Fungal Contamination of Sandpits from Recreational Parks and Schools: A Potential Risk for Human Health
C. Viegas1, 2*, J. Brandão3, R. Sabino1,3, M. Meneses1, C. Veríssimo3
1Environment and Health RG, Lisbon School of Health Technology, Polytechnic Institute of Lisbon, Lisbon.
2Centro de Investigação em Saúde Pública, Escola Nacional de Saúde Pública, Lisbon
3Mycology Laboratory, National Institute of Health Dr. Ricardo Jorge, Lisbon.
Sandpits used by children are frequently visited by wild life which constitutes a source of fungal pathogens and allergenic fungi.
Potentially pathogenic fungi were isolated from all samples besides one. Fusarium dimerum (32.4%) was found to be the dominant species in one park and Chrysonilia spp. in the other (46.6%). Fourteen different species and genera were detected and no dermatophytes were found. Of a total of 14 species and genera, the fungi most isolated from the samples of the elementary schools were Penicillium spp. (74%), Cladophialophora spp. (38%) and Cladosporium spp. (90%). Five dominant species and genera were isolated from the kindergartens. Penicillium spp. was the only genus isolated in one, though with remarkably high counts (32500 colony forming units per gram). In the other kindergarten Penicillium spp. were also the most abundant species, occupying 69% of all the fungi found.
All of the samples exceeded the Maximum Recommended Value (MRV) for beach sand defined by Brandão et al. 2011, which are
Keywords: Recreational Parks; Elementary Schools; Kindergartens; Sand; Fungal Contamination
Soils rich in keratin residue and other organic debris constitute a permanent or occasional reservoir for dermatophytes and other potentially pathogenic fungi. Superficial mycoses such as dermatomycoses are the most common human fungal infections produced by dermatophyte fungi that belong to three genera: Trichophyton, Microsporum and Epidermophyton which affect the skin, hair and nails . The frequency of fungal infections are especially high in urban environments where people, waste, wild and domestic life, congregate in larger densities .
Sandpits used by children are frequently visited by animals such as sparrows, pigeons, dogs, cats and rats and constitute as a source for fungi . Despite this knowledge, standards for fungal quality assessment of sandpit sand still lack regulations by international environmental and health agencies or even local and regional agencies. Municipalities in Portugal are, nevertheless, legally obligated to maintain recreational parks at good hygienic and safety levels, which include annual sand replacement, when used.
A number of studies detected a diverse number of species of fungi in beach sand, an equivalent matrix despite the differences in recreational contexts [3-6]. Under natural conditions, UV light affects both bacteria and fungi but fungi appear to be less susceptible and remain viable for longer periods of time . Some fungi remained thus viable in sand under UV light exposure in lab conditions for up to 6 months .
In absence of guidelines and levels of fungal contaminants, the assessing system proposed by Sabino et al., 2011  and by Brandão et al., 2011  was considered as compatible, given the matrix similarities. In this system, three different fungal groups are searched: (1) Dermatophytes, (2) Potentially pathogenic and allergogenic fungi and (3) Yeasts, as explained by the authors. Group 2, the most heterogeneous one include aerial sporulating species such as the Aspergillus fumigatus and Aspergillus niger complexes and the genera Aspergillus spp. (unspecified species). Also Fusarium spp., Neoscytallidium spp., Scopulariopsis spp. and Scedosporium spp. and any other species or genera which counts results are above 500 CFU per gram of sand are included [3, 8].
A pilot study of sandpits was designed in children’s playgrounds to assess fungal contamination levels in sand from two public recreational parks, three elementary schools and two kindergartens in the region of Lisbon and Tagus Valey.
Materials and Methods
The study was designed in children’s playgrounds and includes two public recreational parks, three elementary schools and two kindergartens of the Lisbon and Surrounding Municipalities. The playgrounds were chosen due to their poor hygienic conditions trying to characterize the most critical scenario regarding exposure of children attending.
A composite of three loci samples from each location of up to 10 centimeter deep was collected with sterile gloves into a sterile plastic container and transported to the laboratory for analysis within 5 days (to ensure viability of all fungi present). For the culture method, 40 g of each sample (composite) were suspended in 40 ml sterilized water, followed by agitation during 30 min at 100 rpm. Duplicates of 0.2 ml for each sample were inoculated in malt extract agar (2%) with chloramphenicol (0.05 g/L) and
For quantification purposes, and regardless of the microbe recovery (extraction) levels, 1 ml sand wash was presumed to represent 1g sand, given the 1:1 extraction ratio. An additional incubation at 40ºC was used to specifically select the growth of Aspergillus fumigatus sensu stricto.
Vanbreuseghem’s (1952)  hair-bait technique was used in order to detect dermatophytes .After exposure of sand during 20 days at 27.5ºC baby sterilized hair were transposed to agar mycosel plates with cyclohexamide, at the same temperature for two weeks.
For species identification, microscopic mounts were performed using tease mount or Scotch tape mount and lactophenol cotton blue mount procedures. Morphological identification was achieved through macro- and microscopic characteristics according to de Hoog et al. (2001) . Fungi were identified at the species level whenever possible, since adverse health effects tend to vary within genera [10,11].
The data analysis was performed and used descriptive statistics using frequency, median and graphical representations appropriate to the nature of the data.
The quantification results for fungi obtained from each analyzed sand sample are summarized in Table 1 (recreational parks) and Table 2 (schools).
Fourteen different species/genera were obtained from both recreational parks. In Sample 1 (Recreational Park 1), 10 fungal species were identified in plates incubated at 27.5ºC, being Fusarium dimerum (complex) (32.4%), Cladosporium spp. (23.5%), Fusarium solani (complex) and Phoma spp. (11.8%) the most frequently isolated (Table 1). In addition to these species, the following were also identified: Chrysosporium spp., Penicillium spp., Alternaria spp., Geomyces spp., Fusarium spp. and Aspergillus sydowii. The same samples incubated at 40ºC exhibited only Alternaria spp.
Table 1. Fungal distribution of the samples collected from the two recreational parks (incubation at 27.50C).
From Sample 2 (Recreational Park 2), when incubated at 27.5ºC, 8 fungal species were isolated, Chrysonilia spp. (46.6%) the most prevalent, followed by Cladosporium spp. (28.8%) and Acremonium spp. (15.1%) (Table 1). The same sample, incubated at 40ºC, showed growth of Penicillium spp. as the most prevalent genera (59.3%), followed by Scopulariopsis spp. and Phialophora spp. (14.8%). No dermatophytes were detected.
Fourteen fungal species were isolated from samples collected from elementary schools (Samples 1, 2 and 3 - incubated at 27.5ºC - Table 2). In Sample 1, Penicillium spp. (74%) was the most frequently found, followed by Fusarium spp. (22%) and A. niger complex (4%). In Sample 2 Cladophialophora spp. (38%), Fusarium spp. (23%) and Neoscytalidium spp. (23%) were the most frequent. In addition to these species, Aspergillus ustus complex and Exophiala spp. were also identified. In Sample 3, Cladosporium spp. was the most prevalent (90%). Other species were also found, namely: Fusarium spp., Cladophialophora spp., Phoma spp., Alternaria spp., Rhizopus spp., as well as isolates from A. terreus complex and A. flavus complex.
When samples from kindergartens (Samples 4 and 5) were incubated at 27.5ºC, 5 different fungal species/genera were isolated (Table 2). Penicillium spp. was the only genera isolated from Sample 4 and in higher counts than the other samples (Table 2). In Sample 5, Penicillium spp. was also the most frequently identified (69%), followed by Phoma spp. (13%), Acremonium spp., and Neoscytalidium spp. and Aspergillus nidulans (complex) (6%). Sample 4 was the only one that did not present any fungal species that belongs to the potentially pathogenic fungi group. All samples showed a CFU counting above the Maximum Recommended Value (MRV) described for beach sand in Brandão et al. (2011)  (Figure 1).
Table 2. Fungal distribution in of the analyzed samples collected from schools’ sandpits (incubation at 27.5 0C).
Figure 1. Potential pathogenic fungi distribution from the analysed sand samples.
In urban areas, where there are high concentrations of people and animals, soil rich in organic matter may constitute a permanent or occasional reservoir for fungi. These fungi can be a potential source of skin infections for humans and animals . Therefore, isolation of fungi from public parks sandpits may be a cause for concern. The risk of fungal skin infection is greater for children, a susceptible age group playing in sandy playgrounds . Sandpits are thought to play a role in the epidemiology of human and animal mycoses .
Brandão and colleagues (2011)  proposed a value of 85 CFU/g as MRV for potentially pathogenic fungi, based on averages of an extensive national representation. In the present study a fungal load from all the identified genera exceeds the MRV for potential pathogenic fungi in all but one of the samples analyzed. Moreover, besides this value (MRV) the need for a more demanding value was already stated in Sabino and colleagues (2011)  and the authors of this paper believe an adjustment to a non-coastal beach context is also required, based on extensive data which needs yet to be generated.
Special care needs to be paid to identify potential pathogenic fungal species, such as species from Fusarium (isolated in samples from recreational parks and in all elementary schools), Aspergillus genera (isolated in all schools analyzed besides one kindergarten) and Neoscytalidium genera (isolated from two schools and one kindergarten). In agreement with Rippon (1982) , sand may be considered to be a direct exposure source of geophilic, zoophilic and antropophilic keratinophilic fungi. Some of the isolated species in this study were previously detected in human and animal infections, such as Aspergillus spp., F. solani complex  and Scopulariopsis sp. . Scopulariopsis species are known to induce opportunistic infections, and S. brevicaulis is well-established agent for onychomycosis . This supports the notion of potential risk of skin/nail infections in children due to exposure to sand . Fusarium genera are frequently implicated in ophthalmic keratitis and other superficial mycosis, and their presence in the analyzed sands is also an issue of concern for children health .
The existence of superficial infections caused by non-dermathophytic fungi have been reported in several studies [18- 19]. When infections due to these other fungal agents occur, neglecting can lead to very unfavourable and even serious outcomes, such as opportunistic follow-up infections by bacteria . Moreover, allergic reactions due to fungal exposure, especially due to dematiaceous fungi as Cladosporium spp. are well documented in several studies .
The fungal load found in the sand of some of the playgrounds analyzed in this pilot study suggests that sand has not been replaced nor treated (cleansed). The high fungal loads represent a potential human health risk and therefore, sandpits in such condition need to handle. The fungal species isolated also confirm the potential risk of exposure of children of young ages to keratinophilic fungi. Further studies applying molecular tools are needed to overcome eventual limitations from the methods applied. Despite the absence of epidemiologic studies to reaffirm concerns, this pilot study demonstrates that regular cleaning or replacing of sand needs to be implemented.
Cite this article: Viegas C. et al., Fungal Contamination of Sandpits from Recreational Parks and Schools: A Potential Risk for Human Health. J J Environ Sci. 2016, 2(1): 013.