ORIGINAL PAPER
Medycyna Pracy 2014;65(1):15–32
© Nofer Institute of Occupational Medicine in Łódź http://medpr.imp.lodz.pl/en Justyna Skóra1 Beata Gutarowska1 Łukasz Stępień2 Anna Otlewska1 Katarzyna Pielech-Przybylska1
THE EVALUATION OF MICROBIAL CONTAMINATION
IN THE WORKING ENVIRONMENT OF TANNERIES
OCENA ZANIECZYSZCZENIA MIKROBIOLOGICZNEGO NA STANOWISKACH PRACY W GARBARNIACH
1 Lodz University of Technology / Politechnika Łódzka, Łódź, Poland
Institute of Fermentation Technology and Microbiology / Instytut Technologii Fermentacji i Mikrobiologii
2 Institute of Plant Genetics of the Polish Academy of Sciences / Instytut Genetyki Roślin Polskiej Akademii Nauk, Poznań, Poland
Abstract
Background: Due to their animal material processing, tannery workers may be exposed to biological agents. The aim of the study was
the microbial contamination assessment of tanneries with different production specifications. Health risk was estimated based on par-ticle size distribution. Moreover, indicators of microbial contamination of tanneries were selected. Materials and Methods: The studies were conducted in 2 types of tanneries – processing raw hides and producing chrome-tanned leather. Air was sampled with MAS-100 Eco Air Sampler, leathers using RODAC Envirocheck® contact plates and swab method, microbial numbers were determined by a cul-ture method. For the bioaerosols size distribution analysis, a six-stage Andersen sampler was used; identification was performed using microscopy and biochemical methods. Microbial contamination was identified by 16S RNA and ITS1/2 rDNA analysis for bacteria and fungi respectively. Results: The microbial number in the air ranged between 1.2×103 and 3.7×103 CFU/m3. While on the leather, it ranged
between 7.6×101 and 5.5×105 CFU/100 cm2. Bacteria dominated in the tanneries (air: 51–92%, leathers: 60–100%). Results indicate that
potential health risks arise from the fungal small bioaerosol particles presence (0.65–2.1 µm). Eleven indicator microorganisms were determined: B. pumilus, B. subtilis, B. cereus, C. lubricantis, C. cladosporioides, P. commune, P. echinulatum, P. chrysogenum,
P. crusto-sum C. parapsilosis and C. albidus. Conclusions: Microbial contamination evaluation in the tanneries showed the increased bacteria
and fungi number in the air in relation to the outdoor air, which indicates an occupational inhalation risk to workers. The designated indicators of microbial contamination in the tanneries are associated with their specific and potentially pathogenic working environ-ment. Med Pr 2014;65(1):15–32
Key words: tanneries, bioaerosols, microbial contamination, microbial indicators Streszczenie
Wstęp: Z powodu przetwarzania surowca zwierzęcego w garbarniach pracownicy mogą być narażeni na czynniki biologiczne. Celem
badań była ocena zanieczyszczenia mikrobiologicznego w garbarniach o różnej specyfice produkcji. Określono także ryzyko zdrowot-ne w oparciu o rozkład ziarninowy bioaerozolu. Ponadto wyznaczono wskaźniki zanieczyszczenia mikrobiologiczzdrowot-nego w garbarniach.
Materiał i metody: Badania przeprowadzono w dwóch rodzajach garbarni – przetwarzających skóry surowe i chromowo garbowane
(wet blue). Powietrze pobierano próbnikiem MAS-100 Eco, próby ze skór, używając płytek odciskowych RODAC Envirocheck® i metody tamponowej, a liczbę mikroorganizmów określano metodą hodowlaną. Rozkład cząstek biooaerozolu wykonano z użyciem 6-stopnio-wego impaktora Andersena. Identyfikację drobnoustrojów wykonano metodą mikroskopową i testami biochemicznymi. Wskaźniki za-nieczyszczenia mikrobiologicznego identyfikowano, analizując odpowiednio dla bakterii i grzybów sekwencje 16S RNA i ITS1/2 rDNA.
Wyniki: Liczebność mikroorganizmów w powietrzu w garbarniach kształtowała się w granicach 1,2×103–3,7×103 jtk/m3. Skóry były
zanieczyszczone mikrobiologicznie w granicach 7,6×101–5,5×105 jtk/100 cm2. W garbarniach dominowały liczbowo bakterie (w
po-wietrzu: 51–92%, na skórach: 60–100%). Wskazano na zagrożenie zdrowotne wynikające z obecności cząstek bioaerozolu grzybowego o rozmiarach 0,65–2,1 µm. Wyznaczono 11 gatunków drobnoustrojów wskaźnikowych dla garbarni: B. pumilus, B. subtilis, B. cereus,
C. lubricantis, C. cladosporioides, P. commune, P. echinulatum, P. chrysogenum, P. crustosum, C. parapsilosis i C. albidus. Wnioski: Ocena
zanieczyszczenia mikrobiologicznego w garbarniach wykazała podwyższoną liczebność bakterii i grzybów w powietrzu w stosunku do powietrza atmosferycznego, co świadczy o występowaniu narażenia inhalacyjnego pracowników. Wyznaczone wskaźniki zanieczyszcze-nia mikrobiologicznego w garbarni są związane ze specyfiką środowiska pracy i potencjalnie chorobotwórcze. Med. Pr. 2014;65(1):15–32
Słowa kluczowe: garbarnia, bioaerozol, zanieczyszczenie mikrobiologiczne, wskaźniki mikrobiologiczne
Corresponding author / Autorka do korespondencji: Justyna Skóra, Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wólczańska 171/173, 90-924 Łódź, Poland, e-mail: justyna-skora@wp.pl
Received: 2013, October 15, accepted: 2014, January 3
Studies were realized within the project of Polish National Center for Research and Development coordinated by the Central Institute for Labour Protection – National Research Institute, no. III.B.03 entitled “Development of principles for evaluation and prevention of hazards caused by biological agents in the working environment using indicators of microbial contamination”.
INTRODUCTION
Leather is an important raw material used in the manufacture of footwear, apparel, furniture and leather products. In 2011, the total turnover of the Eu-ropean Union tanning industry reached 7.8 billion euro, corresponding to the production of 224
mil-lion m2 of finished leather and about 44 thousand tons
of sole leather. Currently, it is estimated that leath-er product industries in the European Union have about 24 000 of different-sized enterprises employing a total of 400 000 workers (1).
In order to give the leather adequate durability and aesthetic quality, it is necessary to perform a series of mechanical and chemical operations, wet and finish-ing processes (2,3). These treatments are necessary be-cause fresh animal skins are of limited value, as they contain large amounts of moist and available nutrients (carbohydrates, fats and proteins) that provide a per-fect medium for rapid growth of microorganisms (4). The sources of microorganisms are diverse. The skin may be contaminated with blood, feces or soil; the means of transport, air and equipment in produc-tion and storage facilities should be also considered. Bacteria that are most often isolated from raw hides belong to the species: Escherichia coli, Staphylococcus
epidermidis, Proteus mirabilis, Morganella morganii, P. morgani, P. vulgaris, Bacillus anthracis, B. subtilis
and B. mycoides (4,5). The action of bacterial proteo-lytic enzymes results in partial or complete hydrolysis of leather components, the emission of ammonia and hydrogen sulphide, stains and damage to the leathers surface (6,7).
Leather has a higher biological resistance than raw hide. This is because it contains collagen, which is resistant to proteolytic enzymes (except colla-genase) and a small amount of water (preferably due to the functional characteristics 14–20%) (8). Micro-biological destruction of chrome-tanned leather (wet blue) and leather results mainly from the activ-ity of filamentous fungi (9). The studies of microbial contamination of leather have shown that the domi-nant species are filamentous fungi from the genera
Penicillium (P. commune, P. glaucum, P. wortmannii, P. frequentans) and Aspergillus (A. niger, A. flavus, A. oryzae, A. fumigatus). Apart from those, the
oc-currence of the species from the genera: Alternaria,
Cladosporium, Trichoderma, Fusarium, Aureoba-sidium, Scopullariopsis and also actinomycetes from
the genus Streptomyces was observed on the surface
of tanned hides (10). They can cause a breakdown of fat and loosening of hide fibres, and create differently coloured stains (11,12). The growth of microorgan-isms in raw hides, chrome-tanned leather and finished products may pose a threat to tanners, especially those involved in the first operations of tanning. Potential microbiological hazards in the working environ-ment of tanneries may result from the exposure to high concentrations of pathogens in raw material and in the air of production facilities as well as from the contact with pathogens. Pathogenic microorganisms found in the working environment include: Bacillus
anthracis (anthrax), Leptospira interrogans
(leptospi-rosis), Clostridium tetani (tetanus), Coxiella (Q fever) and Brucella melitensis, B. suis, B. abortus and B. canis (brucellosis) (13).
There have been reports concerning various health problems among workers in tanneries. In the study carried out by Ory et al. (14) the health complaints among 418 labourers in 15 Indian tanneries were test-ed. Asthma (38%), dermatitis (23%) and chronic bron-chitis (14%) were the most frequently reported com-plaints in the 12 months prior to the survey. Moreover,
Cr6+ ions cause skin irritation, eardrum perforation,
nasal irritation, ulceration and lung carcinoma in hu-mans and animals, and are accumulated in placenta impairing the foetal development in mammals (14). Rastogi et al. (15) analysed the health risks of 197 male workers drawn from different sections of 10 leather tanneries in Kanpur. A control group comprising of 117 male subjects belonging to a similar age group and socioeconomic strata, who never had any occu-pational exposure in the leather tanneries, were also examined for comparison. The findings revealed a sig-nificantly higher prevalence of morbidity among the exposed workers in contrast to that observed in the controls (40.1% vs. 19.6%).
Respiratory diseases (16.7%) were mainly responsi-ble for a higher morbidity among the exposed workers, whereas gastrointestinal tract problems were predomi-nant in the control group (15). There has only been a limited number of studies on the microbiological risks in the working environment of tanneries. The aim of the present studies was to analyse the degree of mi-crobial contamination and the type of microorganisms in the working environment of tanneries with different production specifications – those processing raw hides and those producing wet blue leather. Health risk was also estimated basing on a study of particle size distri-bution in a selected facility.
Microbial contamination in tanneries
Nr 1 17
Moreover, indicator microorganisms were selected for the working environments of the tanneries basing on an analysis of their prevalence in a workplace, source of isolation, and health risk determined based on lite-rature data (according to the classification of the Direc-tive 2000/54/EC (16)), the Regulation of the Minister of Health in Poland dated 22nd of April, 2005 (17), the biosafety levels classification (BSL) of the European Confederation of Medical Mycology (18) and the Insti-tute of Rural Health in Lublin (Instytut Medycyny Wsi im. Witolda Chodźki w Lublinie) (13). The taxonomic classification of the selected indicators was confirmed using molecular methods.
MATERIAL AND METHODS Description of the studied rooms
The assessment of microbial contamination was per-formed in 4 tanneries with different production specifi-cations. Two of the studied tanneries (Tanneries I and II) produced wet blue leather, and the production process was mainly based on retanning, dyeing and finishing leather. The 2 other plants (Tanneries III and IV) spe-cialized in the production of leather for shoes and fancy goods; the production process included all stages of raw hide (fresh and salted) processing and ended up obtain-ing leather for the production of leather goods.
Description of the tested rooms is given in Table 1. Temperature and humidity in the tested rooms were determined using a PWT-401 hygrometer (Elmetron, Poland). Samples of the air were also taken in an office room (internal background) and in the atmospheric air (external background).
Determination of microbiological contamination of the air and leathers
Samples for the analysis of microbial contamination of the air and leather surface in the plants were collected during active working hours. Microbiological contami-nation of the air was determined using an MAS-100 Eco Air Sampler (Merck, Germany). Samples of 50 l and 100 l of air, with flow rate: 100 l/min, were taken directly onto MEA (Malt Extract Agar, Merck, Germa-ny) with chloramphenicol (0.1%) for determination of the total number of fungi (including hydrophilic and xerophilic fungi), and onto a TSA medium (Tryptic Soy Agar, Merck, Germany) with nystatin (0.2%) for the determination of the total number of bacteria. Air samples were taken in 2 repetitions on each medium
in 3 places in each room. Table 1
. C ha ra ct er ist ic s o f t he r oo m s e xa m in ed i n t an ne rie s Ta be la 1 . C ha ra kt er ys ty ka p om ie sz cz eń w b ad an yc h g ar ba rn ia ch Tes te d p la nt Bad an y za kład Ro om s Po mieszczeni a Cu ba tur e Ku ba tura [m 3] Tem pera tur e of t he a ir Tem pera tura po w iet rza [°C] Re la tiv e h umidi ty of t he a ir W ilg ot noś ć wzg lę dn a p ow iet rza [%] Ro om s’ des tin at io n Prze zn aczenie p omieszczeni a Pl ace o f s am plin g M iejs ce p ob or u p ró b M SD M SD Ta nn er y I / / G ar ba rn ia I pr od uc tio n ha lls / h ale pr od uk cyjn e (N = 3) 250–560 26.1 4.71 59.8 10.5 ret ann ag e a nd fini shin g o f w et b lue le at her , s ho rt-t er m sto ra ge o f p alet tes o f ra w m at er ia l, vac uum dr yin g o f hides, m ov em en t o f hides u sin g h oi sts / doga rb owa nie i w yka ńcza nie s kó r w et b lue , k ró tk ot er min ow e prze ch ow ywa nie p alet ze s kó ra mi s ur ow ymi , p ró żnio w e su szenie s kó r, t ra ns po rt owa nie s kó r za p om oc ą w ci ąga rk i sa m ples w er e co lle ct ed n ext t o t he p alet te o f w et b lue le at her s, n ext t o t he vac uum dr yin g o ven, n ext t o t he rac ks w ith dr ie d le at her , in t he t ann ed le at her wa re ho us e / / p ró bk i p ob ra no p rzy p ale cie ze s kó ra mi w et b lue p rzy su sza rni p ró żnio w ej, p rzy w yci ągu z w ys uszo ny mi s kó ra mi , w m agazy nie s kó r ga rb owa ny ch Ta nn er y I I / / G ar ba rn ia II pr od uc tio n ha lls / h ale pr od uk cyjn e (N = 3) 370–650 15.9 1.1 48.1 6.1 ret ann ag e o f w et b lue le at her , s or tin g, s ha vin g, fluffin g, dr yin g a nd c ut tin g, fini shin g o f le at her , sh or t-t er m s to ra ge o f hides / doga rb owa nie s kó r w et b lue , s or to wa nie , o dwła sia nie , szlif owa nie , s uszenie i cię cie o raz w yk oń czenie s kó r, k ró tk ot er min ow e prze ch ow ywa nie s kó r sa m pl es w er e t ak en n ex t t o th e p lan in g m ac hi ne , n ex t t o th e ta nn in g dru m , n ex t t o th e s an di ng an d str et ch in g m ac hi ne , ov er th e t an ni ng ra ck s, ne xt to th e l ea th er cu tti ng m ac hi ne , ne xt to th e r ac ks w ith ta nn ed le at he r / p ró bk i p ob ra no p rz y stru ga rc e p rz y b ęb ni e g ar ba rs ki m p rz y s zli fie rc e z n ap in ar ką na d w ies za ka m i z e s kó ra m i, pr zy u rz ąd ze ni u do ci ęc ia sk ór pr zy w ies za ka ch z sk ór ą g ar bo w an ą
Tes te d p la nt Bad an y za kład Ro om s Po mieszczeni a Cu ba tur e Ku ba tura [m 3] Tem pera tur e of t he a ir Tem pera tura po w iet rza [°C] Re la tiv e h umidi ty of t he a ir W ilg ot noś ć wzg lę dn a p ow iet rza [%] Ro om s’ des tin at io n Prze zn aczenie p omieszczeni a Pl ace o f s am plin g M iejs ce p ob or u p ró b M SD M SD Ta nn er y I II / / G ar ba rn ia III pr od uc tio n ha lls / ha le pr od uk cy jn e (N = 2) 300–18 000 16.4 0.8 39.2 4.9 m ov em en t a nd se gr eg at io n of ra w m at er ia l, s oa ki ng , lim in g an d sh av in g of ra w h id es , t an ni ng an d dy ein g hi de s. Sa m pl es w er e t ak en n ex t t o th e s oa ki ng an d lim in g dru m s, ne xt to th e p al let o f r aw h id e s cr ap s / tr an sp ort ow an ie i s eg re ga cja sk ór su ro w yc h, m oc ze ni e, w ap no w an ie i o dw ła sia ni e s kó r s ur ow yc h, g ar bo w an ie i f ar bo w an ie sk ór . Pr ób y p ob ra no p rz y b ęb na ch d o m oc ze ni a i w ap ni en ia , pr zy p al ec ie ze śc in ka m i ś w ie że j s kó ry sa m ples w er e co lle ct ed n ext t o t he rac ks w ith s or te d w et b lue le at her s, n ext t o t he p la nin g m ac hin e, n ext t o th e d yein g dr um s / p ró bk i p ob ra no p rzy w iesza kac h z p os or to wa ny mi s kó ra mi w et b lue , p rzy s tr uga rce , przy b ęb nac h do b ar w ieni a s kó r Ta nn er y I V / / G ar ba rn ia IV wa re ho us es fo r r aw , we t b lu e a nd ta nn ed le at h-er s, pr od uc -tio n ha lls / / m ag az yn y sk ór su ro w-yc h, w et b lu e i g ar -bo wa ny ch , ha le pr od uk cy jn e (N = 6) 285–27 000 20.2 4.4 50.8 17.7 sto ra ge o f s al te d ra w hides, p re limin ar y a nd m ain so ak in g, r in sin g o f hides, limin g, b at in g, p ic klin g, cu ttin g o f w et b lue le at her s, s to ra ge o f w et b lue an d t ann ed le at her s / p rze ch ow ywa nie s olo ny ch s kó r sur ow yc h, m oczenie ws tęp ne i głó w ne , płu ka nie sk ór s ur ow yc h, o dmięśni anie , w yt ra w ia nie , pi klo wa nie , cię cie s kó r w et b lue , p rze ch ow ywa nie sk ór w et b lue i ga rb owa ny ch sa m ples w er e t ak en in t he ra w hide wa re ho us e, n ext t o t he pa llets o f ra w hides a fter addin g s al t, n ext t o t he p re-s oa kin g dr um, n ext t o t he s oa kin g dr um, n ext t o t he p al lets o f s kin s aft er s oa kin g, n ext t o t he r in sin g dr um, n ext t o t he fles hin g m ac hin e, n ext t o t he limin g dr um, n ext t o t he p al lets o f hides imm edi at ely a fter limin g, n ext t o t he p al lets o f hides aft er b at hin g, n ext t o t he p al lets o f hides a fter p ic klin g, n ext to t he c ut tin g m ac hin e f or w et b lue hides, n ext t o t he rac ks w ith t ann ed le at her / p ró by p ob ra no w m agazy nie s kó r sur ow yc h, p rzy p alet ac h ze s kó ra mi p o do da ni u s oli , p rzy bę bnie do m oczeni a ws tęp neg o, p rzy b ęb nie do m oczeni a wła ści w eg o, o bo k p alet ze s kó ra mi p o et ap ie m oczeni a, ob ok p alet ze s kó ra mi p o m oczeni u wła ści w ym, p rzy bę bnie do płu ka ni a, p rzy urządzeni u do o dmięśni ani a s kó r, przy b ęb nie do wa pnieni a, o bo k p alet ze s kó ra mi za raz p o wa pnieni u, o bo k p alet ze s kó ra mi p o w yt ra w ia ni u, o bo k pa let ze s kó ra mi p o p ik lo wa ni u, p rzy urządzeni u do cię ci a sk ór w et b lue , p rzy w iesza kac h ze s kó rą ga rb owa ną In ter na l ba ck gr ou nd / / T ło we wn ęt rz ne offi ce s / b iu ra (N = 4) 75–160 17.6 0.6 36.6 4.3 no n-p ro duc tiv e r oo m s o f t ann er ies, lo ca te d in s ep ara te bui ldin gs, a nd h av in g a s ta nd ar d o ffice e qui pm en t / / p omieszczeni a w za kład ac h nie zw iąza ne z p ro du kc ją, ulo ko wa ne w o ddzie ln yc h b ud yn kac h i p osi ad aj ące sta nd ar do w e w yp os ażenie b iur ow e sa m ples w er e t ak en in 3 r ep res en ta tiv e p laces f ro m e ac h office / p ró by p ob iera no w 3 r ep re zen ta ty w ny ch miejs cac h każdeg o b iura Ex ter na l ba ck gr ou nd / / T ło ze wn ęt rz ne at m os ph er ic air / po wi et rz e at m os fer yc zn e (N = 4) n.t. n.t. n.t. sa m ples w er e t ak en a t a di sta nce o f 5–10 k m f ro m e ac h ta nn er y / p ró by p ob iera no w o dległoś ci 5–10 k m o d każdej ga rb ar ni M – m ea n / ś re dn ia ; S D – s ta nd ar d d ev ia tio n / o dc hy le ni e s ta nd ar do w e. n.t . – n ot t es te d / n ie b ad an o. Ta ble 1 . C ha ra ct er ist ic s o f t he r oo m s e xa m in ed i n t an ne rie s – c on t. Ta be la 1 . C ha ra kt er ys ty ka p om ie sz cz eń w b ad an yc h g ar ba rn ia ch – c d.
Microbial contamination in tanneries
Nr 1 19
For the analysis of the size distribution of vi-able bioaerosols, a six-stage Andersen sampler (model WES-710, Westech Instruments, UK) was used. The use of this instrument made it possible to divide the bioaerosols into 6 fractions, in accordance with their aerodynamic diameters, as follows: ≥ 7.0 µm (1st stage), 7.0–4.7 µm (2nd), 4.7–3.3 µm (3rd), 3.3–2.1 µm (4th), 2.1–1.1 µm (5th) and 1.1–0.65 µm (6th). The air was sampled with a vacuum pump at a constant 28.3 l/min flow rate. Samples were taken over 5 min (141.5 l of air) onto MEA with chloramphenicol (0.1%) (fungi) or TSA medium with nystatin (0.2%) (bacteria) in 3 repetitions on each medium in the production hall in Tannery I.
The materials processed in tanneries were also ana-lyzed for microbiological content. The names of leather have been adapted from Thanikaivelan et al. (2004) (3):
n hide – skin of a large animal, such as cow or buffalo,
n leather – a general term for hide or skin with its
original fibrous structure more or less intact, and that has been treated so as to be imputrecible, the hair or wool might or might not have been removed,
n wet blue – chrome-tanned leather in wet condition.
Samples from leathers were taken using RODAC
Envirocheck
®
plates (Replicate Organism DetectionAnd Counting, Merck, Germany) with TSA medium for bacteria and with Sabouraud medium (Merck, Germany) for fungi. For the analysis of the microbial contamination of hides, a traditional swab method was applied, using saline solution (0.85% NaCl), swabs and
metal frames of surface area 25 cm2 and media: MEA
with chloramphenicol (0.1%) (fungi) or TSA medium with nystatin (0.2%) (bacteria). Samples were taken from 3 surfaces on each medium.
The media samples from the air and leather mate-rials were incubated at 30±2°C for 48 h (bacteria) or at 27±2°C for 5–7 days (fungi). After incubation, col-onies were counted, and the results were expressed
in CFU/m3 air, CFU/100 cm2 materials. The final result
was the arithmetic mean of all of the repetitions. Identification of microorganisms
The pure cultures of bacteria and yeast were charac-terized macroscopically, and then, selected diagnos-tic features were tested: Gram-staining, catalase test and oxidase test (Microbiologie Bactident Oxydase, Merck, Germany). For isolates of bacteria whose fre-quency of occurrence was greater than 25%, API tests were performed (BioMérieux, France): API 50 CH, API STAPH and API 20 NE, for yeasts, diagnostics was performed using the API C AUX test. Bacteria
identi-fied as indicators of microbiological contamination at workplaces in tanneries were determined according to the procedure described below, underwent the ge-netic identification based on the nucleotide sequence of gene 16S rRNA (19).
Identification of all of the isolated filamentous fungi was performed basing on macroscopic and microscop-ic observations after culture on CYA medium (Difco, USA) and YES medium (Yeast Extract with Supple-ments), with the use of a taxonomic method (17–23).
Identification of moulds and yeast which were specified as the indicators of microbial contamination at workplaces was performed basing on the ITS1/2 se-quence of the rDNA region (24). Genomic DNAs of indicators strains were extracted using the described method (25). The resulting nucleotide sequences of the studied microorganisms were analysed and com-pared with the sequences published in the National Center for Biotechnology Information (NCBI) data-base, using the BLASTN 2.2.27+ program (26). The se-quences obtained for microorganisms were deposited in the NCBI GenBank database.
Selection of indicators
of microbiological contamination at workplaces In order to determine the indicators of microbiological contamination at workplaces in tanneries, protocols or characteristics were established according to Skóra et al. (2012) (27) and modified (frequency of strains isolation, source of isolation and harmfulness to human health) as given in Table 2. A scale of evaluation was also devel-oped for identifying indicators of microbiological con-tamination at workplaces as given in Table 3.
Mathematical calculations
The frequency of occurrence of a species (ƒ) was calcu-lated by dividing the number of samples in which the strain occurred and the total number of samples.
Statistical analyses were performed using STATIS-TICA 6.0 software (Statsoft, USA). The obtained re-sults of microbiological contamination of the air and leathers in tanneries with various production profile were evaluated using one-way Analysis of Variance (ANOVA) at the significance level 0.05. When statis-tical difference was detected (p < 0.05), means were compared by the post-hoc Tukey’s test at significance level 0.05.
Linear regression analysis was used to determine the effect of air humidity and air temperature on microbio-logical contamination of the air in tested tanneries.
Ta ble 2 . C rit er ia f or s el ec tin g i nd ic at or s o f c on ta m in at io n w ith h ar m fu l b io lo gi ca l a ge nt s a t t an ne rie s* Ta be la 2 . K ry te ria t yp ow an ia w sk aź ni kó w z an ie cz ys zc ze ni a s zk od liw ym i c zy nn ik am i b io lo gi cz ny m i n a s ta no w isk ac h p ra cy * No. Lp. Cr iter ia Kr yt er ium D es cr ip tio n O pi s Sc ale Ska la Po in ts Pun kt y 1 fre quen cy o f s tra in iso la tio n f ro m t he en vir onm en t / częs toś ć izo lac ji szczep u ze śr odo w iska th e f re quen cy [%] w ith w hic h t he o rga ni sm wa s i so la te d f ro m t he a ir a nd/o r s ur face a t t he w or ks ta tio ns / częs toś ć [%], z j aką izo lo wa no da ny dr ob no us tró j z p ow iet rza i/l ub p ow ierzc hni w p omieszczeni ac h n a s ta no w iskac h p rac y 0–20% 1 21–40% 2 41–60% 3 61–80% 4 81–100% 5 2 stra in i so la tio n s our ce / / źr ódło p oc ho dzeni a szczep u th e p res en ce o f micr oo rga ni sm in in do or a ir an d/o r o n t he s ur faces a t w or ks ta tio ns w ith rega rd t o i ts p res en ce in t he o ut do or a ir (b ac kg ro un d) / w ys tęp owa nie mi kr oo rga nizm u, w p ow iet rzu w ew nęt rzn ym i/l ub n a po w ierzc hni ac h, n a s ta no w iskac h p rac y w o dniesieni u do j eg o w ys tęp owa ni a w p ow iet rzu a tm osf er yczn ym (tło) pr es en ce in t he in do or a ir a nd o ut do or a ir, a bs en ce o n s ur faces / o be cn oś ć w p ow iet rzu w ew nęt rzn ym oraz w p ow iet rzu a tm osf er yczn ym, b ra k n a p ow ierzc hni ac h 1 pr es en ce o n s ur faces a nd o ut do or a ir, a bs en ce in t he in do or a ir / o be cn oś ć n a p ow ierzc hni ac h o raz w p ow iet rzu a tm osf er yczn ym, b ra k w p ow iet rzu w ew nęt rzn ym 2 pr es en ce in t he in do or a ir, a bs en ce in t he o ut do or a ir a nd o n s ur faces / o be cn oś ć w p ow iet rzu w ew nęt rzn ym, b ra k w p ow iet rzu a tm osf er yczn ym i n a p ow ierzc hni ac h 3 pr es en ce o n s ur faces, a bs en ce in t he in do or a ir a nd o ut do or a ir / o be cn oś ć n a p ow ierzc hni ac h, b ra k w p ow iet rzu a tm osf er yczn ym i w ew nęt rzn ym 4 pr es en ce in t he in do or a ir, o ut do or a ir a nd o n s ur faces / o be cn oś ć w p ow iet rzu w ew nęt rzn ym, po w iet rzu a tm osf er yczn ym i n a p ow ierzc hni ac h 5 pr es en ce in t he in do or a ir a nd o n s ur faces, a bs en ce in t he o ut do or a ir / o be cn oś ć w p ow iet rzu w ew nęt rzn ym i n a p ow ierzc hni ac h, b ra k w p ow iet rzu a tm osf er yczn ym 6 3 ha rmf uln es s t o h um an he al th / szk od liw oś ć zdr ow ot na d la l udzi** acco rdin g t o c la ssific at io n: t he Dir ec tiv e UE 2000/54/EC (16), t he R egu la tio n o f t he M ini ster o f H ea lth in P ol an d d at ed A pr il 22, 2005 (17), b ios af et y le ve ls o f t he E ur op ea n C onf edera tio n o f M edic al M yco log y (BS L) (18), I ns tit ut e o f R ura l H ea lth in L ub lin (IMW ) cla ssific at io n (13), li tera tur e d at a / wg k la sy fikac ji przyj ęt ej w D yr ek ty w ie UE 2000/54/WE (16), Ro zp orządzeni u M ini stra Z dr ow ia z dni a 22 k w iet ni a 2005 r . (17), p ozio m ów bio be zp ie czeń stwa E ur op ea n C onf edera tio n of M edic al M yco log y (BS L) (18), k la sy fikac ji In styt ut u M ed yc yn y Wsi im. W ito ld a C ho dźk i w L ub linie (IMW ) (13), da ny ch li tera tur ow yc h no d at a o n h ea lth r isks / b ra k d an yc h n a t em at szk od liw oś ci zdr ow ot nej 1 ha rmf uln es s o f s pe cies acco rdin g t o li tera tur e / szk od liw oś ć zdr ow ot na w edług d an yc h li tera tur ow yc h 2 cla ss 2 acco rdin g t o IMW c la ssific at io n (13) o r c la ss BS L1 (18) o r h ar mf uln es s acco rdin g t o li tera tur e an d co nfir m ed in l ab ora to ry s tudies o n s tra in i so la te d f ro m w or k en vir onm en t / k la sa 2 wg k la sy fikac ji IMW (13) l ub k la sa BS L1 (18), a lb o szk od liw oś ć w yni ka jąc a z d an yc h li tera tur ow yc h i p ot w ier dzo na w b ad ani ac h l ab ora to ryjn yc h d la szczep u w yizo lo wa neg o ze śr odo w iska p rac y*** 3 cla ss 2 acco rdin g t o Dir ec tiv e UE 2000/54/EC (16) o r c la ss 2 acco rdin g t o t he R egu la tio n of t he M ini ster o f H ea lth d at ed A pr il 22, 2005 (17) o r c la ss BS L2 (18) / k la sa 2 wg D yr ek ty w y UE 2000/54/WE (16) l ub k la sa 2 wg R ozp orządzeni a M ini stra Z dr ow ia z dni a 22 k w iet ni a 2005 r . (17), l ub k la sa BS L2 (18) 4 cla ss 3 acco rdin g t o Dir ec tiv e UE 2000/54/EC (16) o r c la ss 3 acco rdin g t o t he R egu la tio n o f t he M ini ster o f H ea lth d at ed A pr il 22, 2005 (17) o r c la ss BS L 3 (18) o r hig her c la ss es / k la sa 3 wg D yr ek ty w y UE 2000/54/WE (16) l ub k la sa 3 wg R ozp orządzeni a M ini stra Z dr ow ia (17), lu b k la sa BS L3 (18), l ub k la sy w yższe 5
21 * B as ed o n / N a p od st aw ie : S kó ra e t a l. H ar m fu l b io lo gi ca l a ge nt s a t m us eu m w or ks ta tio ns ( 27 ). ** N ec es sa ry f or b el on gi ng t o a I II g ro up o f i nd ic at or s i s o bt ai ni ng ≥ 3 p oi nt f ro m c ri te ri a: h ar m fu ln es s t o h um an h ea lth / N ie zb ęd ne d o z ak w al ifi ko w an ia d o I II g ru py w sk aź ni kó w j es t u zy sk an ie ≥ 3 p un kt ów z k ry te riu m : sz ko dl iw oś ć z dr ow ot na d la l ud zi . ** * P ro du ct io n o f m yc ot ox in s, a lle rg en s, e xo tox in s, h ae m ol ys in s, p ol ys ac ch ar id e c ap su le s, a gg lu tin in s a nd o th er v ir ul en ce f ac to rs d ep en di ng o n t he s pe ci es / W yt w ar za ni e m yk ot ok sy n, a le rg en ów , e gz ot ok sy n, h em ol iz yn , ot oc ze k, a glu ty ni n i i nn yc h c zy nn ik ów w ir ul en cj i w z al eż no śc i o d g at un ku . BS L1 – s ap ro ph yt es o r p la nt p at ho ge ns o cc up yi ng n on v er te br at e e co lo gi ca l n ic he s o r f un gi u til iz in g d ea d a ni m al p ro du ct s. I nf ec tio ns a re c oi nc id en ta l, s up er fic ia l a nd n on -in va siv e o r m ild / s ap ro fit y l ub p at og en y r oś lin za jm uj ąc e n isz e e ko lo gi cz ne b ez kr ęg ow có w lu b g rz yb y w yk or zy st uj ąc e p ro du kt y r oz kł ad u m ar tw yc h z w ie rz ąt . Z ak aż en ia s ą p ow ie rz ch ow ne , n ie in w az yj ne l ub ł ag od ne . BS L2 – s pe ci es p ri nc ip al ly o cc up yi ng n on ve rt eb ra te e co lo gi ca l n ic he s, b ut w ith a r el at iv el y p ro no un ce d a bi lit y t o s ur vi ve i n v er te br at e t is su e. I n s ev er el y i m m un oc om pr om is ed h um an s, t he y m ay c au se d ee p, o pp or tu ni st ic m yc os es . P at ho ge ns c au si ng s up er fic ia l i nf ec tio ns a lso f al l i nt o t hi s c at eg or y / g at un ki z aj m uj ąc e n isz e e ko lo gi cz ne b ez kr ęg ow có w, a le z r el at yw ni e d uż ą z do ln oś ci ą d o p rz eż yc ia w t ka nk ac h k rę go w có w. U lu dz i z o bn iż on ą od po rn oś ci ą m og ą w yw oł yw ać g łę bo ki e z ak aż en ia . D o t ej k at eg or ii n al eż ą t ak że p at og en y p ow od uj ąc e z ak aż en ia p ow ie rz ch ow ne . BS L3 – p at ho ge ns p ot en tia lly a bl e t o c au se s ev er e, d ee p m yc os es i n h ea lth y h um an s / p at og en y p ot en cj al ni e z do ln e d o w yw oł yw an ia p ow aż nyc h g łę bo ki ch g rz yb ic u z dr ow yc h lu dz i. Ta ble 3 . Th e e va lu at io n s ca le f or s el ec tin g i nd ic at or s o f m ic ro bi al c on ta m in at io n a t t he e xa m in ed t an ne rie s, w or ks ta tio ns Ta be la 3 . S ka la d o w yz na cz an ia w sk aź ni kó w z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o n a s ta no w isk ac h p ra cy w b ad an yc h g ar ba rn ia ch G ro up o f in dic at or s G ru pa ws kaźni kó w Po in ts Pun kt y Eva lu at io n O cen a D es cr ip tio n O pi s I 3–6 micr oo rga ni sm s n ot a ss oci at ed w ith th e s pe cifici ty o f t ann er ies / mi kr oo rga nizm y nie zw iąza ne ze s pe cy fiką ga rb ar ni micr oo rga ni sm s o cc ur rin g in t he o ut do or a ir a nd o cc asio na lly in r oo m s w ith lo w micr ob io log ic al co nt amin at io n, n ot co ns tit ut in g h ea lth h aza rd t o w or ker s / mi kr oo rga nizm y w ys tęp uj ące w p ow iet rzu at m osf er yczn ym i s po rad ycznie w p omieszczeni ac h o ni sk im s to pni u za nie czyszczeni a mi kr ob io log iczn eg o, nies ta no w iące za gr ożeni a zdr ow ot neg o d la p raco w ni kó w II 7–10 micr oo rga ni sm s co ns ta nt ly p res en t in t he ta nn er ies / mi kr oo rga nizm y s ta le o be cn e w ga rb ar ni ac h micr oo rga ni sm s o cc ur rin g in t he in do or a ir o r o n s ur faces in t he r oo m s w ith lo w micr ob io log ic al co nt amin at io n, m ay a lso b e p res en t in t he o ut do or a ir, n ot co ns tit ut in g / co ns tit ut in g h ea lth h aza rd t o w or ker s / mi kr oo rga nizm y w ys tęp uj ące w p ow iet rzu l ub n a p ow ierzc hni ac h w m uze ac h o ni sk im s to pni u za nie czyszczeni a mi kr ob io log iczn eg o, m ogące w ys tęp ować r ów nie ż w p ow iet rzu a tm osf er yczn ym, nies ta no w iące za gr ożeni a / s ta no w iące za gr ożenie zdr ow ot ne d la p raco w ni kó w III* 11–16 in dic at or s o f micr ob ia l co nt amin at io n at w or kp laces in t ann er ies / ws kaźni ki za nie czyszczeni a szk od liw ymi czy nni ka mi bio log iczn ymi n a s ta no w iskac h p rac y w ga rb ar ni ac h micr oo rga ni sm s o cc ur rin g w ith hig h f re quen cy in a re as o f hig h micr ob ia l co nt amin at io n, b ot h in t he air a nd o n s ur faces, o cc asio na lly a lso o cc ur rin g in t he o ut do or a ir, co ns tit ut in g a h ea lth h aza rd t o w or ker s / / mi kr oo rga nizm y w ys tęp uj ące z d użą częs toś ci ą w p omieszczeni ac h si lnie za nie czyszczo ny ch mi kr ob io log icznie za ró w no w p ow iet rzu , j ak i n a p ow ierzc hni ac h, s po rad ycznie w ys tęp uj ące r ów nie ż w p ow iet rzu a tm osf er yczn ym, s ta no w iące za gr ożenie d la zdr ow ia p raco w ni kó w * N ec es sa ry f or b el on gi ng t o a I II g ro up o f i nd ic at or s i s o bt ai ni ng ≥ 3 p oi nt f ro m c ri te ri a: h ar m fu ln es s t o h um an h ea lth / N ie zb ęd ne d o z ak w al ifi ko w an ia d o I II g ru py w sk aź ni kó w j es t u zy sk an ie ≥ 3 p un kt ów z k ry te riu m : sz ko dl iw oś ć z dr ow ot na d la l ud zi .
RESULTS
The number of microorganisms in the air of the
tannery rooms ranged between 1.2×103 and 3.7×
×103 CFU/m3. The number of bacteria in the tanneries
producing chrome-tanned leather, the so-called wet
blue (Tanneries I and II) was in the range from 5.1×102
to 6.1×102 CFU/m3, while in Tanneries III and IV,
where leather production begun from the raw
mate-rials stage, it was higher, and reached 2.3×103 to 3.3×
×103 CFU/m3 (p < 0.05).
Contamination with filamentous fungi in the air of the tanneries remained at a lower level, reaching
3.1×102–1.4×103 CFU/m3 (Table 4).
The prevalence of bacteria in the air was reported in 3 out of 4 tested plants (51–92% of all of the iso-lated microorganisms). The number of
microor-ganisms detected on leathers ranged from 7.6×101
to 5.5×105 CFU/100 cm2. On the surface of raw hides,
the number of bacteria was at the level of 5.5×105
to 1.2×106 CFU/100 cm2, and it was lower on wet blue
leathers by about 4 orders of magnitude on the
loga-rithmic scale (from 7.6×101 to 5.9×102 CFU/100 cm2).
Bacteria constituted 60–100% of the total number of microorganisms isolated from test leathers (Table 5).
A qualitative analysis revealed the presence of 80 strains isolated in the tanneries, the highest spe-cies diversity was found for filamentous fungi (46 iso-lates), bacteria (25), and the lowest for yeasts (9).
Bacteria most frequently isolated from the air of the tanneries belong to the genera: Bacillus, Brevibacillus,
Brevundimonas, Corynebacterium, Kocuria, Micrococ-cus, Staphylococcus. Bacteria from the genera of Micro-coccus, StaphyloMicro-coccus, and Pseudomonas were the most
common bacteria species on leather surfaces (Table 6). Moulds included Cladosporium, and Penicillium, and as for yeasts, Cryptococcus spp. were most fre-quently isolated from the leather processing plants. The leather surface was most often inhabited by Penicillium,
Cryptococcus, Kodamaea and Pichia fungi (Table 6).
Basing on the developed criteria, taking into ac-count the prevalence, source of isolation and health hazard, 11 species of indicator microorganisms for the tanneries were determined (risk group 3 was confirmed in the literature or by laboratory tests conducted by the authors in previous studies): B. pumilus, B. subtilis,
B. cereus, Corynebacterium lubricantis, C. cladospo-rioides, Penicillium commune, P. echinulatum, P. chry-sogenum, P. crustosum, Candida parapsilosis and Cryptococcus albidus (Table 6). The analysis of viable Table 4
. Q ua nt ita tiv e a na ly sis o f m ic ro bi al c on ta m in at io n i n t he a ir i n t he e xa m in ed t an ne rie s Ta be la 4 . A na liz a i lo śc io w a z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o p ow ie tr za w b ad an yc h g ar ba rn ia ch Pl ace o f s am plin g M iejs ce p ob or u p ró b Fun gi in t he a ir G rzy by w p ow iet rzu [CFU/m 3] Bac ter ia in t he a ir Ba kt er ie w p ow iet rzu [CFU/m 3] M icr ob es in t he a ir Dr ob no us tro je w p ow iet rzu to ta l og ółem [CFU/m 3] fun gi grzy by (N = 6) [%] bac ter ia ba kt er ie (N = 6) [%] M±S D min.–m ax M±S D min.–m ax M±S D min.–m ax Ta nn er y I / Ga rb ar ni a I (N = 3) 7.3×10 2±3.0×10 2 1.7×10 2–2.2×10 3 5.1×10 2±2.5×10 2 1.5×10 2–1.5×10 3 1.2 ×10 3±5.4×10 2 4.1×10 2–3.6×10 3 59 41 Ta nn er y II / Ga rb ar ni a II (N = 3) 5.8×10 2±3.9×10 2 2.0×10 1–2.0×10 3 6.1×10 2±3.0×10 2 2.0 ×10 1–2.9×10 3 1.2×10 3±6.9×10 2 4.0×10 1–4.9×10 3 49 51 Ta nn er y III / Ga rb ar ni a III (N = 2) 1.4×10 3±1.9×10 2 2.0×10 2–2.8×10 3 2.3×10 3±1.4×10 4 3.0×10 2–4.0×10 3 3.7×10 3±4.6×10 2 5.8×10 2–6.1×10 3 37 63 Ta nn er y IV / Ga rb ar ni a IV (N = 6) 3.1×10 2±2.1×10 2 6.0×10 1–1.7×10 3 3.3×10 3±2.4×10 3 2.6×10 2–1.1×10 4 3.6×10 3±2.6×10 3 3.0×10 2–1.2×10 4 8 92 In ter na l b ac kg ro un d / Tło w ew nęt rzn e (N = 4) 1.6×10 2±2.0×10 2 2.5×10 1–4.5×10 2 8.6×10 2±8.0×10 2 1.6×10 2–1.6×10 3 1.0×10 3±9.7×10 2 1.8×10 2–2.0×10 3 14 86 Ext er na l b ac kg ro un d / Tło ze w nęt rzn e (N = 4) 3.5×10 2±4.3×10 2 7.0×10 1–9.8×10 2 5.9×10 2±4.7×10 2 1.7×10 2–1.0×10 3 9.4×10 2±7.8×10 2 2.4×10 2–2.0×10 3 36 64 m in . – m in im um v alu e / w ar to ść m in im al na ; m ax – m ax im um v alu e / w ar to ść m ak sy m al na . O th er a bb re vi at io ns a s i n T ab le 1 / I nn e s kr ót y j ak w t ab el i 1 .
23 Ta ble 5 . Q ua nt ita tiv e a na ly sis o f m ic ro bi al c on ta m in at io n o n l ea th er s a nd h id es i n t he e xa m in ed t an ne rie s Ta be la 5 . A na liz a i lo śc io w a z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o s kó r g ar bo w an yc h i s ur ow yc h w b ad an yc h g ar ba rn ia ch Pl ace o f s am plin g M iejs ce p ob or u p ró b Fun gi o n le at her/hides G rzy by n a s kó rac h ga rb owa ny ch/s ur ow yc h [CFU/100 cm 2] Bac ter ia o n le at her/hides Ba kt er ie n a s kó rac h ga rb owa ny ch/s ur ow yc h [CFU/100 cm 2] M icr ob es o n le at her/hides Dr ob no us tro je n a s kó rac h ga rb owa ny ch/s ur ow yc h to ta l og ółem [CFU/100 cm 2] fun gi grzy by (N = 6) [%] bac ter ia ba kt er ie (N = 6) [%] M±S D min.–m ax M±S D min.–m ax M±S D min.–m ax Ta nn er y I / Ga rb ar ni a I (N = 3) 1.5×10 1±1.5×10 1 5.1×10 0–4.6×10 1 6.1×10 1±6.3×10 1 1.0×10 1–2.0×10 2 7.6×10 1±5.8×10 1 1.5×10 1–2.1×10 2 20/0 80/0 Ta nn er y II / Ga rb ar ni a II (N = 3) 2.3×10 2±2.1×10 1 1.2×10 2–4.5×10 2 2.3×10 2±1.9×10 2 1.8×10 2–3.1×10 2 5.9×10 2±1.4×10 2 4.5×10 2–7.3×10 2 39/0 61/0 Ta nn er y III / Ga rb ar ni a III (N = 3) 1.5×10 2/0±1.3×10 2/0 5.6×10 1/0–2.4×10 2/0 2.3×10 2/1.2×10 6± ±5.4×10 1/3.0×10 5 1.9×10 2/8.6×10 5– –2.6×10 2/1.6×10 6 3.7×10 2/1.2×10 3± ±7.6×10 1/3.0×10 5 3.2×1 2/8.6×10 5– –4.3×1 2/1.6×10 6 40/0 60/100 Ta nn er y IV / Ga rb ar ni a IV (N = 3) 1.0×10 1/0±5.1×10 0/0 5.1×10 0/0–1.5×10 1/0 2.9×10 2/5.5×10 5± ±2.0×10 2/3.7×10 3 5.6×10 1/5.0×10 5– –4.4×10 2/5.8×10 5 3.0×10 2/5.5×10 5± ±2.1×10 2/3.7×10 4 6.1×10 1/5.0×10 5– –4.5×10 2/5.8×10 5 3/0 97/100 A bb re vi at io ns a s i n T ab le s 1 a nd 4 / I nn e s kr ót y j ak w t ab el i 1 i 4 . Ta ble 6 . C la ss ifi ca tio n o f m ic ro or ga ni sm s f ro m t he w or k e nv iro nme nt i n t an ne rie s i nt o g ro up s o f i nd ic at or s o f m ic ro bi al c on ta m in at io n Ta be la 6 . M ik ro or ga ni zm y ś ro do w isk a p ra cy w g ar ba rn ia ch v s g ru py w sk aź ni kó w z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o M icr oo rga ni sm M ik ro or ga nizm Fr eq uen cy o f o cc ur ren ce in a ll o f t he sa m ples in t ann er ies (a ir/s ur faces) Częs toś ć w ys tęp owa ni a w e wszys tk ic h pr ób ac h w ga rb ar ni ac h (p ow iet rze/p ow ierzc hnie) [%] Pr es en ce in t he ou tdo or a ir O be cn oś ć w p ow iet rzu ze w nęt rzn ym In dic at or s o f micr ob ia l co nt amin at io n in t ann er ies Ws kaźni ki za nie czyszczeni a mi kr ob io log iczn eg o w ga rb ar ni ac h iso la tio n fre quen cy częs toś ć izo lac ji so ur ce źr ódło ha rmf uln es s to h um an szk od liw oś ć dl a l udzi to ta l og ółem gr ou p of in dic at or s gr up a ws kaźni kó w A B Bac ter ia / B ak ter ie Ba cill us c er eu s 0/0 0/67 l – 4 4 3 a 11 III* Ba cil lu s l ich en ifo rm is 0/60 l 0/0 + 3 2 1 6 I Ba cil lu s m yc oi de s 0/30 l 79/0 – 4 6 1 11 III Ba cill us p um ilu s 0/67 l 57/0 – 4 6 3 13 III* Ba cil lu s s p. 67/0 33/0 + 4 1 1 6 I Ba cill us s ub til is 27/60 l 0/0 – 3 6 3 12 III* Br ev ib aci llu s s p. 100/0 0/0 – 5 3 1 9 II
M icr oo rga ni sm M ik ro or ga nizm Fr eq uen cy o f o cc ur ren ce in a ll o f t he sa m ples in t ann er ies (a ir/s ur faces) Częs toś ć w ys tęp owa ni a w e wszys tk ic h pr ób ac h w ga rb ar ni ac h (p ow iet rze/p ow ierzc hnie) [%] Pr es en ce in t he ou tdo or a ir O be cn oś ć w p ow iet rzu ze w nęt rzn ym In dic at or s o f micr ob ia l co nt amin at io n in t ann er ies Ws kaźni ki za nie czyszczeni a mi kr ob io log iczn eg o w ga rb ar ni ac h iso la tio n fre quen cy częs toś ć izo lac ji so ur ce źr ódło ha rmf uln es s to h um an szk od liw oś ć dl a l udzi to ta l og ółem gr ou p of in dic at or s gr up a ws kaźni kó w A B Co ry ne ba ct er iu m a cc ol en s # 100/0 0/0 – 5 3 3 11 III* Co ry ne ba ct er iu m p ro pi nq uu m 0/0 79/0 – 4 3 3 10 II Ko cu ria r os ea 23/0 79/67 l + 4 5 1 10 II Ko cu ria v ar ia ns 100/0 40/0 + 5 1 1 7 II M icr oc oc cu s l ut eu s 0/0 93/0 + 5 1 1 7 II M icr oc oc cu s l yl ae 0/0 100/100 l + 5 5 1 11 III M icr oc oc cu s s p. 81/100 l 43/0 + 5 5 1 11 III Pa en ib aci llu s p ol ym yx a 31/0 0/0 – 2 3 1 6 I Ps eu do m on as a lca ligen es 0/0 33/0 – 2 3 3 8 II Ps eu do m on as fl uo re sc ens 0/0 57/0 – 3 3 3 9 II Ps eu do m on as l ut eo la 0/0 0/100 h + 5 2 3 10 II Ps eu do m on as p ut id a 0/0 0/100 h + 5 2 3 10 II St ap hy lo co ccu s c oh ni i 0/0 71/0 + 4 1 1 6 I St ap hy loc oc cu s h om in is 95/30 l 0/0 + 5 5 2 12 III St ap hy lo co ccu s l en tu s 50/0 86/0 + 5 1 1 7 II St ap hy lo co ccu s s ciu ri 0/60 l 0/100 l + 5 2 1 8 II St en ot ro ph om on as m al to ph ili a 22/0 0/0 – 2 3 1 6 I Fi la m en to us f un gi / P leśnie Acr em on iu m sp . 33/0 0/0 + 2 1 1 4 I Al ter na ria a lter na ta 39/0 0/0 + 2 1 3 6 I As per gi llu s c la va tu s 11/0 0/0 – 1 3 3 7 II As per gi llu s fl av us 4/0 7/0 – 1 3 4 8 II As per gi llu s n iger 15/0 0/0 + 1 1 3 5 I As per gi llu s s yd ow ii 19/0 0/0 – 1 3 3 7 II Ta ble 6 . C la ss ifi ca tio n o f m ic ro or ga ni sm s f ro m t he w or k e nv iro nme nt i n t an ne rie s i nt o g ro up s o f i nd ic at or s o f m ic ro bi al c on ta m in at io n – c on t. Ta be la 6 . M ik ro or ga ni zm y ś ro do w isk a p ra cy w g ar ba rn ia ch v s g ru py w sk aź ni kó w z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o – c d.
25 As per gi llu s u stu s 0/0 2/0 – 1 3 3 7 II As per gi llu s v er sic ol or 0/0 15/0 – 1 3 3 7 II Au re ob as id iu m p ul lu la ns 15/0 0/0 – 1 3 3 7 II Be av ren ia sp . 0/0 15/0 – 1 3 1 5 I Bo tr yt is ci ner ea 22/0 24/0 + 2 1 1 4 I Ch ae to m iu m gl ob us om 0/0 0/50 l – 3 4 3 10 II Cl ad os po riu m c la do spo ri oi de s 83/10 l 79/0 + 5 5 3 13 III* Cl ad os po riu m h er ba ru m 100/0 2/0 + 5 1 3 9 II Cl ad os po riu m m acr oc ar pu m 78/0 78/0 + 4 1 1 6 I Cl ad os po riu m s ph aer os per m um 0/0 43/0 + 3 1 3 7 II Eu ro tiu m a ms tel od am i 4/0 0/0 – 1 3 1 5 I Fu sa riu m o xys po ru m 0/0 29/0 – 2 3 3 8 II H um ico la sp . 12/0 0/0 – 1 3 1 5 I M uc or p lu m beu s 22/0 0/0 – 2 3 3 8 II Pa eci lo m yc es l ila cin us 0/0 5/0 – 1 3 3 7 II Pa eci lo m yc es v ar io tii 35/0 10/0 – 2 3 4 9 II Pe nc ill iu m a tr am en to su m # 92/0 20/33 l – 5 6 3 14 III* Pen cil liu m b re vi co m pa ct um 0/0 0/33 l + 2 2 3 7 II Pen cil liu m i m pl ica tu m 15/0 0/0 – 1 3 3 7 II Pen ici lli um c an es cens 0/0 0/50 l – 3 4 3 10 II Pen ici lli um c hr ys ogen um 0/0 64/0 + 4 1 3 8 II Pen ici lli um c om m un e 0/0 2/0 + 1 1 3 5 I Pen ici lli um e ch in ul at um 15/0 0/0 – 1 3 3 7 I Pe ni cill iu m f re ii # 19/10 l 86/0 + 5 5 3 13 III* Pen ici lli um gl ab ru m 0/0 2/0 – 1 3 3 7 II Pen ici lli um g ris eo fu lv um 73/0 0/0 + 4 1 3 8 II Pen ici lli um h irs ut um # 54/0 0/50 l – 3 6 3 b 12 III* Pe ni cill iu m l iv id um # 88/0 0/0 – 5 3 3 11 III* Pen ici lli um o xa licu m 0/0 36/0 + 2 1 3 6 I Pen ici lli um p al ita ns 33/0 0/0 – 2 3 3 8 II Pen ici lli um p ol on icu m 0/0 46/0 + 3 1 3 7 II
M icr oo rga ni sm M ik ro or ga nizm Fr eq uen cy o f o cc ur ren ce in a ll o f t he sa m ples in t ann er ies (a ir/s ur faces) Częs toś ć w ys tęp owa ni a w e wszys tk ic h pr ób ac h w ga rb ar ni ac h (p ow iet rze/p ow ierzc hnie) [%] Pr es en ce in t he ou tdo or a ir O be cn oś ć w p ow iet rzu ze w nęt rzn ym In dic at or s o f micr ob ia l co nt amin at io n in t ann er ies Ws kaźni ki za nie czyszczeni a mi kr ob io log iczn eg o w ga rb ar ni ac h iso la tio n fre quen cy częs toś ć izo lac ji so ur ce źr ódło ha rmf uln es s to h um an szk od liw oś ć dl a l udzi to ta l og ółem gr ou p of in dic at or s gr up a ws kaźni kó w A B Pen ici lli um s ol itu m 17/0 0/0 + 1 1 3 5 I Pe ni cill iu m v er ru co su m # 0/40 l 86/100 l – 5 6 3 14 III* Pen ici lli um v er ru cu lo su m 0/0 0/50 l – 3 4 3 10 II Rh iz op us n ig ric ans 19/0 5/0 + 1 1 3 5 I Sc ler oc lei sta o rn at a 0/0 0/33 h – 2 4 1 7 II St em ph yli um sp . 0/0 0/50 l – 3 4 1 8 II Tr ich od er m a k on in gi i 0/0 17/50 l + 3 5 1 9 II Tr ich od er m a v iri de 19/0 0/0 – 1 3 3 7 II Ye as t / Dr ożdże Ca nd id a pa ra ps ilo sis 62/30 l 0/0 – 4 6 3 13 III* Cr yp toc oc cu s a lb id us 78/100 l 0/0 – 5 6 3 12 III* Cr yp toc oc cu s n eo fo rm an s # 72/100 l 0/0 – 5 3 3 11 III* Cr yp to co ccu s t er reu s 83/0 0/0 – 5 3 1 9 II Kl oe ck er a sp p. 0/0 30/67 l – 4 6 1 11 III Ko da m ae a o hm er i 27/100 l 0/0 – 5 6 1 12 III Pi ch ia a ng us ta 0/100 l 0/0 – 5 4 1 10 II Rh od ot or ul a gl ut in is 17/0 0/0 + 1 1 3 5 I Rh od ot or ul a m in ut a 0/0 10/0 + 1 1 1 3 I A – t an ne ri es p ro ce ss in g r aw h id es / g ar ba rn ie p rz et w ar za ją ce s kó ry s ur ow e; B – t an ne ri es p ro du ci ng c hr om e-ta nn ed l ea th er s / g ar ba rn ie p rz et w ar za ją ce s kó ry c hr om ow o g ar bo w an e. l – l ea th er / s kó ry g ar bo w an e; h – h id es / s kó ry s ur ow e. # S ee T ab le 7 / P at rz t ab el a 7 . a N on -h em ol yt ic e nt er ot ox in ( N H E) a nd h em ol ys in B L ( H BL ) w er e d et ec te d i n a l ab or at or y t es t / N ie he m ol ityc zn a e nt er ot ok sy na ( N H E) i h em ol iz yn a B L ( H BL ) z os ta ły w yk ry te w b ad an ia ch l ab or at or yj nyc h. b O ch ra tox in A a nd o th er m yc ot ox in w er e d et ec te d i n a l ab or at or y t es t / O ch ra to ks yn a A i i nn e m yk ot ok sy ny z os ta ły w yk ry te w b ad an ia ch l ab or at or yj nyc h. In di ca to rs o f m ic ro bi al c on ta m in at io n a t w or ks ta tio ns i n t an ne ri es a re b ol de d / W sk aź ni ki z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o w g ar ba rn ia ch z os ta ły p og ru bi on e. Ta ble 6 . C la ss ifi ca tio n o f m ic ro or ga ni sm s f ro m t he w or k e nv iro nme nt i n t an ne rie s i nt o g ro up s o f i nd ic at or s o f m ic ro bi al c on ta m in at io n – c on t. Ta be la 6 . M ik ro or ga ni zm y ś ro do w isk a p ra cy w g ar ba rn ia ch v s g ru py w sk aź ni kó w z an ie cz ys zc ze ni a m ik ro bi ol og ic zn eg o – c d.
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Nr 1 27
bioaerosol particle size distribution in the air of Tan-nery I demonstrated that its working environment was dominated by small particle sizes of 1.1–2.1 µm. Both bacterial and fungal bioaerosol particles were the ma-jority of all (24–37%).
In the case of bacterial particles, a fraction of an aer-odynamic diameter of 2.1–3.3 µm (17–23%) was also significant. The smallest share in the air of up to 10% was observed for bacterial particle size of 0.65–1.1 µm (Figure 1). Percentages of individual bacterial bioaero-Table 7. Genetic analysis of indicators of harmful biological agents contamination in the examined tanneries
Tabela 7. Analiza genetyczna wskaźników zanieczyszczenia szkodliwymi czynnikami biologicznymi w badanych garbarniach
Microorganisms Mikroorganizmy Identification by conventional methods* Identyfikacja konwencjonalnymi metodami* Identification by genetic methods Identyfikacja metodami genetycznymi
Similarity of nucleotide sequences to the compared strains Podobieństwo sekwencji nukleotydowych
do porównywanych szczepów strain
szczep %
Bacteria / Bakterie Bacillus pumilus Bacillus pumilus B. pumilus ATCC 7061 99
Bacillus subtilis Bacillus subtilis B. subtilis NBRC 104443 99
Bacillus cereus Bacillus cereus B. cereus CMCC 63305 99
Corynebacterium accolens Corynebacterium lubricantis C. lubricantis KSS-3Se 100
Fungi / Pleśnie Cladosporium cladosporioides Cladosporium cladosporioides C. cladosporioides ATCC 11275 100
Pencillium atramentosum Penicillium echinulatum P. echinulatum FRR 1151 99
Penicillium freii Penicillium commune P. commune ATCC 10428 100
Penicillium hirsutum Penicillium chrysogenum P. chrysogenum ATCC10106 99
Penicillium lividum Penicillium commune P. commune CBS 311.48 100
Penicillium verrucosum Penicillium crustosum P. crustosum ATCC 90174 99
Yeast / Drożdże Candida parapsilosis Candida parapsilosis C. parapsilosis ATCC 22019 99
Cryptococcus albidus Cryptococcus albidus C. albidus CBS 969 99
Cryptococcus neoformans Rhodotorula glutinis R. glutinis LEMI 150 99
* Conventional identification of filamentous fungi was carried out on the basic of macro- and microscopic observation based on taxonomic keys; bacteria and yeast identification was carried out with Analytical Profile Index (API) tests / Identyfikacja konwencjonalna pleśni prowadzona była na podstawie obserwacji makro- i mikroskopowych w oparciu o klucze taksonomiczne; identyfikację bakterii i drożdży przeprowadzono z użyciem testów bazujących na Indeksie Profilu Analitycznego (API).
Fig. 1. The contribution of bioaerosol fraction of bioaerosol at workstations in tannery I Ryc. 1. Rozkład frakcji bioaerozolu w bioaerozolu stanowisk pracy w garbarni I
bacteria – hall I / bakterie – hala I
bacteria – control / bakterie – kontrola Aerodynamic diameters of bioaerosol particles / Średnica aerodynamiczna cząstek bioaerozolu [µm]
7.0–11.0 4.7–7.0 3.3–4.7 2.1–3.3 1.1–2.1 0.65–1.1
The contribution of bioaerosol fraction / Udział frakcji bioaer
ozolu [%] 0 5 10 15 20 25 30 35 40
fungi – hall I / grzyby – hala I fungi – control / grzyby – kontrola
sol fractions were similar in the production room and in the control sample (outdoor air). The fungal bioaer-osol particle distribution was different for the indoor air of the tanneries and in the atmospheric air. Control samples were dominated by fractions of particles with sizes ranging from 1.1 to 4.7 µm. The smallest parti-cles with diameters of 0.65–1.1 µm and 1.1–2.1 µm were dominant in the production air, constituting 24–37% of the total fungal bioaerosol. Also, a higher percentage of large particles (7.0–11.0 µm) in the production room was observed with respect to the control group.
DISCUSSION
Microclimate conditions prevailing in the working en-vironment of tanneries (low temperature: 13–26°C and high humidity of the air: 40–90%) are conductive to the growth of microorganisms.
The microbial contamination of the air in tanneries, depending on humidity and temperature of the air in both types of tanneries (producing hides and chrome-tanned leathers) were described with linear regression equations (Table 8). The regression analysis of the cor-relation between humidity and temperature in the tan-neries and microbial contamination of air gave the de-termination coefficients in the range of 0.6013 to 0.9193, indicating the significant influence of air humidity and temperature on the total number of microbes in the analysed air samples. The designated Pearson correla-tion coefficient for the examined correlacorrela-tions showed a positive value in the range of 0.78 to 0.96, therefore, it can be concluded that with the increase of humidity
and the temperature of the air the total number of mi-crobes increases.
These studies demonstrated that microbial con-tamination of the air in the tanneries varied and de-pended on the characteristics of the plant and the used raw materials. The tanneries which processed raw hides and conducted a complete technological process in-volving advanced stages were characterized by about tenfold higher bacterial air contamination (p < 0.05), while the microbial contamination of leather surfac-es was 10 000 timsurfac-es higher there than in the wet blue tanneries (p < 0.05).
One-way analysis of variance with post-hoc Tukey’s test confirmed the significant influence of tanner-ies profile production on microbiological contamina-tion of the surfaces of hides and leathers as well as the air samples (expressed as the total number of micro- organisms).
The maximum levels of air pollution reported in the tanneries implementing a full technological
process (bacteria: 4.0×103–1.1×104 CFU/m3, fungi:
1.7×103–2.8×104 CFU/m3) indicate high inhalation
exposure to biological agents. This confirms the sta-tus of the control air (outdoor air) which, due to the number of microorganisms, in accordance with PN-89/Z-04111/02 and PN-89/Z-04111/03 (28,29), was
clean air (less than 1.0×103 CFU/m3 of bacteria and less
than 3.0×103 CFU/m3 of fungi) (Table 4) (p < 0.05).
These results clearly show that the level of pollution in plant premises is influenced by the used technological process. However, the number of microorganisms in the air of the test plants did not exceed the quantitative Table 8. Regression coefficients defining the correlation between microbiological contamination of air in the examined tanneries
and microclimate conditions (humidity and temperature)
Tabela 8. Współczynniki regresji opisujące zależność między liczbą drobnoustrojów w powietrzu w badanych garbarniach
a warunkami mikroklimatu (wilgotność i temperatura)
Parameter Parametr
Linear parameters of regression equations Współczynniki liniowe równań regresji
(y = ax+b) R2 r p
a b
Humidity
tanneries processing raw hides / garbarnie przetwarzające skóry surowe 154.2 –3 484.9 0.9 0.9 < 0.05
tanneries producing chrome-tanned leathers / garbarnie przetwarzające skóry
chromowo garbowane 1 153.0 –45 272.2 0.6 0.9 < 0.05
Temperature
tanneries processing raw hides / garbarnie przetwarzające skóry surowe 551.0 –6 825.0 0.9 0.9 < 0.05
tanneries producing chrome-tanned leathers / garbarnie przetwarzające skóry
chromowo garbowane 3 237.6 –53 620.3 0.7 0.7 < 0.05
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rial and the working conditions in the tanneries. They included 4 bacteria: B. pumilus, B. subtilis, B. cereus,
Corynebacterium lubricantis, 5 moulds: C. cladospo-rioides, Penicillium commune, P. echinulatum, P. chrys-ogenum, P. crustosum and 2 species of yeasts: Candida parapsilosis and Cryptococcus albidus. Genetic
iden-tification of yeasts initially classified as Cryptococcus
neoformans showed that they belonged to the species of Rhodotorula glutinis.
No data is available on the adverse effects of this yeast to human health, which is why it is not present-ed as an indicator species. The genetic analysis also indicated differences in the taxonomic classification of 1 bacteria species from genus Corynebacterium and even 5 mould species belonging to the genus of
Penicil-lium selected as indicators of microbial contamination
in the tanneries. The usefulness of molecular biology methods in the diagnostics of moulds isolated from the air has been proven (35,36).
Microorganisms designated as indicators of contami-nation with harmful biological agents in the tanneries are described in the literature as potentially pathogenic and dangerous to human health. The indicators in-cluded up to 3 species of the genus Bacillus (B. subtilis,
B. cereus, B. pumilus). The presence of these bacteria
had been previously identified as typical for leather (4) and air microflora in tanneries (37). B. subtilis has aller-genic effects particularly burdensome for the employees of the biotechnology industry (production of enzymatic proteins) (38). This species is classified by the Regula-tion of the Minister of Health, 2005 (Journal of Laws of 2005, No. 81, item 716, as amended and the Journal of Laws 2008, No. 48, item 288) as a risk group 2 (17).
Another bacterium of this type, B. cereus, may exhibit toxic effects (it produces enterotoxins causing food poisoning), and infectious diseases – pneumonia, in exposed tanners through contact with food, airborne dust and airborne droplets (39). The ability to induce α-haemolysis and toxicity was detected for the strain iso-lated from the tanneries – laboratory tests showed a non-hemolytic enterotoxin (NHE) and hemolysin BL (HBL) (unpublished data). Bacillus pumilus is also considered an etiologic agent of food poisoning, while in the case of direct contact, it can cause skin infections (40).
Corynebacterium lubricantis bacteria are
respon-sible for opportunistic infections in humans – Direc- tive 2000/54/EC classifies all of the species of this genus as risk group 2 (16).
Designated as an indicator of microbial contamina-tion in the tanneries, C. cladosporioides is a common reference values established by the Polish Commission
for Maximum Admissible Concentrations and Inten-sities for Agents Harmful to Health in the Working Environment (30) (for the total number of mesophilic
bacteria of 1.0×105 CFU/m3, and the total number
of fungi 5.0×104 CFU/m3).
The percentage of microorganisms in the air was dif-ferent, depending on the type of the plant. In Tanner-ies III and IV, processing raw hides, bacteria frequen-cies ranged from 63 to 92%, compared to the other test plants: 41–59%. This fact is most likely the result of processing material (raw hides) being susceptible to bac-terial attack and high humidity caused by the processes conducted in tanneries. The analysis of bacteria on the leather surface showed a significantly higher contami-nation of raw hides than wet blue leathers. Raw hides, during the study, were only contaminated with bacteria.
The results of the bioaerosol particle size distribu-tion measurement in the tannery air are especially noteworthy. The bacterial particle size distribution in the indoor air was similar to that in the outdoor air. In the case of fungi, there were significant differen-ces (p < 0.05) between the production and control air. It was found that the smallest (fractions: 0.65–1.1 µm and 1.1–2.1 µm) and the largest test particles (7.0–11.0 µm) were found dominant in the produc-tion hall. This phenomenon is likely to arise due to the grouping of fungi in units of dust created during pro-duction (large particles) or the lifting of single mould spores with the movement of air (small particles).
The analysis of fungal bioaerosol distribution in the tannery indicates that the dominant fractions of aero-dynamic diameters of 0.65–1.1 µm and 1.1–2.1 µm (to-gether accounting for 67%) may penetrate the pulmo-nary alveolus of tanners, which may consequently lead to the occurrence of unspecific airways inflammation and allergic disorders (31). Moreover the exposure to the mould’s β-D-glucan mays causes inflammation re-actions in lymphocytes, affects interleukin-1 secretion via T-lymphocytes, stimulates tumour defence mecha-nisms, causes a decrease in the numbers of pulmonary macrophages and inhibits phagocytosis (32). Conse-quently, the effects of glucan can lead to fatigue, head-aches and other neurological symptoms in exposed workers, especially in the municipal waste industry and in different branches of agriculture (33,34).
Twelve species of microorganisms were selected as indicators for the working environment of the tanner-ies. They indicated a potential health hazard to workers and were associated with the specificity of raw
