This scoping review does have some limitations. First, our search was limited to articles written in or translated to English language, so there is a possibility that relevant articles written in other languages were left out. Additionally, the included studies were not assessed for risk of bias, which is critical to identify the quality of evidence. Nevertheless, this review provides a comprehensive overview of the recent prevalence of dietary supplement use among athletes.

#### **5. Conclusions**

In conclusion, the lack of homogeneity among studies regarding the definition of dietary supplements, reporting timeframes, and data collection methods complicates the attempt to compare the findings. Results from this review may contribute to future educational intervention program initiatives to increase the knowledge of users on dietary supplements, their benefits, and their risks and on the importance of their use under professional guidance. Given the higher prevalence of use of dietary supplements among athletes, more research and initiatives are indispensable in this population as proper nutrition is an extremely important factor in ensuring a high quality of life and optimum performance for athletes.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nu14194109/s1, Table S1: Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.

**Author Contributions:** Conceptualization, D.E.K. and J.D.; investigation, J.D.; resources, J.D. and M.M.; data curation, J.D. and M.M.; writing—original draft preparation, J.D., and M.M.; writing review and editing, D.E.K., J.D., and M.M.; supervision, D.E.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

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

#### **References**

## *Article* **Analysis of Sport Supplement Consumption by Competitive Swimmers According to Sex and Competitive Level**

**Berta Moreno 1,2, Santiago Veiga 2, Antonio J. Sánchez-Oliver 3, Raúl Domínguez 3 and Esther Morencos 1,\***

**Abstract:** Sports supplements (SS) are commonly used by athletes to improve their performance. SS use by competitive swimmers is reported to be prevalent but there is no evidence of such use by elite swimmers, either male or female. The objective of this research was to study the patterns of SS use by competitive swimmers based on sex and competitive levels (national and international); Methods: Using the categories of the Australian Institute of Sport (AIS), a total of 102 competitive swimmers (59 men and 43 women) completed a validated self-administered questionnaire on the use of SS; (3) Results: Overall, 86.9% of swimmers had consumed SSs with no differences observed between males and females (*p* = 0.247) or between competitive levels (*p* = 0.597). The SS that were most consumed by swimmers were caffeine (53.5%), sport drinks (52.5%), sport bars (51.5%), and vitamin C (43.4%). SSs categorized as medical supplements were consumed significantly more frequently by international swimmers (*p* = 0.012), with significant differences also found in the level—sex interaction (*p* = 0.049); (4) Conclusions: Compared to other sports disciplines, the prevalence of SS consumption is high in competitive swimmers regardless of performance level or gender. However, the consumption of medical supplements was greater in swimmers at a higher performance level.

**Keywords:** nutrition; swimming; ergogenic aids; performance; elite swimmers

#### **1. Introduction**

The main objective of competitive swimming is to complete a set distance (ranging from 50 m to 1500 m in official swimming pool events) in the shortest possible time using the front crawl, breaststroke, backstroke, or butterfly technique [1]. Swimming events can last from approximately 20 s to 16 min approximately, and depending on the characteristics of the event, the efforts require different energy systems, such as high-energy phosphagen, glycolytic and oxidative phosphorylation of carbohydrates, fats, or proteins [2].

The fact that swimming takes place in the water poses unique challenges for the swimmer, with the obligation to reduce drag forces and to maximize propulsive forces becoming a decisive factor in dictating the physiological and energetic demands rather than the duration of the test itself [3]. Training programs for elite competitors usually consist of a large amount of high-intensity training [4,5], although specific training demands depend on the characteristics of the sprint, middle-distance, or distance event. For example, the aerobic involvement in a 400 m front crawl event is 81%, while in the shorter events, such as 50 m and 100 m front crawl, the aerobic contribution is 15.3% [2]. This also has consequences on the different nutritional requirements for success in each discipline [2,6,7].

Previous studies have investigated strategies to increase performance by means of better recovery [2,3,8–10] through the use of nutritional measures, such as sports supplements [8,11,12]. The SSs should be considered as a supplement to the usual diet, which is consumed with the aim of achieving specific performance benefits [13]. Despite this,

**Citation:** Moreno, B.; Veiga, S.; Sánchez-Oliver, A.J.; Domínguez, R.; Morencos, E. Analysis of Sport Supplement Consumption by Competitive Swimmers According to Sex and Competitive Level. *Nutrients* **2022**, *14*, 3218. https://doi.org/ 10.3390/nu14153218

Academic Editor: Joerg Koenigstorfer

Received: 18 July 2022 Accepted: 4 August 2022 Published: 6 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

only a small portion of current SSs on the market have been shown to produce significant improvements in performance [13–16]. Some SS, such as sodium citrate or phosphate, have been reported to provide a negligible benefit to exercise capacity [17–19]. Thus, it is necessary to carry out a cost—benefit analysis based on the criteria for SS safety, efficacy, and legality [15] prior to introducing supplements, providing strategies that allow us to resist in the face of external agents [20].

Different international institutions periodically publish position stands that are elaborated to guide the practice of supplementation, such as the Australian Institute of Sport (AIS) which created the ABCD system, whereby each SS is classified according to the level of scientific evidence [21]: SSs in group A present a high level of scientific evidence; those in group B can have a positive effect in certain circumstances, but more evidence is necessary; those in group C are supplements for which evidence is lacking; and those in group D are prohibited substances. At the same time, SSs are subdivided into sports foods, medical supplements, and ergogenic aids. Several studies have reported the effects of supplementation in athletes based on this classification system [14,22–25], and athletes following a SS program under the AIS guide have displayed a greater consumption of SSs with a high level of scientific evidence [26].

The reported range of SS consumption in sports is very wide and comprises between 30–95% of athletes [23,25,27]. Sex and level of performance are among the most important variables for SS consumption, as SS consumption increases with the competitive level of athletes and in men compared to women [13,23,25,28]. A higher rate of SS consumption has been reported among swimmers (in comparison to other disciplines) as swimming was ranked among the top four sports in terms of the SS use prevalence at the 2000 Sydney Olympic Games [29,30]. The SS consumption patterns in competitive swimmers indicated that all 23 swimmers surveyed in Sri Lanka had consumed SSs and had a daily intake of 3.4 different supplements [26]. However, these data were collected from swimmers with a low competitive level, and, to the best of our knowledge, no other evidence on the SS consumption prevalence or SS characteristics in elite swimmers has been observed.

 It was hypothesized that swimmers of a higher level of performance and male swimmers would present higher SS consumption.

#### **2. Materials and Methods**

#### *2.1. Participants*

A total of 102 competitive swimmers (59 men and 43 women) voluntarily participated in this investigation. All participants belonged to a federated swimming club and were competing in national (*n* = 60) and/or international (*n* = 39) events at the time. Swimmers of international level had taken part in international-level competitions with the national team for at least two years. Three swimmers from the original sample were eliminated from the study because they did not have the required competitive national level; therefore, 99 swimmers were included in the final sample. All participants had an average of six days of training per week, four hours of training per day, and an average of three days per week of out-of-water training.

#### *2.2. Instruments*

The questionnaire used contained three main sections: the first one collected anthropometric, personal, and social data of the respondent; the second section covered the practice of the sport activity and included 10 questions about, for example, the number of years federated, the competitive level, years competing in the national team, the hours of water training by week, the sessions of water training by week or dry-land training sessions by week. The last and most extensive section was related to SS consumption. This section contained three questions on the type of diet the swimmers had, 12 questions on the use and consumption of SS (i.e., what supplements are taken, for what reasons

are they taken, who advises to take them, when are they taken, what were the reasons or where can the supplements be purchased), and, finally, two questions on the use of banned substances. In addition, this part included the definition of sports supplements (SS) proposed by Knapik [28] and an updated list of SSs. The response options included a time frame such as whether swimmers took SSs for training, competition, or both and whether they took them before, during, or after training and/or competition.

The questionnaire was previously validated in terms of its content, application, structure, and presentation [31] and its quality was evaluated in a systematic review [28]. According to an eight-point scale that included assessments of sampling methods, sampling frame, sample size, measurement tools, bias, response rate, statistical presentation, and description of the participant sample, the quality of the questionnaire reached a 54% score (57 out of 164 questionnaires evaluated) and it was considered adequate to obtain accurate information on supplement use by athletes. It is worth noting its use in different studies that have analyzed the consumption of SS in athletes [14,24,25,27,32].

#### *2.3. Process*

All swimmers who participated in this research did so voluntarily and only had to complete a questionnaire on the use of supplements [31]. Swimmers completed the questionnaire between April and October 2019 in a web format that was prepared to facilitate its distribution (Google Forms, Google, Mountain View, CA, USA). The information recorded in the questionnaires did not allow the identification of the swimmers, all of whom remained anonymous. The participants were recruited with the help of the National Swimming Federation, which distributed an e-mail with instructions for completion and an online version of the questionnaire to all the teams with a national license. Thus, when the swimmers went to their usual training pool, the characteristics of the study were explained to them, and consent was obtained from all of them. The protocol complies with the Declaration of Helsinki for human research and was approved by the Ethics Committee of the local university committee.

#### *2.4. Statistical Analysis*

The Kolmogorov–Smirnov test and the Levene test were applied to check for normality and homoscedasticity. Quantitative variables were presented as an average (M) ± standard deviation (SD), while qualitative variables were in percentages. For the analysis of possible differences in the level of performance (international vs. national) and of the possible differences in sex with regard to the motivation, expectations, and contextualization of the use of SS, a chi-square test (χ2) was performed. If statistical differences were reported, an odds ratio (OR) was also performed. As to the total SSs ingested, a Student's T-test for independent samples was carried out to analyze possible differences between the levels of performance or between performance based on sex. The statistical level of significance was set at *p* < 0.05. The statistical analyses were performed using the Statistical Package for Social Sciences (version 18.0 for Mac, SPSSTM Inc., Chicago, IL, USA).

#### **3. Results**

The sample characteristics are reported in Table 1. Male swimmers presented as taller (*p* < 0.001) and heavier (*p* = 0.005) than female swimmers, although no anthropometric differences were reported for level or level—sex. Additionally, no statistically significant differences were found for the number of weekly training days or the number of strength training days in relation to the competitive level, sex, or level—sex (*p* > 0.05). However, national female swimmers performed more strength training days than did international female swimmers (*p* = 0.044). No differences were reported for level (*p* = 0.522) and sex (*p* = 0.384) in the years of federated swimming with 20.2% of the sample federate for less than 5 years, 39.4% between 5 and 8 years, and 40.4% for more than 9 years.

**Table 1.** Characterization of sample data divided by sex and competitive level (international and national).

Data expressed mean ± standard deviation (SD). λ Sex differences of the same competitive level; \* Level differences of the same sex. Statistical significance fixed at *p* < 0.05.

#### *3.1. SS Consumption Characteristics*

In relation to SS consumption characteristics, 6.1% of the sample said they were against SS consumption in swimming, while 80.8% said they were in favor and 13.1% either did not know or did not answer, with no statistically significant differences observed according to sex (*p* = 0.841) or competitive level (*p* = 0.417). When asked if they had ever consumed SSs, 86.9% of the sample answered affirmatively, with no differences observed between men and women (*p* = 0.247) or between swimmers at international and national levels (*p* = 0.597). The main motivations for SS consumption were performance improvement (59.2%), health care (14.6%), or to cover dietary deficits (10.0%), with no statistically significant differences between sexes (*p* = 0.109) or between the level of swimmers (*p* = 0.530).

As shown in Figure 1, SSs were most frequently purchased at specialized SS stores (32.0%) and pharmacies (27.2%), followed by shopping centers (10.9%) and the Internet (10.9%), with no differences between sexes (*p* = 0.193). There were also no differences between swimmers at the national and international levels (*p* = 0.822).

As can be seen in Figure 2, the coach was the person who most frequently advised the consumption of SSs (40.3%), followed by the dietitian—nutritionist (20.1%), the physician (10.0%), the physical trainer (8.1%), family (6.7%), teammates (6.0%), friends (4.7%), or others (4.1%). No significant differences were observed between sexes (*p* = 0.421) or levels (*p* = 0.227).

Regarding when SSs were consumed within the season, most subjects reported consuming SSs during the competitive period (66.0%), followed by those who consumed them only during the training period (23.0%), throughout the year (7.0%), or at other times (4.0%), with no differences found according to sex (*p* = 0.702) or competitive level (*p* = 0.838). In addition, no differences were found between men and women (*p* = 0.284), although differences were observed according to level (*p* < 0.001). International level swimmers presented a greater mode of SS consumption before, during, and after exercise (39.5% vs. 18.6%), whereas national level swimmers had a greater SS consumption mode exclusively before (42.4% vs. 13.2%; OR = 2.56 [1.05–6.27]) or after (27.2% vs. 21.1%; OR = 1.29 [0.60–2.74]) exercise as compared to international level swimmers.

#### *3.2. Type of SS Consumption*

Group-level evidence of SSs (A, B, and C; see Table 2) showed no statistical differences in SS consumption for competitive level, sex, or level—sex (*p* > 0.05). As for the different subgroups of group A, a higher consumption of medical supplements was found in international level swimmers (*p* = 0.012), reaching statistically significant differences in men (*p* = 0.001) but not in women (*p* = 0.709). In addition, higher consumption was found in

women at the national level as compared to men at the same competitive level (*p* = 0.013). Finally, the subgroup "sport performance" showed higher consumption for national level men as compared to national level women (*p* = 0.036).

185

**Figure 2.** Sources of information or advice on the use of supplements by elite swimmers according to sex (**up**) and competitive level (**down**).

As can be seen in Table 3, there was a consumption rate higher than 10% for 17 SSs in total, with caffeine (53.5%), sports drinks (52.5%), sports bars (51.5%), and vitamin C (43.4%) as the most consumed substances. No differences were observed based on sex for supplements with a consumption rate of at least 10%. Males were observed to have significantly lower consumption of vitamin complexes compared to females (*p* = 0.029; OR = 0.33 [0.12–0.87]), but they had a higher consumption of creatine (*p* = 0.041; OR = 2.92 [1.10–7.72]), taurine (*p* > 0.001), and a trend toward significance in the case of whey protein (*p* = 0.059; OR = 2.77 [0.99–7.7]). On the other hand, international level athletes presented a greater iron intake (*p* < 0.001; OR = 5.77 [2.32–14.32]) than those at the national level.

**Table 2.** Number of SSs used by swimmers of both sexes and at different competitive levels according to AIS categories [21].

Data expressed mean ± standard deviation (SD). \*: Significant difference for a determined factor; #: Significant difference between international and national of a same sex; λ Significant difference between males and females of a same competitive level; Statistical significance at *p* < 0.05.

**Table 3.** Most used supplements according to sex and competitive level based on AIS categories [21].

**Table 3.** *Cont*.

\* Statistical difference in the consume between groups (*p* < 0.05).

#### **4. Discussion**

The aim of this study was to analyze SS use in international and national level swimmers, describing the pattern of consumption according to sex and competitive level. Although previous studies [8,26,33–35] have examined SS consumption in competitive swimmers, to our knowledge, this is the first study to report on SS consumption in an extensive sample of elite level swimmers. The results of the present study show a high prevalence of SS use in competitive swimmers (86.9%) compared to other disciplines (30–95%), such as elite sailors (52%) [14] or rugby players (65.3%) [25]. These data support a higher use of SS in individual versus team sports (81% versus 58%) [36] and a prevalence similar to that of previous research on swimming [8,28,30,33,35,36].

#### *4.1. Characteristics of SS Consumption in Competitive Swimmers*

High training volume of competitive swimmers including the large number of weekly training sessions in and out the water [4] could explain the high prevalence of SS use in competitive swimmers. Contrary to expectations, no significant differences were observed between national and international level swimmers in the rate of SS consumption as has been shown in other studies [14,22]. This could be explained by the similar training volume of national and international caliber athletes, as defined by Mckay et al. [37] who distinguished the elite-international and the highly trained-national by the competitive standard. Finally, no differences were observed in the prevalence of SS use according to sex, contrary to other recent studies [14,25,36] but in line with similar training volume in swimming between gender.

The main reason for SS consumption in the present sample was to improve sports performance (59.2%), regardless of gender or competitive level. This motive for SS consumption is common (45–77.8%) among elite athletes in different sports [14,32,36,38], and it is consistent with the most used type of SS in the sample, that is, caffeine, which has previously been reported to improve sports performance [21,39]. The most frequent places of purchase of SS in this study, which were well beyond the rest, were specialized shops (32%) and pharmacies (27.2%), a finding that is consistent with a recent study of elite sailors by Caraballo et al. [14]. It is worth noting that online shopping, a current trend reported in some studies [25,36], was not one of the most frequent places of purchase considering the cases of biased and unreliable information, inclusion of undeclared pharmacological substances on the label, and lack of specific legislation [27,40–43].

Although the source of information about SS should be professionals in the field (sports doctors and/or dieticians—nutritionists), swimmers in the present research chose coaches as the main source of information (40.3%) as in a very recent study in gym users by Finamore [44] and the findings in a study of German athletes [45]. This demonstrates some areas of improvement in SS consumption by competitive swimmers, since athletes who receive advice from a professional have a higher consumption of SS for which there is a high level of evidence [25]. It is noteworthy that most of the SS consumed were of category A, which suggests that the coaches of our swimmers were very well informed and instructed to advise on responsible consumption of SS to elite swimmers.

Most subjects reported consuming SS during the competitive period (66.0%), regardless of gender or level of competition. This is related to the three most consumed SS substances of caffeine, sports drinks, and sports bars as they tend to be used during competitive periods. The timing of SS consumption coincided with data reported in similar studies of elite athletes [14], although differences were observed between international and national swimmers. Higher level competitive swimmers had higher SS intake during exercise compared with the higher pre-exercise SS intake of lower level swimmers. The social environment, training regimens, and competition place great demands on elite athletes [4]; perhaps for this reason, added to the demands of the competition itself, means that the highest-level swimmers decide to resort to supplementation as an aid at the moment of greatest sporting demand. This finding in the present investigation is interesting since none of the other studies on swimmers has measured the moment of SSs [2].

The amount of SS consumed in the sample of competitive swimmers (5.45) was higher than what has been reported on fencers (3.33) [23], rugby players (3.9) [25], and sailors (3.9) [14] but lower than studies conducted with squash players (8.4) [32], rowers (16.28) [22], all elite athletes. These data support the hypothesis that there is a difference in consumption depending on the sport or sport modality [13,28,31], although we did not detect a lot of differences in SS consumption by sex [25] or competitive level [28].

#### *4.2. Type of SS Consumption in Competitive Swimmers*

When dividing SSs according to the level of evidence (AIS classification), national level men in group A had higher consumption than did women. Although women consumed more SSs in medical supplement subgroup A (0.4 vs. 1.0), these differences in consumption could be explained by the biggest differences in the sport performance subgroup (1.5 vs. 0.8). On the other hand, within the medical supplement subgroup, higher consumption was observed at the international versus the national level for men and at the national level for women as compared to men. The reason for these differences could be the inclusion of iron in the medical supplement subgroup as it has been reported to be the most consumed supplement by elite athletes in different sports [22,23,28,32,36] and specifically by women [28,36]. Iron deficiency is one of the most prevalent nutritional deficiencies in the athlete population, especially in women due to the increased demand for iron during menstruation [46,47]; it is also prevalent at the end of the competitive period [48], which is more demanding for international athletes. In addition, this supplement requires individual dispensing and appropriate sports physician/scientist supervision, so it is reasonable that international swimmers would have a higher intake.

Regarding the most consumed SS in the sample, differences were found according to sex, with men consuming more creatine and taurine and showing a tendency in the case of whey protein, and women consuming more vitamin complexes. These findings are related to those obtained in previous studies in which iron and vitamin consumption were higher for women and protein supplements (including creatine) were higher for men [28,49,50]. It is worth noting that most of the SSs consumed in the present research belonged to group A (more than 10% prevalence), which may suggest that the sample of swimmers was well informed about the most effective supplements in their sport, even if coaches were the main advisers about SSs, as mentioned above. For this reason, a good coach's training-education in this subject (with documents such as the IOC, AIS, or ISSN [13,21,51]) could be a key aspect to correctly advise their swimmers at these levels.

Finally, the present study may present certain limitations that need to be noted for a better interpretation of the results. First, the questionnaire which was used to assess the consumption of dietary supplements in elite athletes collected information retrospectively, and this could lead to inaccurate information on the number and/or type of supplements reported. Even so, although the questionnaire collects the information retrospectively, a validated and reliable questionnaire was used to assess the use of dietary supplements in athletes, as was conducted in other studies of other sports with such a questionnaire [31]. These studies are proof that, despite these small limitations, such a questionnaire should not

affect your answers, guaranteeing their accuracy. Second, it is possible that swimmers could have employed supplements but did not recognize their use at that time and, therefore, did not report their use in the questionnaire, resulting in a false negative.

The analysis of SS consumption in this study was analyzed per unit, not per quantity. The quantity should be subject to the weight of the athlete, so it is a very interesting topic for future studies.

#### **5. Conclusions**

The present study indicated a high prevalence of SS consumption in competitive swimmers compared to other disciplines, with caffeine, sports drinks, and sports bars being the most consumed SSs. Swimmers reported performance enhancement as the primary motivation for SS consumption and, consequently, SS intake was most common during the competitive period. Differences in SS consumption were observed according to the competitive level, with international level swimmers exhibiting (i) a greater consumption of SSs during exercise (and not only before or after exercise) and (ii) a greater intake of medical supplements (like iron) with a higher level of scientific evidence. In addition, differences were observed by gender in the consumption of certain SSs such as creatine and taurine or the vitamin complexes. These results provide the first evidence of SS consumption in elite swimmers and demonstrate some specific patterns according to the high demands of volume and intensity in the elite swimming training programs.

**Author Contributions:** Conceptualization, B.M., E.M., S.V. and A.J.S.-O., methodology, B.M., E.M., S.V., A.J.S.-O. and R.D., validation and formal analysis, A.J.S.-O. and R.D., investigation, B.M., E.M., S.V., A.J.S.-O. and R.D., data curation, B.M., E.M., S.V., A.J.S.-O. and R.D., writing—original draft preparation, B.M., E.M., S.V. and A.J.S.-O., supervision, E.M., S.V. and A.J.S.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The protocol complies with the Declaration of Helsinki for human research and was approved by the Ethics Committee of the local university committee.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

**Acknowledgments:** The authors would like to thank the swimmers and coaches participating in the experiment for their valuable collaboration. The authors have no conflicts of interest to declare. The data sets generated during and/or analyzed during this study are not available to the public but can be requested from the corresponding author, who was the organizer of the study. The experiments comply with the laws in force in the country in which they were performed.

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

#### **References**

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**4.6 Grain and flour beetles**

The grain and flour beetles are very common stored grain pests feeding on almost all stored grains available throughout the world.

*4.6.1 Cadelle (*Tenebroides mauritanicus *(Linnaeus, 1758) Coleoptera: Tenebrionidae)*

*Distribution:* Cadelle is present in almost all parts of the world. *Host range:* its main host is grain and grain products.

*Bionomics:* Cadelle is the longest-lived pests, frequently found in granaries, mills, storehouses containing grains, flour, meal, pulses, etc. These are elongated flattened, oblong, black, or blackish beetle that resembles mealworms but, differ from them in size and body texture. These are smaller than mealworms and have loosely joined thorax and abdomen. Larvae are white, fleshy, long, with head, thoracic shield, having two black horny points on the abdomen, and are one of the largest of the stored grain pests. Both larvae and adults are very much destructive and move from grain to grain and devour the embryo.

*Damage symptoms*: It is a very serious pest of grains and bores inside wood for pupation. The presence of small holes in grains and in wood is the primary indication of its infestation.


**5. Conclusions**

The STAR ASA model was able to integrate the characteristics of the wastewater and the soil, as well as the nutritional requirements of the crops and the environmental regulations, by means of mathematical models selected to evaluate the feasibility of the application of wastewaters with a high content of organic matter with agricultural soil irrigation. The model allowed the estimation of the hydraulic load rate, the time lap between applications, and the amount of nutrients provided to the soil.

Based on the theoretical analysis of the used models, it was determined that the controlled application of wastewater allows the filtration and degradation of the constituents with a high content of organic matter that were applied to the soil, as long as the parameters recommended by the STAR ASA model are fulfilled.

Finally, considering the characteristics of the analysed wastewaters, the characteristics of the selected soils, and the nutritional requirements of the sugar cane, it was determined that for the test cases, the application of the effluents in the soil will contribute to fertilization, specif‐ ically nitrogen-related fertilization. However, it needs to be clarified that the results obtained in this study cannot be generalized to other type of effluent without previous analysis. The wastewaters, the agricultural soils, the climatic conditions, and the crops requirements may be different.


*Case Report* **Sustainable Urban Development for Older Australians: Understanding the Formation of Naturally Occurring Retirement Communities in the Greater Brisbane Region**

**Jiaxuan E 1 , Bo Xia 1,\* , Laurie Buys 2 and Tan Yigitcanlar 1**

**Abstract:** As most older Australians prefer to age-in-place, providing sustainable and age-friendly communities poses a significant challenge to urban policymakers. The naturally occurring retirement communities (NORCs) have organically emerged as a collaborative model of care to support older adults to age-in-place, but neither academic research nor government policies recognise this housing option for older Australians. This paper aims to analyse the distributions and temporal patterns of NORCs in the Greater Brisbane Region, Australia, to understand the formation and development of NORCs. The geovisualisation method was employed to identify the distribution changes of NORCs between 2006 and 2016. The Global Moran's I and Local Moran's I measures were utilised to analyse the spatial correlation and the clusters of NORCs. The results show that NORCs increased significantly from 2006 to 2016, and their distribution was mainly clustered or co-located along the coastline and Brisbane River areas. The evolvement of NORCs reflected the change of aggregation pattern of older population between 2006 and 2016. Understanding the distribution trend of NORCs informs government policy and decisions in addressing issues of service delivery and community cooperation, and eventually leads to sustainable urban development and successful ageing in place for older Australians.

**Keywords:** age-in-place; ageing communities; naturally occurring retirement communities; sustainable urban development; age-friendly cities; older population; Brisbane; Australia


**4. Conclusions**

The disappearance of insecticides after treatment could be related to many different paths. The main effects in the field are related to sunlight photodegradation and the development of the fruits during fruit growing leading to a decrease of the residues below the legal limit and sometimes the analytical detectable levels. Run-off and washing could affect only the residues adsorbed in the adhering dust on fruits and vegetable surfaces not influencing the residues adsorbed in the epicuticular waxes. Drying causes a reduction in weight, theoretically the residue could increase giving a value that is a function of the concentration factor, however the results showed a decrease in insecticide residues in foodstuffs. The different processing steps affect insecticides residues involving partition, microbiological and chemical degradation, and adsorption on the waste such as lees, marc, vegetation water and pomace.


**2. Microsatellite typing**

#### **2.1 Material and methods**

The 123 *C. albicans* (64 [52%] of them biofilm positive) strains for this study were collected during a study in biofilm forming abilities of yeast on indwelling devices (Paulitsch et al., 2009). The strains were stored at -70°C until examination.

Strains were subcultured on Sabouraud agar plates for 24 h at 35°C. For DNA extraction the PrepManTM Ultra Kit (Applied Biosystems [AB], Foster City, California) was used. For the microsatellite typing three different primer pairs were used (Botterel et al., 2001). The unmarked primers were HIS3R, CDC3R, and EF3 (Invitrogen, Lofer, Austria). The fluorescence labeling of the primers HIS3 (NEDTM, yellow), CDC3 (VICTM, green), and EF3R (6-FAMTM, blue) (all AB) was fitted to the DyeSet DS-33 (AB) which is recommended for 5-dye custom primer analyses. PCR was performed using the 96 well GeneAmp PCR System 9700 or the 96 well 2700 Thermal Cycler (both AB). PCR reactions were carried out as singleplex reactions for each primer pair. The samples were initially incubated for 2 minutes at 94°C to activate the Taq Polymerase (Eppendorf, Hamburg, Germany) and to denature the DNA. After thermal cycling (30 cycles; 94°C for 45 s, 48°C for 45 s, 68°C for 90 s) samples were kept at 68°C for another 5 minutes to complete partial polymerization.

Sample preparation for the injection in the 3100 Automatic Sequencer (AB) was done following the instructions. For analysis 1 µL of PCR product, 0.3 µL of size standard (GeneScanTM 500-LIZ®, AB) and 10 µL Hi-DiTM Formamide (AB) were mixed and transferred into a 96 well plate. The samples were denatured for 4 minutes at 94°C in a thermal cycler and immediately placed on ice. In every run three samples were used as internal control. The plate was transferred in the sequencer and processed using the Foundation Data Collection 3.0 software of the sequencer.

Data analysis was done with the GeneMapper® v3.7 software. Therefore it was necessary to set up the microsatellite analyses following the instructions of the manual (AB, 2005). The peaks were automatically detected (Auto Binning) with the created bin set, low quality data were checked manually and corrected. The results were exported in a Microsoft Excel sheet for documentation.

#### **2.2 Results**

4 Epidemiology Insights

strains was based on scanning electron microscopical findings (Paulitsch et al., 2009). Different primer pairs and also different combinations of primer pairs for the subtyping of *C. albicans* were reported elsewhere, see e.g. the works of Sampaio et al. (2005) or Fan

For each marker and for a given isolate one or two bands were observed, and each observed band was assigned to an allele. Because *C. albicans* is diploid each strain can be characterized

The discriminatory power (DP) is a numerical index to describe the probability that two unrelated samples of a test group are placed in two different typing groups. The DP of EF3 is 0.86, the DP of CDC3 is 0.77, and the DP of HIS3 is 0.91 (Botterel et al., 2001). The combined DP of all three markers was 0.97. In order to get reliable results, this index has to

The 123 *C. albicans* (64 [52%] of them biofilm positive) strains for this study were collected during a study in biofilm forming abilities of yeast on indwelling devices (Paulitsch et al.,

Strains were subcultured on Sabouraud agar plates for 24 h at 35°C. For DNA extraction the PrepManTM Ultra Kit (Applied Biosystems [AB], Foster City, California) was used. For the microsatellite typing three different primer pairs were used (Botterel et al., 2001). The unmarked primers were HIS3R, CDC3R, and EF3 (Invitrogen, Lofer, Austria). The fluorescence labeling of the primers HIS3 (NEDTM, yellow), CDC3 (VICTM, green), and EF3R (6-FAMTM, blue) (all AB) was fitted to the DyeSet DS-33 (AB) which is recommended for 5-dye custom primer analyses. PCR was performed using the 96 well GeneAmp PCR System 9700 or the 96 well 2700 Thermal Cycler (both AB). PCR reactions were carried out as singleplex reactions for each primer pair. The samples were initially incubated for 2 minutes at 94°C to activate the Taq Polymerase (Eppendorf, Hamburg, Germany) and to denature the DNA. After thermal cycling (30 cycles; 94°C for 45 s, 48°C for 45 s, 68°C for 90 s) samples were kept at 68°C for another 5 minutes to complete partial

Sample preparation for the injection in the 3100 Automatic Sequencer (AB) was done following the instructions. For analysis 1 µL of PCR product, 0.3 µL of size standard (GeneScanTM 500-LIZ®, AB) and 10 µL Hi-DiTM Formamide (AB) were mixed and transferred into a 96 well plate. The samples were denatured for 4 minutes at 94°C in a thermal cycler and immediately placed on ice. In every run three samples were used as internal control. The plate was transferred in the sequencer and processed using the

Data analysis was done with the GeneMapper® v3.7 software. Therefore it was necessary to set up the microsatellite analyses following the instructions of the manual (AB, 2005). The peaks were automatically detected (Auto Binning) with the created bin set, low quality data were checked manually and corrected. The results were exported in a Microsoft Excel sheet

et al. (2007).

by six alleles with the method used.

be greater than 0.90 (Botterel et al., 2001).

2009). The strains were stored at -70°C until examination.

Foundation Data Collection 3.0 software of the sequencer.

**2. Microsatellite typing 2.1 Material and methods**

polymerization.

for documentation.

Typing of 123 *C. albicans* strains was done with the above mentioned three primer pairs. Only from strain number 85 (sample W60) no data from the EF3 locus was producible. Although the DNA was isolated a second time and several PCR reactions were done for this locus, no peaks could be generated. In table 1 detailed information of all three loci for each strain is listed.

 (a) Biofilm of *C. albicans* (W65) in catheter lumen. (b) Biofilm detail of *C. albicans* (W91). : yeast cells; +: bud scars; m: matrix material; arrow: hyphae. SEM micrographs were taken with a Philips XL30 ESEM scanning electron microscope using the high vacuum mode (emission electrons detection, acceleration voltage 20 kV, operating distance 10 mm).

(samples in italic letters: biofilm positive)

Table 1. Microsatellite data for 123 *C. albicans* strains.

A comparison of the results did not reveal information of typical microsatellite models for *C. albicans* strains which produced biofilms in this study. Only 41 of the investigated strains showed a similarity with one or up to six other strains (Table 2).

(+: biofilm positive; -: biofilm negative)

Table 2. Microsatellite models.

The most convergent data were generated with the CDC3 primer pair, only 12 different allele pairs were; found, with the EF3 primer pair 25 different pairs were located, and HIS3 primers provided 50 different pairs of alleles.

From six patients two strains were available, each of them originated from different samples and showed *C. albicans* infections in routine diagnostics. Both samples from one patient were biofilm positive, from another patient both samples were negative. The microsatellite data of these catheters are listed in table 3. When only HIS3 and CDC3 alleles were compared, five out of the six patients showed the same strain two times, when they were also compared with EF3 primer alleles, only one patient had the same strain two times.

The comparison of the genotyping of biofilm forming *C. albicans* strains (e.g. see figure 1) with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes.

**CDC3/CDC3R EF3/EF3R HIS3/HIS3R**

120 W107 117 129 129 139 154 158 121 W108 117 129 123 133 162 202 122 *W110* 121 125 123 137 154 166 123 *W113* 117 125 129 133 178 178

A comparison of the results did not reveal information of typical microsatellite models for *C. albicans* strains which produced biofilms in this study. Only 41 of the investigated strains

**CDC3/CDC3R EF3/EF3R HIS3/HIS3R n allele 1 allele 2 allele 1 allele 2 allele 1 allele 2 + - total**  113 117 123 129 150 162 2 2 117 129 129 139 154 154 1 4 5 117 129 130 139 154 154 7 4 11 121 129 130 139 154 154 2 2 121 125 123 137 154 166 1 3 4 117 125 120 129 162 162 2 2 117 125 126 126 162 186 1 1 2 117 125 120 120 162 198 2 2 117 125 120 129 162 198 2 2 4 117 125 120 129 162 214 2 2 125 125 123 133 166 182 3 3 125 125 126 133 166 182 1 1 2 23 18 41

The most convergent data were generated with the CDC3 primer pair, only 12 different allele pairs were; found, with the EF3 primer pair 25 different pairs were located, and HIS3

From six patients two strains were available, each of them originated from different samples and showed *C. albicans* infections in routine diagnostics. Both samples from one patient were biofilm positive, from another patient both samples were negative. The microsatellite data of these catheters are listed in table 3. When only HIS3 and CDC3 alleles were compared, five out of the six patients showed the same strain two times, when they were also compared with EF3 primer alleles, only one patient had the same strain two

The comparison of the genotyping of biofilm forming *C. albicans* strains (e.g. see figure 1) with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes.

(samples in italic letters: biofilm positive)

(+: biofilm positive; -: biofilm negative) Table 2. Microsatellite models.

times.

primers provided 50 different pairs of alleles.

Table 1. Microsatellite data for 123 *C. albicans* strains.

showed a similarity with one or up to six other strains (Table 2).

**sample allele 1 allele 2 allele 1 allele 2 allele 1 allele 2**

Fig. 1. (a) Biofilm of *C. albicans* (W65) in catheter lumen. (b) Biofilm detail of *C. albicans* (W91). : yeast cells; +: bud scars; m: matrix material; arrow: hyphae. SEM micrographs were taken with a Philips XL30 ESEM scanning electron microscope using the high vacuum mode (emission electrons detection, acceleration voltage 20 kV, operating distance 10 mm).

Microsatellite Typing of Catheter-Associated *Candida albicans* Strains 11

Totally different data were provided from Shi et al. (2007) who collected isolates by female and male patients with genital infection, rectal and oral samples. The authors reported 54.9% of the strains investigated to show the same multilocus genotype, these results were

The CDC3 locus showed 12 different allele pairs, the EF3 locus 25 allele pairs, and the HIS3 locus 50 allele pairs. This is convergent with the data within the three loci and leads to 94 multilocus genotypes. When compared with the results of Botterel et al. (2001) who reported 65 different multilocus genotypes with different allele associations of 10 for CDC3, 22 for EF3, and 25 for HIS3, it is obvious that the HIS3 locus was clearly more divergent within the current study. However, it remains unclear whether this variation is typical for *C. albicans* strains collected from BSI, or if the discriminatory power (DP) of the HIS3 locus (0.91) is not strong enough. The calculated overall DP for the CDC3, EF3, and HIS3 multilocus genotyping was 0.97. It is worth noting that the DP of HIS3 alone was the highest of the three loci (CDC3: 0.77, EF3: 0.86) (Botterel et al., 2001). Nevertheless, a comparison of the typing information without the HIS3 locus showed that the groups of strains sharing the same genotype do not increase significantly (data

The comparison of the genotyping of biofilm forming *C. albicans* strains with non-biofilm forming *C. albicans* species shows also a consistent distribution of genotypes. There is no literature to compare these specific results with, but as aforementioned, a consistent contribution of genotype data collected with the CDC3, EF3, and HIS3 multilocus

The collected information about strains from the same patients are worth a closer look: Only one patient out of six showed 2 strains sharing the multilocus genotype. Using the same typing system, Beretta et al. (2006) investigated 14 isolates of eight patients and reported 4 strains with the same genotype for one patient out of three. Another patient had 2 of 3 strains sharing the genotypes (Beretta et al., 2006). When only HIS3 and CDC3 alleles were compared, five out of the six patients in the current study show the same strain twice. Because of these findings, the typing was done without EF3 locus information, and as it is mentioned above for the typing without HIS3 allele information, no significant increase in the numbers of strains sharing the same multilocus genotype could be seen (data not

Recapitulating the multilocus genotyping with the CDC3, EF3, and HIS3 system during this study, the data presented here is in good agreement with the authors mentioned

The multilocus genotyping with the CDC3, EF3, and HIS3 system during this study did work well and provided data comparable to former studies. Therefore it is strongly indicated that the genotyping of *C. albicans* strains should be continued in future studies. Aditionally the results give possible evidence that genotypes do not matter in the connection to biofilm forming abilities, so that potentially all *C. albicans* strains are able to

genotyping system seems to be normal for *C. albicans* strains.

clearly different from all other studies.

not shown).

shown).

above.

**4. Conclusion**

(+: biofilm positive; -: biofilm negative)

Table 3. Microsatellite models of 12 strains from six patients (two strains each).


*Edited by Dhastagir Sultan Sheriff*

With the emerging newer trends in sexuality and clashes with the traditional practice of human sexuality, there is a need for introspection and a framework of ethical principles that may guide human sexuality and its practice. Human sexuality is more than a biological phenomenon. Open discussions about sexual identity and sexual practices will help people better understand themselves, others, and the world around them. This book gives a panoramic view of certain aspects of human sexuality in health and disease.

ISBN 978-1-83962-441-4

Psycho-Social Aspects of Human Sexuality and Ethics

Published in London, UK © 2021 IntechOpen © Mybona / iStock


**6. Future perspectives**

EVs are endogenous carriers that facilitate intercellular communication. Although their existence has been known for a long time, they have attracted recent renewed interest because of their possible participation in the spread of particularly cancer initiating or metastasis promoting agents from tumour cells, which appear to produce them in excessive amounts. By the same token, EVs from normal cells may be able to reverse the malignant characteristics of cancer cells by transfer of tumour suppressors or pro-apoptotic molecules, providing more 'natural' therapy. In the drug delivery field, they are causing much excitement as potential therapeutics because of their efficient transfer of proteins, mRNA and miRNA, as well as existing drugs, into selective targets. They have obvious advantages over artificial liposomes or other nanoparticles. However, this requires more knowledge of EV content and how they are released, their stability and how they target cells. There is also a need for clearer quanti‐ tative and qualitative analysis of EVs in terms of their classification and production from normal and cancerous cells.


**3. Laboratorial studies, field studies, and biological outcomes**

Studying the effects of infrasonic or lower-frequency airborne pressure waves on biological structures is a very complex undertaking, whether it be on cell cultures, on animal models, or on human populations. Laboratorial studies, occupational field studies, and residential field studies all have their own strengths and weaknesses. When the latter go unrecognized, however, experimental design flaws can ensue. In this section, the attributes of these different experimental setups are discussed, and their weaknesses and strengths are explored. Together

with the preceding section, this serves as a preamble to Section 4, where the results of experimental studies are described in detail.


*Article* **The Relationship between Physical Activity, Mobile Phone Addiction, and Irrational Procrastination in Chinese College Students**

**Mengyao Shi 1, Xiangyu Zhai 2, Shiyuan Li 1, Yuqing Shi 1 and Xiang Fan 1,3,\***

**Abstract:** The aim of the current study was to examine the associations between physical activity, mobile phone addiction, and irrational procrastination after adjustment for potential confounding variables. The participants were 6294 first- and second-year students recruited as a cluster sample from three public universities in Shanghai, China. Physical activity, mobile phone use, and irrational procrastination were assessed using the International Physical Activity Questionnaire-Short Form (IPAQ-SF), the mobile phone addiction index scale (MPAI), and the irrational procrastination scale (IPS). The participants were divided into four groups according to their mobile phone usage status and physical activity level. The binary logistic regression model was used to predict the probability of serious irrational procrastination among different groups. The emergence of serious of irrational procrastination under physical activity of different intensity and different mobile phone addiction statuses was predicted by a multiple linear regression model. In this study, the combination of insufficient physical activity and mobile phone addiction is positively associated with high levels of irrational procrastination. Furthermore, students who exhibited both mobile phone addiction behaviors and insufficient physical activity tended to have significantly higher odds of reporting high levels of irrational procrastination than those students who exhibited one behavior or neither behavior. After adjusting for the effects of age, BMI, tobacco, alcohol use, and sedentary time, the result is consistent with previous outcomes. These findings suggest that intervention efforts should focus on the promotion of physical activity and reduction of mobile phone addiction.

**Keywords:** physical activity; mobile phone addiction; procrastination; college students; China