## 5 Conclusions  
Poly(styrene) cups showed a relatively low level of styrene migration with the highest being 0.110 \(\upmu\)g/mL, whereas food containers showed a much higher level of styrene leaching of up to 6 \(\upmu\)g/mL. This could be due to an increase in the hydrophobicity of the simulants' characteristics from low to high fat content and the increase in the testing temperatures from 5 \({}^{\circ}\)C to 70 \({}^{\circ}\)C. Statistically, results showed that the migration level of styrene has increased, the oil content being 95% ethanol. This means increasing the amounts of fat content in food, combined with increasing temperatures and longer exposure to the packaging, significantly increased the level of migration and thus ingestion from food. ANOVA statistical analysis is used to compare the means of three or more groups of data, whereas t-test analysis is used to compare the means of two groups. This was carried out on each individual sample to determine the significance of changing the temperature, simulant type, or both, on the level of migration observed in the results. All significant values were tested at 95% confidence level \(p<0.05\), concluding that fat content and high temperatures were found to significantly increase the level of styrene migration. HPLC was used to detect styrene migration in the range of (0.0004 \(\upmu\)g/mL to 6.423 \(\upmu\)g/mL) across all food and drink packaging samples tested. Although the migration levels in this investigation did not exceed the safety limits of (10 mg/dm\({}^{2}\)) set out in regulatory documents by the European Union 10/2011 legislation, the cumulative exposure to styrene from food packaging and other routes should not be overlooked. From the HPLC chromatograph of methanol solution spiked with 10 \(\upmu\)g/mL styrene, RT seems to be consistent at around 2.1-2.3 for styrene. (See Figures S5 and S6 in SI). Other peaks could be assigned to 4-_tert_-butylcatechol, a styrene stabilizer used to inhibit polymerization into (PS). Contamination, polymerization, or change of column can also change the elution time and add extra peaks. Moreover, styrene can behave differently in different solvents which may lead to different retention times (RT). Another difficulty faced with HPLC analysis is that distorted peaks can also be a result of a mix up with background noise. Moreover, introducing new columns or stationary phases species can interact with the analyte in an unintentional manner in what is called'silanol' species forming. The ability of Nile red to detect leaching Poly(styrene) microplastic particles was also seen to be a successful method. Nile Red stain was reported [26] to be adsorbed to polymeric materials and fluorescences under specific wavelengths of incident light. These particles were measured at between 6 \(\upmu\)m and 104 \(\upmu\)m in size, with the highest migration (2-10 per cm\({}^{2}\)) observed from a Poly(styrene) in simulant 50-95% ethanol at 70 \({}^{\circ}\)C.  
This did not include any consideration of additional microplastic migration due to behaviour, such as use of cutler, so in practice levels could be much higher. The desirability of phasing out Poly(styrene)-based packaging is well understood, but in reality, it is likely to be in use for many more years, hence more detailed exposure estimates are still of great relevance and microplastic shedding is an emerging issue that should be evaluated much more extensively, particularly as alternatives to Poly(styrene) are sought and introduced. The following are available online at [[https://www.mdpi.com/article/10.3390/foods10051136/s1](https://www.mdpi.com/article/10.3390/foods10051136/s1)]([https://www.mdpi.com/article/10.3390/foods10051136/s1](https://www.mdpi.com/article/10.3390/foods10051136/s1)), Table S1. Nile red staining indicative of plastic leaching in Samples 5-7 tested after 10 days at 5 \({}^{\circ}\)C, Table S2. Nile red staining indicative of plastic leaching in Samples 5-7 tested after 2 hours at 60 \({}^{\circ}\)C, Table S3.