substantially concentrated in the sediments below the water [21]. In sediment samples of the coast of Hong Kong, NPs were found in higher concentrations than in water samples, with average values ranging from 3–8 times higher [22]. Because these species experience direct contact with sediments, they are likely to absorb a considerable amount of NPs. In kelps and seaweeds, NPs would be enriched due to the absorption of these compounds, which is a similar tendency to the concentration of seaweed samples from China [23].

The present study performed NP analysis for each individual part of the cod and pollock fish, which revealed different concentrations. The NP contents in the intestines and eggs of cod and pollock were significantly higher than those in their flesh. The concentration distributions of each sample type are shown in Figure 3. A recent study found that the average content in flounder livers was 10.7 times higher than that in flounder muscles, while it was 4.3~19.1 times higher in eel livers than in eel muscles [18], which was a similar tendency to that found in the present study.

Our results indicate that NPs in fish flesh, which is primarily composed of muscles, are more stable and less varied than in the intestines, suggesting that these chemicals do not readily metabolize in the muscles. Low concentrations in the flesh may be attributed to their effective absorption and elimination by the liver and kidneys, as well as a lack of blood flow to the muscles. A smaller deviation in the concentrations of these compounds was observed in the flesh, which possibly indicates that NPs in fish muscles mostly come from the water. Chemical absorption from water is constant and unaffected by the diet or the availability of food for the fish. Because NPs are pollutants metabolized by fish, their levels in tissues vary depending on various factors, such as the routes and metabolic rates of NPs and the reproductive maturity and feeding behaviors of the fish [18]. This makes comparisons of the levels of these compounds between fish species or fish captured in different aquatic basins challenging.

**Figure 3.** Average concentration of NPs in flesh, intestine, and roe of fish.

In a previous study, the total concentration of NP compounds analyzed in freshwater fish from other contaminated sites was as high as 5 μg/g (wet), while NPs in carps and yellow perches ranged from 18 to 2075 ng/g (wet). The presence of high quantities of NP compounds in fish suggest that they are persistent, hydrophobic, and bioaccumulative in aquatic organisms [24]. Once NPEOs enter the sediment, their degradation half-life is estimated to be approximately 60 years [25]. The presence of NPs in the environment will, therefore, continue to affect living organisms, which will consequently remain in the food chain as people consume food.

For livestock products, two key factors are expected to contribute to contamination of NPs: (1) bioaccumulation of compounds throughout the food chain and (2) migration from plastics used for packaging food products. The NP concentrations differed between the types of livestock and poultry products, and the differences were relatively large even within the same food type. Exposure to NPs of animals would mostly occur from food consumption. The amount of these compounds will, therefore, inevitably differ due to variations in the types and amounts of crops which each animal ingests. Furthermore, due to pollutant biomagnification in the food web, animals at the top of the chain are thought to have high levels of exposure to xenobiotics [26]. These chemicals are ingested mostly through their diets, especially for ruminants. A large proportion of cellulose can be found in crop residue and agroindustrial by-products such as straw in cereal and maize stover. These fibrous by-products are difficult for the rumens of ruminants to break down. Several feed additives capable of altering the fiber fermentation and digestion of ruminants are produced to enhance the ruminal environment for the purpose of promoting the efficiency of consuming roughage. As a result, nonionic surfactants can be used as feed additives for livestock. Tween 80, which contains NPEOs, is an example of a typical feed additive [27]. Our results indicated that beef had the highest average NP concentration among the analyzed livestock products. Since cows were the only ruminant in the livestock products analyzed in the present study, using NPs as feed additives was likely related to their high beef content.

Another route of NP contamination in foods is their packaging process. From plastic packaging materials in which NPs are added as antioxidants, for example, tris(nonylphenol) phosphate could migrate into foods [28]. Kawamura et al. investigated how much PVC stretch film migrated into fatty and nonfatty food types, and found that NPs moved into fatty foods at a higher rate than into nonfatty foods due to their lipophilic properties [29].

In addition to the lean meat portions of livestock, the intestines of cattle and pigs were also analyzed in the present study, considering their high rate of consumption by Koreans. NP levels were higher in intestines than in lean tissue, indicating a similar tendency to aquatic products. This could be explained by NPs having a stronger and greater affinity to tissues than intestines. Results of the distribution of concentrations for different edible portions of livestock are presented in Figures 4 and 5.

**Figure 4.** Average concentration of NPs in different edible parts of beef.

**Figure 5.** Average concentration of NPs in different edible parts of pork.

No previous study has compared the NP levels in the intestines and lean tissue of livestock. However, different internal tissues of birds, including ducks, were found to have a specific affinity to NPs. NPs accumulated the most in the muscles of each bird species, followed by the livers and kidneys. The various levels that accumulated in their livers and kidneys could possibly reflect differing degrees of excretion from the body due to a varied affinity to fatty tissues [26]. That tendency for bird intestines to accumulate NPs is expected to occur similarly in livestock products. When NPs are consumed through food or water, the intestine acts as a first barrier, absorbing and glucuronidating them. However, because the alkylphenol transport mechanism is hindered by its long alkyl chain, NP removal from the intestine is delayed, resulting in its accumulation in intestinal tissue. This NP accumulation causes its steady release into the bloodstream, which eventually reaches the liver. Although the liver can metabolize NPs, it also has a transport system that is restricted by long alkyl groups [30].

The lipid contents and NP concentrations of chicken and duck meat are listed in Table 5. Chicken breast had the lowest NP level, as well as the lowest lipid content, among types of chicken meat, with other portions with larger lipid contents having six-fold to ninefold higher average NP levels. Additionally, the highest average NP concentration among poultry products was found in duck meat, which had the highest lipid content, possibly due to the aquatic environment being contaminated, and ducks being an aquicolous species [31].


**Table 5.** Lipid contents and NP concentrations for chicken and duck meat.

\* Lipid contents were referred to the food nutrition database of the Ministry of Food and Drug Safety (2019).

The presence of NPs in agricultural products purchased from markets has been observed to vary between regions, with its concentration differing between and within vegetable species. The distribution of concentrations for each food category of agricultural products is shown in Figure 6. The main factors expected to contribute to this large variation are as follows: the differences in the degree of contamination of sludge and soil used for cultivation, and the application of pesticide products containing varying amounts of NPEOs during cultivation.

**Figure 6.** Concentration of NPs in agricultural products.

Applying sludge to agricultural land is a cost-effective technique for sewage disposal, and is one of the most common methods of sludge management. NPEOs are biodegraded in sewage treatment by the hydrolysis of ethoxylate groups, producing short-chain ethoxylates and NPs. Significant amounts of these breakdown products have been found in sewage effluents, surface waters, and sediments. Accumulation in sewage sludge induced the release of high NP concentrations into the environment once the sludge is applied to agricultural land. NPs may contact crops after being applied to soils, where they might be absorbed and accumulated through their root systems. A greater NP persistence in soil means a higher potential for crop uptake [32]. Furthermore, because of its hydrophobic property, NPs adsorb into solid organic particles in soil, and eventually bioaccumulate over time to produce large amounts [33].

NP breakdown in soil and water is influenced by factors such as oxygen availability and microbial activity, resulting in observations of substantial differences in NP persistence. The degradation of long-chain NPEOs begins with ethoxylate chains being cleaved into short chains, such as NP1EO and NP2EO. These short-chain ethoxylates further degrade into NPs, indicating that NPs are likely to accumulate due to polyethoxylate breakdown. The physical, chemical, and biological properties of individual soils ultimately control NP degradation and plant availability [32]. These results suggest that NP concentration

depends on the soil where crops are grown, which can have a wide range even within the same food type of agricultural products.

Another possible route for NPs reaching agricultural products is through adjuvants in pesticides. NPEOs are commonly used as nonionic surfactants in pesticide formulations due to their low cost and high performance. These surfactants are used in agricultural pesticides for various reasons, including increasing droplet spreading, lowering surface tension, lowering solvent evaporation rates, increasing residence duration on plant surfaces, and improving pesticide suspension and emulsion stability [33]. Using pesticides and fertilizers in agriculture can lead to NPEO degradation product accumulation with shorterchain ethoxylate groups, such as NPs, on crop surfaces. Ethoxylate degradation to NPs could result in NP accumulation in fruits, vegetables, and cereals treated with pesticides. Furthermore, NPs can remain in the soil for months after sludge application, resulting in its uptake in plants [34]. NP residues are particularly shown to be primarily generated by pesticides used in the cultivation of leafy vegetables. High NP concentrations were observed in commercial pesticide products commonly used in China, significantly differing within a range of 138~1245 mg/kg [35].

Due to the varied concentrations of these compounds in different vegetables and crops, most samples were considered to be contaminated by NPs via diverse pathways at various stages of the food production process. Some of this contamination would derive from alkylphenol ethoxylates, which are utilized in disinfectants and pesticide formulations as nonionic surfactants and emulsifiers. NPEO degradation products could induce NP accumulation on the roots and other portions of vegetables and crops after being used in agriculture. Another possible source is degradation products such as NP residues from tris(nonylphenol) phosphate, a component of plastic, which could migrate into vegetables when applied by methods involving food contact [7]. Tris(nonylphenol) phosphate is produced when NPs react with phosphorous trichloride, which is applied as an antioxidant stabilizer. As a result, when the plastic is utilized in ways where it contacts food, NP residue degradation products or impurities from tris(nonylphenol) phosphate in the plastic may migrate into agricultural products [36].

#### **4. Conclusions**

Analytical methods were developed and validated for four different matrices to determine the NP concentrations in various foods. The linearity, sensitivity, accuracy, and precision results demonstrated that the methods established in this study are efficient for determining analytes in four different matrices with varied food compositions.

The results of the analyzed NPs in foods indicate that these compounds are ubiquitous in food products in Korea, suggesting the possibility of disrupting the endocrine systems of organisms. The monitoring of these substances should, therefore, be updated regularly in order to evaluate dietary exposure in humans. The separation and identification of branched NPs in a single run still represent a great challenge due to the coexistence of many different NP isomers. Further research should, therefore, be conducted, with a focus on increasing the separation of these isomers using sophisticated analytical methods, such as GC-MS/MS and GCxGC/MS.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/foods12020269/s1, Table S1: Concentration of NPs in aquatic products; Table S2: Concentration of NPs in livestock products; Table S3: Concentration of NPs in agricultural product.

**Author Contributions:** Conceptualization, Y.-S.K., S.M.L. and M.K.; methodology and formal analysis, D.C.; data curation, Y.-S.K., S.M.L. and M.K.; writing—original draft preparation, S.M.L. and D.C.; writing—review and editing, Y.-S.K. and S.M.L.; visualization, D.C.; supervision, Y.-S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by a grant (20162MFDS116-2) from Ministry of Food and Drug Safety in 2020-2021 and BK21 FOUR (Fostering Outstanding Universities for Research, No.4299990914600) funded by the Korea government (MSIT), and Inha University Research Grant.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

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