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ZETASIZER

In this section we weill discuss the fact that the most consumed brand in central Mexico is the one with the highest concentration of microparticles thoughout the experiments we carried out, our analysis and collaborations. So, with the aim of detecting the presence of nano and microparticles in bottled water for determining their intake by consumers, we used the Zetasizer device at our university to carry out an analysis of water samples from bottles of different brands at the conditions presented below:

 

• Condition A: No treatment (water bottles as purchased)

• Condition B: 37°C for 7 days

• Condition C: Sun radiation for 7 days ~ 35-38°C

• Condition D: Impact test*

• Condition E: Agitation Test for 7 days at oscillatory agitation (250 RPM)*.

 

*These conditions weren’t applied to the glass and aluminum bottles

 

The studied water bottles were from the following brands:

 

• Brand #1

• Brand #2

• Brand #3

• Brand #4

• Brand #5

• Brand #6

• Brand #7

• Brand #8

• Brand #9

 

To make a comparison between the particles liberated in water from bottles of different materials, some of these bottles were made of plastic and others were made of glass and aluminum.

 

The results and the conditions applied for each bottle are shown in the graphs presented below (the X axis represents the size of the particles in nm and the Y axis represents the intensity of the signal in percentage):

PET bottles
Brand #1

Analysis of Brand #1 bottled water demonstrated the presence of particles at all conditions, even when no treatment was applied (condition A). Also, a change in particle dimensions and amount occurred when the bottle was subjected to heat (condition B); the same thing happened for the impact test (Condition D). With sun radiation (condition C) and agitation (condition E), two groups of particles of different sizes appeared, with a distinct distribution for each condition. This showed us that the conditions that had a major effect on the particles release were the solar radiation and agitation. 

Brand #2

For water samples of Brand #2, large particles were detected in condition A; however, for conditions B and C there was presence of particles of smaller sizes, which indicated degradation. In condition D, two groups of particles appeared, but the larger group didn’t have a significant change in dimension respecting the particles from condition A. For condition E, two groups of particles were present as well, but with different dimensions and distribution than those from condition D. This revealed that heat,  radiation and agitation affected most the particle liberation.

Brand #3

In the water samples of Brand #3, two groups of particles were present in water for conditions A and C; nevertheless, they were different in distribution and amount, showing a change between the two conditions. The impact test performed for condition D only caused the appearance of one group of particles. For condition E, three groups of particles were detected, all of different amounts and sizes. This demonstrated that the particles’ distribution and amount changed when the bottle was subjected to treatment. For condition B, the device indicated “error”; thus, no measurements could be made. 

Brand #4

Analysis of Brand #4 bottled water showed presence of particles for every condition. For condition B, the particles were of a considerably smaller size than those from condition A, which implied degradation. Also, two groups of particles of different amounts and dimensions appeared for condition C, as well as for condition E. The particles detected in condition D were larger in size than most of the particles from the other conditions. This revealed a greater effect of sun radiation and agitation for the particle liberation.

Brand #5

No particles were detected in Brand #5 water samples for conditions A and D, which indicated a higher quality of the bottle; however, when heat was applied (Condition B), a group of particles appeared in water; the same thing happened when the bottle was subjected to agitation (condition E). The sun radiation exposure (Condition C) caused the liberation of even more particles, presenting three different groups of them; thus, being the major condition for particle generation.

Glass and aluminium bottles

Brand #6

Brand #6 water samples featured particles in all three conditions. Particularly, for condition A two groups of particles were detected and just one for conditions B and C. This was unexpected to us, since water is contained in a glass bottle; however, we believe that the plastic cap might be responsible for the particles’ release.

Brand #7

The Brand #7 of water is presented in an aluminium container, the company stating themselves that the container is plastic free. Nonetheless, every metallic container needs a thin layer of protective coat, similar to soda cans. A polymer plastic lining is added to these cans to avoid the soda and aluminum reacting with each other. This may be the reason why this brand of water presents the same kind of nanoparticles throughout the tests.

Brand #8

Brand #8 of water showed no particles throughout the three tests made. The principal reason this brand may not show any particles according to our study is because the water is contained in a glass bottle, not a plastic one. Demonstrating another great advantage of preferring glass over plastic, apart from is greater simplicity to be recycled.

Brand #9

Brand #9 of water, just as Brand #8, showed no particles throughout the three tests made. The principal reason this brand may not show any particles according to our study is because the water is contained in a glass bottle, not a plastic one. Demonstrating another great advantage of preferring glass over plastic, apart from is greater simplicity to be recycled.

Overall analysis

Presence of particles was observed in all PET water bottles at most conditions. In every case, when subjected to heat or heat with solar radiation, a change in dimensions and distribution of the particles occurred. Specifically, a population of smaller particles appeared, which is related to degradation. Also, in general for all brands, no significant changes were observed in either quantity, distribution, or particle size for the impact test. Regarding the agitation test, more than one group of particles was detected for most cases, presenting particles of larger dimensions than those from the other conditions.

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On the other hand, there was no presence of particles for two of the glass bottles (Brand #8 and Brand #9) at any condition. We hypothesize that particles detected in the remaining glass bottle (Brand #6) were due to the plastic bottle cap. Also, the presence of particles in the aluminum bottle water sample could be caused by some of the materials used in the bottle manufacturing. 

These results could be explained due to the effects of UV light, high temperatures and agitation on plastic. Particularly, we have found in literature that UV exposure can be an important source of plastic degradation, acting through autocatalytic thermal oxidation, and leading to an increase in the amount of microplastics and microfibers [1]. Furthermore, it has been shown that “plastic polymers have viscoelastic properties that can be altered by temperature…” [2]; in fact, hot water might exacerbate degradation, contributing to the release of microplastics [3]. Additionally, fragmentation of plastic is likely to occur because of agitational and mechanical forces, which might produce cracks in the material, especially if the bottle has been previously exposed to sunlight, becoming weak and brittle [4].

All those findings will allow us to estimate the approximate intake of EDCs considering the amount of microplastics present in each sample, and determine which factors (temperature, sun exposure, brand, etc.) favor the liberation of these substances in bottled water.
 

References:

[1] L. Sørensen, A. S. Groven, I. A. Hovsbakken, O. Del Puerto, D. F. Krause, A. Sarno, and A. M. Booth, “UV degradation of natural and synthetic microfibers causes fragmentation and release of polymer degradation products and chemical additives”, Science of The Total Environment, vol. 755, no. 2, February 2021
 

[2] A. I. Catarino, R. Thompson, W. Sanderson, and T. B. Henry, “Development and optimisation of a standard method for extraction of microplastics in mussels by enzyme digestion of soft tissues”, Environmental Toxicology and Chemistry, vol. 36, no. 4, pp. 947–951, August 2016

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[3] M. Godoy. (2020, October 19). Study: Plastic Baby Bottles Shed Microplastics When Heated. Should You Be Worried? [Online]. Available: https://www.npr.org/sections/goatsandsoda/2020/10/19/925525183/study-plastic-baby-bottles-shed-microplastics-when-heated-should-you-be-worried

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[4] INTERNATIONAL MARITIME ORGANIZATION (n.d.). Sources, fate and effects of microplastics in the marine environment: a global assessment. [Online]. Available: https://ec.europa.eu/environment/marine/good-environmental-status/descriptor-10/pdf/GESAMP_microplastics%20full%20study.pdf

Collaboration

Monterrey Collaboration

To widen our study about the presence of microplastics in bottled water, we collaborated with the iGEM TEC MTY team, designing a protocol to use the Zetasizer device (read more here) and asking them to analyze samples of water bottles commonly found in their region. This would allow us to make a comparison between the microplastic particles' presence at different conditions in the bottles they analyzed, and the bottles our team analyzed at the same conditions. Furthermore, as the iGEM TEC MTY team only used plastic bottles, their results could help us to validate the data we obtained regarding the conditions that have a major effect in particle liberation from this material. On the other hand, both teams belong to the same country; thus, our collaborative study could set the path for acknowledging the microplastics problematic in our community and establishing preventive measures to diminish their intake.

The iGEM TEC MTY team applied the following conditions to their samples:

• No treatment (normal conditions at which it was purchased)

• Constant sunlight exposure for 12-16 hours, with daily exposure for 2 weeks (14 days)

• Exposure to 37 ° C for 1 week (7 days)

• Constant agitation of 250 RPM for 1 week

• 1 m impact resistance

They analyzed bottled water from the brands presented below:

• Brand #1

• H2O BioLeve

• Wonu Agua

 

The results for each brand are shown below:

Similar to the trend pointed out for the data obtained by the team, the data shown in the previous five graphs of “Intensity” vs “Size” suggest that, independently from the nature of the treatment or condition to what the bottled water was exposed, there were particles smaller than 2780 nm in diameter (the smallest ones that were detected are of 0.6470 nm) (keep in mind that the bottles were made of plastic). It is worth mentioning that there were two peaks in almost every graph (for almost every tested condition). The only scenario where there was just one peak was the fourth scenario, where the bottle was constantly shaken (250 rpm, for seven days). 

Additionally, it is interesting to note that the condition number one, which is no treatment at all, “yielded” two peaks, and one of them corresponds to particles of 0.6470 nm and the other one to particles of 2780 diameter. Considering this, and the predicted sizes for the particles detected in the other scenarios, it could be hypothesized that some particles of 2780 nm were reduced in size and lead to the apparition of particles of 171, 265.6, 110.1, 1477 and maybe even particles of 2400 nm. However, with the data at hand it is not possible to determine whether the smaller particles come from the particles that had a diameter of 2780 nm or if they are newly detached particles (recently detached from the inner wall of the bottle).

Finally, and considering the data obtained from the experiments conducted by iGEM TecCEM team, even though it corresponds to the same brand of bottled water, and although the conditions tested by this team and iGEM TEC MTY were virtually identical, the particle sizes were of different sizes; as the biggest particles detected by iGEM TecCEM (for Brand #1 bottles) were of 405.1 nm in diameter. Nevertheless, more data is needed to statistically determine the difference between these two datasets.

The five previous graphs correspond to the brand “H2O BioLeve” and the water from the bottles that was analyzed was contained in plastic bottles. 
For three out of five conditions, two groups of particles were identified and, for the other two conditions, just one group of particles was identified. Once again, in the cases where two groups of particles were detected, one group corresponded to particles of a diameter greater that 2000 nm (more specifically, either 2780 or 5560 nm) and the other groups is made up of particles that have a diameter of one order of magnitude smaller that the diameter of the bigger particles.

Recalling the data shared by iGEM TEC MTY for Brand #1 bottles, it is interesting that, among these two brands (Brand #1 and H2O BioLeve), BioLeve has the biggest particles of all, regardless of the condition applied to the bottled water. Talking about this maximum value registered, this value is associated with the bottled (H2O BioLeve) water that was analyzed after a 1 meter drop. Keeping in mind the previous relation, it would be useful to have more data (repetitions) to determine if this condition has in fact a statistical significance diameter of the measured particles. 

Regarding the five previous graphs, they are the graphical representation of the size distribution of the particles detected for samples of “Woni Agua” brand of water. The bottle is made of cardboard.

As with the analysis derived from the dataset for Brand #1 shared by iGEM TEC MTY, for this brand of bottled water, four out of five conditions are associated with two populations of particles. And, once again, these two populations differ by one order of magnitude between them (comparing the data from “Peak 1” column with the data from “Peak 2” column). By the way, the condition of a meter drop for this brand of water also led to particles as big as the biggest ones detected for “H2O BioLeve” when the bottle was left in free fall (1 m). These two maxima represent the scenarios where the biggest particles (in diameter) were found, not just for the dataset shared by iGEM TEC MTY, but also for the dataset we obtained (iGEM TecCEM).

Overall analysis

In conclusion, “Wonu Agua” and “H2O BioLeve” were the brands that, after a drop of 1 meter, theri bottled water had the biggest particles of all, even considering the data obtained by iGEM TecCEM. Nevertheless, it is important to keep in mind that “Wonu Agua”’s bottles are made of a different material than “H2O BioLeve”’s bottles; as the first ones are made of cardboard/paper and the latter ones are made of plastic. 
In spite of apparently being related to the biggest particles of all, the one meter impact test did not lead to a “local” maximum particle size for Brand #1 water (just considering the dataset shared by iGEM TEC MTY). Actually, for Brand #1 water yielded the “local” biggest particles just for the scenario where the bottled water was exposed to sunlight. However, if we consider the dataset obtained by iGEM TecCEM, it is also true that the biggest particles for the Brand #1 brand are the ones that were obtained after exposing the bottle to sunlight. 

Portugal Collaboration

We also collaborated with the NOVA Portugal team, sending them the same protocol as we did to the iGEM TEC MTY team (read more here). Their analysis would help us to identify the differences between the microplastic presence in bottled water from distinct countries and various brands, when subjecting the bottles to the same conditions; however, we are still in the process of interpreting the results that the team sent us, so we can’t make a comparison yet.

Microplastics obtained from sanding bottles experiment.

PET bottles were sanded to emulate the detachment of microplastics from bottles that are normally sold commercially and expose them to daily conditions, such as solar radiation, heat, agitation and impact.

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We divided the experiment into 3 parts to make an analysis using UV-spectrophotometer scanning and measure them to see if it remains the same value (average of 0.070 Absorbance at 290nm corresponding to microplastics) or with exposicion if there's any emergent bodies and which sizes are them. EDCs must be smaller than the 0.070A.
 

A- Samples without any exposure and no Laccase.

Remains of the sanding were placed in a citrate buffer suspension 1 mg/mL. Were kept in a 50 ml erlenmeyer flask under conditions with no sunlight, no agitation, nor impact. 

Result: no other values of interest were observed. Meaning no smaller emerging bodies were present in these samples. 

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​Pure BPA solution that makes measurements where 290 nm is determined as detection, that is why we do absorbance detection. Meaning at 290 nm there's presence of this type of EDC.
Image shows the highest peak on the UV spectrophotometer screening samples without sun exposure.
 

B - Samples with microplastics left in direct sunlight without Laccase.

Prepared in a buffer solution at a concentration of 1 mg/mL and direct sun light.

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This is to validate the point of the Zeta sizer where it indicates that with sun exposure, there is a shift of peaks to smaller populations in the absorbance measurements, hypothetically corresponding to EDCs. For further analysis we are going to focus on BPA since we already have the Standard curve. 

The readings exposed to the sun give us some small peaks with an average value of 0.045A (concentration of  0 mg/mL according to BPAs std curve)and 0.070A (concentration of  0.36 mg/mL according to BPAs std curve) of emerging components. 

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In the UV spectrophotometer screening of samples, focusing on 290 nm, in fig A and fig B we can find the absorbance of 0.104A and 0.170A respectively. Showing us that the sun exposure does have an effect on the degradation of microplastics increasing compounds like BPA in this case. 

 

The increase and appearance of smaller species than the inicial is watched with the only difference in exposure to sunlight, at 290 nm we confirm one it's about BPA.
 

Samples with microplastics with Laccase.

In this part, the hypothesis is that Laccase can work on those elements that appeared in the suspension due to the degradation of microplastics.

Here we run into the noise that protein makes since Lacase wavelength is at 280 nm.
At the moment it was seen in absorbance data, that in control of microplastics there is a lower concentration of small components than those exposed to the sun. In other cases where there is no laccase present and there is no noise due to its wavelength the opposite as in this case.

Conclusion

Small peaks relate, EDCs not only degrade in BPA but a good amount of EDCs. We focus on BPA because we studied it in our BPA degradation reaction by Laccase Trametes versicolor then from sample 1 with an average of 0.070A (concentration of 0.36 mg / mL according to the curve) to sample 2 with an average of 0.045A, with the only difference that sample 2 was exposed to sunlight. This difference and values ​​tell us that new small species were created by the sun, just as the resolution of the Zetasizer shows us the same in all plastic bottles.
Also in this case, figures A and B confirm the increase of BPA after sun UV exposure.  And the decrease of BPA with Laccase depending on the concentration.

This experiment reinforces that when there is no exposure to something there are larger species and when heat or some method is applied, small species appear in the readings. Which, in all plastic bottles appear, either with shaking, heat or impact.

This strengthens our hypothesis for the zetasizer assay.
If more smaller species are found when the plastic bottle is subjected to conditions of daily life such as leaving the bottle in the sun, or in the backpack, or in the car, among many other cases.