Odour concentration measurement:Dynamic olfactometry allows to determine the odour concentration (cod) of an odorous air sample, which is expressed in European odour units per cubic metre (ouE m−3), and represents the number of dilutions with neutral air that are necessary to bring the odorous sample to its odour detection threshold concentration.
The olfactometric method is based on the identification of the so-called ‘odour detection threshold’, i.e. of the threshold at which an odour is perceived by 50% of the assessors (panel).
To bring a sample to this threshold, a suitable dilution device must be used, which allows to dilute the gas sample according to given ratios with neutral air, i.e. with air made odour- and humidity-free by filtration through active carbon and silica gel. This dilution device is called ‘olfactometer’.
Odour concentration measurements are carried out in a specific room, called ‘olfactometric chamber’, at the Department of Chemistry, Materials and Chemical Engineering ‘Giulio Natta’ at the Politecnico di Milano. The olfactometric chamber is specifically equipped and meets the requirements of the EN 13 725:2003. The atmosphere inside the olfactometric chamber is controlled to minimize the risk that undesired external factors may affect the measurement. Odours coming from the equipment and other materials are eliminated and the room is ventilatedr to keep the environment odour-free. The ventilation air is filtered through active carbon before introduction into the olfactometric chamber to be deodorized. A minimum of 20 air exchanges/hour are guaranteed inside the olfactometric chamber. An olfactometer Ecoma Mod. TO8 equipped with four odour sniffing ports controlled by a PC was used as a dilution device (Fig. 1).
The measurement sessions start with a high dilution rate, as to keep the first sample presentations below the odour threshold concentration. The assessors smell the gas from a sniffing port and at each presentation, they shall indicate if they perceive an odour (‘yes/no’ method). After each round, the concentration of the sample is increased by reducing the dilution ratio. A button shall be pressed as soon as the sample odour is detected. The assessors are not asked to recognize or to describe the perceived odour, but just to indicate the moment at which they perceive a difference from neutral air. In some cases (at least 20%), reference air is presented instead of the sample (blanks), as to keep the assessors' concentration high. The measurement finishes when all the panel members have answered ‘yes’ correctly for at least two consecutive times.
Hedonic tone measurement: The hedonic tone (H.T.) describes the pleasantness/unpleasantness of an odour sample on a 9-point scale, ranging from −4 (extremely unpleasant) to +4 (extremely pleasant) .
Hedonic tone measurements are performed using the same dilution apparatus used for the determination of the odour concentration, i.e. the olfactometer.
The range of concentrations that has to be presented to the panel members comprises six dilution steps, which differ by a factor of two, the lowest dilution step corresponding to the sample odour threshold concentration. For this reason, it is mandatory to know a sample odour concentration before evaluating its hedonic tone.
In the case of low odorant concentrations, a smaller number of dilution steps may be presented. If necessary, the test may be confined to the undiluted test gas. The presentation of odour stimuli is done at random. Adaptation effects must, moreover, be minimized by ensuring that an above-threshold odour stimulus is not presented for longer than 15 s, with an allowance of an additional decision time of 5 s. The minimum break between two different stimuli should be at least 1 m. To check possible contamination of the olfactometer or to minimize possible guessing tendencies on the part of the panel members, the presentations are interspersed with blank samples (neutral air samples).
A series of measurements must not commence either with a blank sample or with a maximum stimulus.
For every sample presentation at the different dilution steps, the panel members shall evaluate the hedonic tone of the perceived concentration in accordance with the above-mentioned 9-point scale (Fig. 2).
Panel selection: For odour concentration measurements, the panel members are selected by means of specific sensitivity tests, in conformity with the criteria of the EN 13 725 . According to the norm, the panel sensitivity must be tested towards a reference odorant (n-butanol) as to choose a panel representative of the population average sensitivity. The following requirements must be satisfied: the sensitivity towards n-butanol must be included within a given range; the standard deviation with respect to the reference value must not exceed a given value and no errors on ‘blanks’ shall be committed. According to the EN 13 725, assessors having an odour threshold to n-butanol between 20 ppb and 80 ppb can be accepted as examiners. At our Laboratory, as a higher quality criterion, a narrower range is adopted, thus selecting as panel members, only people having an odour threshold to n-butanol comprised between 30 ppb and 60 ppb.
For hedonic tone measurements, the panel member's sensitivity (level of individual odour threshold) should not be a criterion for panel selection, as it is the case for the determination of odour concentration. The procedure for panel selection described in the German VDI, which was adopted by our Laboratory, provides to test the assessors using specific reference odorants.
The odorants employed for panel selection are vanillin (1 g to 200 ml dipropylene glycol) and guaiacol (1 μl to 200 ml distilled water), representing a pleasant and an unpleasant odour respectively. More in detail, panel screening is done by offering each panel member reference odorants in bottled and asking him or her to evaluate them according to the above-mentioned scale (Fig. 2), and the mean value of the results obtained from the entire panel must lie within a given range, i.e. between +1.9 and +2.9 for vanillin (pleasant) and −0.8 and −2.0 for guaiacol (unpleasant)) .
Measurement sessions: For the odour concentration evaluation, three consecutive measures for each sample have to be performed, with a minimum of four panellists for each measure, as a minimum of 12 individual detection thresholds are necessary to obtain the odour concentration of each analysed sample . The geometric mean of these 12 individual detection thresholds is calculated at the end of each test session. If one of the values obtained by one panellist is too far from the geometrical mean (according to EN 13 725, the ratio between an individual threshold estimate and the geometrical mean of all individual threshold estimates in a measurement should be between +5 and −5), the data from that panellist are excluded, and the geometric mean is recalculated using only the remaining measures .
For hedonic tone evaluation, the panel has to consist of at least 15 persons on account of the interindividual differences which may occur . The hedonic odour tone is expressed as the arithmetic mean of the hedonic odour tone values evaluated by each of the 15 panel members at the six different concentrations, thus representing the average of 90 assessments. This procedure is not reported in the above-mentioned German guideline, which limits the presentation of the hedonic tone measurements to a graphical representation of the panel responses with a statistical evaluation of their dispersion. We considered this procedure not to be suitable for comparing the odour pleasantness/unpleasantness released from different products, whereby the use of an averaged value, supported by suitable statistical analysis, is more significant.
All statistical analyses were conduced applying the Z-test method, considering a confidence interval of 95%. In general, the Z-test compares two groups of values (A and B) to determine if there is a significant difference between them . The Null Hypothesis considered is that the difference between A and B is not significant, whereas the Alternative Hypothesis is that the two mean values are significantly different. Using data of each group of values, the variances are calculated. Successively, relevant ‘z’ value is calculated. The obtained ‘z’ value is compared with the critical ‘z’ value relevant to the confidence interval chosen (95%) to establish if the Null Hypothesis has to be rejected or not. If the Null Hypothesis has to be rejected, the Alternative Hypothesis is proved and the two mean values (A and B) are significantly different. Moreover, the uncertainty related with the averaged data was calculated considering a confidence interval of 95%. Calculated uncertainties are reported on graphs as error bars.
Biological urine is proved to have extremely variable composition [17-21]. Urine composition may be affected by several different factors, such as the studied subject (sex, age), its diet and health conditions (e.g. infections or other pathologies), and the hour of the day (typically, the first morning urine is the most concentrated, then concentration decreases during the day).
Such variability is in contrast with the requirement of reproducibility of the tests for the sensorial evaluation of the products to be tested. This requirement can be fulfilled by the use of a reproducible synthetic urine-like fluid having a constant and stable composition over time. For this reason, the first step of the work consisted of the identification and the production of artificial urine, which, besides being stable, shall also be similar to real urine from the point of view of olfactory perception.
The identification of a synthetic urine-like fluid to be used for the tests is also necessary to prevent the sensorial assessors (panel) from any risk of biological contamination.
The preparation of artificial urine was based on a wide bibliographical research focused on the scientific studies describing the volatile and odorous substances contained in biological urine [22-24]. The recipe of artificial urine was thus identified as a mixture of volatile compounds to be added to a solution of water and salts.
Moreover, microbiological activity occurring in the biological fluid was simulated by addition of an enzyme (urease) to the mixture.
The enzyme is added to the liquid solution just before the pad loading, so that the enzymatic reaction starts as soon as the products are filled with the artificial urine.
Sensorial measurements were performed to optimize the similarity between artificial and biological urine, especially as far as the odour properties are concerned.
The dosage of the single substances is therefore a crucial aspect associated with the preparation of artificial urine for sensorial odour testing. The artificial urine used for this study was prepared as to be comparable, from the point of view of the emitted odour (quality and quantity), to biological urine after 24 h ageing. This time interval was chosen as it may reasonably represent the worst-case scenario for the final disposal of an absorbent pad.
Tests for the comparison of synthetic urine vs. biological urine
The comparison of synthetic vs. biological urine was based on sensorial measurements of both odour concentration and hedonic tone.
Biological urine was collected from 20 healthy individuals, aged in a range between 50 and 90 years, over a 24-h period. Samples were combined from each person over a 24-h collection and analysed individually. This procedure aimed to obtain a reasonable representation of inter- and intra-individuals variability of urine composition.
The samples to be analysed were prepared by storing a known quantity of fluid into special sampling bags at a fixed temperature. In more detail, 200 ml of fluid was used for each sample, and stored at a temperature of 37°C, this being the body temperature. The sampling bags are in Nalophan™, with a Teflon inlet tube and a volume of about 6 litres (Fig. 3).
The olfactometric evaluations were run on the gaseous phase above the liquid (headspace).
The analyses for the determination of both the odour concentration and the hedonic tone were repeated at different time intervals after sample preparation, to account for the variability of urine over time. The first test was performed at 15 min after sample preparation, then at 6 h and finally at 24 h.
These time intervals were chosen as to account for three different moments of the use of urine absorbing products: urination (time ‘0’, 15 min was left as to allow the realization of equilibrium conditions between liquid and gas phase), change (6 h wearing) and final disposal (24 h).
Tests for the comparison of different urine absorbing products
As previously explained, the sensorial tests for the comparison of different urine are performed using synthetic urine.
The absorbent articles are prepared flat on the bench and loaded with an amount of synthetic urine that is representative of the average loading of urine that can be present in urine absorbent products of a specified size. For instance, for products used for light incontinence, usual loading is below 30 mL of urine, for products specific for severe incontinence, it is common that the users load for each product is around 300 mL.
The chosen amount of synthetic urine is loaded in the centre of each product sample. Each sample is then positioned into a sampling bag in Nalophan™ and in contact with 6 L of neutral air. Also in this case, it is important to fix suitable storage conditions, i.e. time and temperature, to simulate the conditions of use of the products to be tested.
For this reason, a storage temperature representative of the body temperature (37°C) was chosen, and a storage time in a range between 4 and 8 h, which is estimated to be the average usage time of the products.
After storage, the bags are wrapped into additional black bags to prevent prejudice against the products and proceed to the olfactometric analyses for the evaluation of both the odour concentration and the hedonic tone [11, 13].
Also in this case, the tests were repeated on three different samples prepared in triplicate and stored for three different times: 15 min, 6 h and 24 h after sample preparation, for the same reasons discussed above.