How to correctly report your blanks in microplastic research?

After twenty years of microplastic research, the quest for data harmonization calls for rigorous quality controls. The first quality aspect that instantly springs to mind is, of course, blanks.

Blanks serve the purpose of informing us how much microplastic we are putting into our sample during the manipulation process. This includes sampling, digestion, How to correctly report your blanks in microplastic research?density separation, transfer steps, and so on. For this reason, we can all agree on how important blanks are to accurately reporting microplastic content in any given sample matrix.

A blank isn't just a blank

However, take note that there's a difference between different 'blanks'. When I started working with microplastics in 2020, you would often see the use of so-called 'air-blanks'. With air-blanks, you simply put a petri dish or similar, with a surface area close to that of your analysis filter, inside the laboratory for the duration of the experiment. Recently, air-blanks have been phased out and replaced with 'procedural blanks'.

A procedural blank, as the name suggests, provides a more comprehensive image of the contamination profile. The principle is very simple; take a 'microplastic-free' analyte and put it through your entire protocol, from sampling to analysis. In my opinion, this is the better blank, but it is also more difficult to conduct. A 2023 study by Noonan et al. found that only 3/4 of microplastic studies used and reported blank data, while only 1/3 reported their blank data (Fig. 1).

Fig. 1 - A 2023 study by Noonan et al. found that only 3/4 of microplastic studies used and reported blank data, while only 1/3 reported their blank data (Fig. 1). Figure reproduced from Noonan et al. (2023). Owner: Springer Nature.

While some argue that blank values should only be reported and not subtracted from the original data, I personally disagree with this notion - I think you should always subtract your blank! I was once presented with the argument that if you have a higher number of microplastics of a certain polymer type in your blank than in your original sample, will you then report negative values? Well, of course not! That would not make sense - you report zero then. You know then that the background contamination exceeds the true signal.

What does it even mean to subtract a blank?

However, be cautious. Blank subtraction should be performed on a polymer-to-polymer basis. Just because you find 'loads' of polyethylene in your blank doesn't automatically invalidate your polystyrene findings, for example.

At Microplastic Solution, we have defined a set of blank subtraction guidelines grounded in a particle-pairing approach. Here, microplastics in the blank and in the original sample (also known as the primary sample) are paired once, and consequently removed from the pool. These are rules:

Pair identification: When two particles in the primary sample and the blank match in polymer type and size they form a 'pair'. Once a pair is identified, the microplastic particle in the primary sample is subtracted, and its matching particle in the blank can no longer be used for subtraction.

Priority of subtraction: Microplastic particle pairs that are closest in size should be subtracted first.

Size limitation: If the diameter of any particle in the primary sample exceeds twice the size of the largest microplastic particle (of any polymer type) detected in the blank, it cannot be subtracted.

The size limitation rule is important, though it may seem counterintuitive at first. Let me explain the reasoning behind it.

Why don’t we subtract a microplastic particle more than twice the size of any found in the blank? Larger particles require more energy to move, making it easier for smaller microplastics to contaminate a sample. In natural environments, energy levels fluctuate significantly more than they do in a controlled laboratory setting. Consequently, if no microplastic particle twice the size of one in your original sample appears in the blank, it is unlikely that your protocol could have inadvertently introduced such a large particle into the sample. Fig. 2 visualizes these ground rules.

Fig. 2 - Visualization of Microplastic Solution blank subtraction rules. Details are described above and at: https://microplasticsolution.com/microplastic-detection#negative-control.

In practice, we segment all individual microplastic particles into size- and polymer groups for both the original sample and the blank. We then compare and subtract the number of microplastic that are closests in size, within the present polymer groups. This can typically be done inside an excel sheet (Fig. 3).

Fig. 3 - A true example of how we subtract blank data from the original sample using Microsoft Excel.

We encourange you to use these blank subtraction gudelines for your next project. If you want to cite the guidelines, cite our website using the following MLA formatting style:

Hagelskjær, Oskar. "Microplastic Solution". www.microplasticsolution.com. Accessed [insert date].

How to reduce your contamination profile?

It is important to subtract your procedural contamination, but ideally you would prefer to remove it alltogether. Unfortunately, this is close to impossible (depending on your analytical microplastic size cut-off, as larger particles are easier to control).

If I was to give one tip on how to reduce your procedural contamination, that you might not have considered, it would be to kiln sterilize your glassware. Kiln sterilization (also known as muffling) is the process of 'burning' your glassware.

This is usually done using a kiln (hence the name) at 500°C for at least one hour. This process ensures that those pesky little microplastics are removed from your glassware for good - Remember to wrap your glassware in aluminium foil as soon as you evacuate it from the kiln - But let the glassware cool of first!

What is your outlook on microplastic blanks and subtraction?

Oskar Hagelskjær, Ph.D.

Founder and CEO, Microplastic Solution

Oskar@microplasticsolution.com