Behaviour of methyl tert-butyl ether in marine water in relation to some species of bacteria Текст научной статьи по специальности «Биологические науки»



ПОВЕД1НКА МЕТИЛ ТРЕТ-БУТИЛОВОГО ЕФ1РУ У МОРСЬК1Й ВОД1 ЩОДО ОКРЕМИХ ВИД1В БАКТЕР1Й

Гаркавий С.С., Папапрепошс П., Каландзiс Т.

BEHAVIOUR OF METHYL TERT-BUTYL ETHER IN MARINE WATER IN RELATION TO SOME SPECIES OF BACTERIA

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GARKAVY S., PAPAPREPONIS P., KALANTZIS T.

National O. Bohomolets Medical University, Department of Hygiene and Ecology, Ukraine; Aristotle University of Thessaloniki, Medical School, Laboratory of Hygiene, Greece; University of Thessaly, Department of Ichthyology and Aquatic Environment, Greece

УДК

[614.771:628.19:665. 633.002.35]

Keywords: fuel oxygenates, MTBE, water, pollution, bacteria.

© Garkavy S., Papapreponis P., Kalantzis T. СТАТТЯ, 2011.

ethyl tert-butyl ether (MTBE) is a known fuel oxygenate, which is added to gasoline to improve combustion efficiency and to reduce air pollution. Due to its high octane level, ease blending with gasoline and low cost, MTBE is the most widely used oxygenate. The production of MTBE was started in the 1970s and, by the end of the century, world demand of MTBE has reached 22 million tons per year [1]. Rapid growth of MTBE usage has resulted in wide environmental pollution. Nowadays, MTBE has frequently been detected in water, as result of accidental releases and due to its high water solubi-

lity, low soil adsorption and poor biode-gradability [2-4]. It is also difficult to eliminate MTBE from water using traditional treatment methods [5, 6]. Moreover, MTBE has become a concern for human health; MTBE exposure can cause skin irritation, central nervous system depression and mental confusion as well as cancer [7]. US EPA considered MTBE as a suspected human carcinogen [8].

Studies on bacteria were mostly concentrated on toxicity of MTBE in standard tests, i.e. Ames test with Salmonella typhimurium [9]. Others were devoted to biodegradation ability of bacterial

ПОВЕД1НКА МЕТИЛ ТРЕТ-БУТИЛОВОГО ЕФ1РУ У МОРСЬКЙ ВОД1 ЩОДО

ОКРЕМИХ ВИД1В БАКТЕР1Й

Гаркавий С.С., Папапрепошс П., Каландзс Т.

Нац'юнальний медичний ушверситет iM. О.О. Богомольця, м. Кив, Украша; Унверситет Аристотеля, медична школа, лаборатор'я ппени, Салонки, Грещя;

Унверситет ФессалП, кафедра iхтiюлюпi та акватичнюгю середювища, Грешя Метил трет-бутилювий еф'р (МТБЕ) (С5Н12О) е сучасним оксигенатором бензину (ОБ), який пдвищуе його октанове число та зменшуе забруднення атмосферного повтря, знижуючи юльюсть шюдливих викидв автомобльних двигунв. Об'еми виробництва та споживання МТБЕ у сучасних умовах становлять близько 13,5 млн. тонн щюрiчню, а його дя на довклля та здоров'я людини е недостатньо вивченою. Метою роботи було встановити вплив МТБЕ на юкремi види бактерй морськоi води. Бльшсть вдомих мiкрюбiюлюгiчних досл'1джень присвячена видленню специфiчних мiкрююрганiзмiв, здатних до б'юдеструкц'й МТБЕ, або токсиколопчним досл'1дженням (тест Еймса). Разом з тим даних щодо впливу МТБЕ на сантарно-показову бактер'альну флору об'еЫв довк'шля, зокрема водойм, юпублiкюваню недостатньо. У пюпереднiй робот нами була зроблена спроба встановити вплив МТБЕ на санiтарню-пюказювi мiкрююрганiзми морськоi води, проте результати виявилися суперечливими. У цй робот наведено результати додаткових досл'1джень поведнки зростаючих концентрашй МТБЕ (вд 0,015 до 150 мг/л) у морсьюй вод щодо окремих вид'в бактерй. Досл'1дження проводилися у мiкрюбiюлюгiчнiй лабораторИ кафедри ппени Унверситету Аристотеля м'юта Салонки (Грешя) у рамках виконання проекту бвропейського Союзу Еразмус Мундус. Об'ектом дюслiдження була морська вода, забрана з припортовоi зони термального заливу Егейського моря. Псля приготування 10% розведень з використанням пептонно)' води проби морськоi води змшувалися з МТБЕ та мiкрюбiюлюгiчню досл '1джувалися зпдно з '( стандартними методами — ISO 6222 (визначення загальноi клькост аеробних мезофльних мiкрююрганiзмiв); APHA 9222 (визначення бактерй групи кишковоi палички (БГКП) та '¡хшх фекальних форм), ISO 7899-2 (визначення ентерокоюв) та ISO 16266 (визначення Pseudomonas aeruginosa). Отриманi результати засвдчили, що серед досл'1джуваних мiкрююрганiзмiв найбльш чутливими до МТБЕ виявилися БГКП. Данi кореляшйнот анал'\зу та анал'\зу множинноi лЫйно/ регресИ свдчили про статистично дюстювiрне зменшення чисельност БГКП з '1 збльшенням концентрацП МТБЕ. Коливання температури води та повтря пд час в'щбору зразюв не мали дюстювiрнюгю впливу на ютриманi результати. Механ'1зм дИ МТБЕ на бактер 'альнi клтини нами не вивчався, проте е припущення, що окр 'м МТБЕ бактерицидну дю виявляють продукти його розпаду — трет-бутиловий спирт (ТБС), трет-бутиловий фюрмiат (ТБФ) та формальдепд.

Ключовi слова: оксигенатори бензину, МТБЕ, вода, забруднення, бактерп.

31 Environment & Health № 3 2011

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strains extracted from MTBE polluted sites. Available data suggest that the following microorganisms are able to completely consume MTBE: Achromobacter xylosoxi-dans MCM1/1 [10], Methylibium petroleiphilum PM1 [11], Mycobacterium austroafricanum IFP 2012 [12], Hydrogenophaga flava ENV735 [13], Aquincola tertiari-carbonis [14]. Also, some strains of bacterial species, such as Methylobacterium, Rhodococcus, Arthrobacter, Bacillus, Pseudomonas are able to partly degrade MTBE [15-18].

In the present study, we aim to investigate whether MTBE has got any influence on the microflora of sea water, by studying standard sanitary microbiological indicators of water contamination, which indicate general pollution by organics. Our previous study on sea water sanitary microflora and MTBE showed contradictory results with no statistical evidence [19] and directed us to carry out an extended research to confirm or refute or previous findings. MTBE invokes strong interest to the scientific community, technocrats, and policy makers, in order to understand its fate in aquatic environment and consequently sustain ecological balance and protect public health.

Materials and methods

Sampling and preparation of samples. Samples were collected from the port of Thessaloniki, Greece. Thessaloniki maintains the second largest seaport of Greece located in a closed gulf (Gulf of Thessaloniki), which is the edge of the largest Thermaicos Gulf at the north-west of the Aegean Sea. The gulf is characterized by a massive ship transport and, thus, sea water is expected to be burdened continuously with several chemicals released by vessels.

Sampling occurred at 7 different days between January and April 2010. Weather conditions were recorded at sampling dates. We used a bathometer, with an attached sampling bottle of 1L capacity, to collect water from a depth of at least one meter; in this way, we

avoided sampling from the surface, where the number of microorganisms is expected to be remarkably influenced by environmental factors, such as solar radiation.

Samples were delivered immediately to the Laboratory of Hygiene, Medical School, Aristotle University of Thessaloniki and were assessed for the detection and enumeration of indicator bacteria by applying membrane filtration method. We spiked the sea water samples with pure MTBE chemical (Merck KgaA, Darmstadt, Germany). Prepared spiked aliquots included MTBE at the following concentrations: 0.015 mg/L, 0.15 mg/L, 1.5 mg/L, 15 mg/L, and 150 mg/L; we also tested a non-spiked aliquot, serving as control. We deliberately used the same concentrations of MTBE as in previous study in order to have the opportunity to compare results of both studies.

Because sea water usually includes a large number of bacterial cells, all aliquots were diluted with peptone diluent at 10% dilution (10 ml aliquot plus 90 ml peptone solution), before membrane filtration. This was necessary for obtaining distinct colonies on the membranes; bacteria counts were multiplied by 10 and the final results were expressed by 100 ml of water (cfu/100ml).

Microbiological analysis (counting and confirmation). We followed international standard methods for enumeration of: total aerobic mes-ophilic microbial flora (ISO 6222); total coliform and faecal coliform bacteria (APHA 9222); bacteria of the genus enterococcus (ISO 7899-2); and Pseudomonas aeru-ginosa (ISO 16266) [20]. For the enumeration of total microbial flora, 1 ml of each aliquot was drawn on a Petri dish and melted yeast extract agar was poured into the dish (pour plate method). For the rest indicators, 100 ml of each diluted aliquot was filtered through a 0.45 |m pore membrane and the membranes were transferred onto mEndo agar LES (Merck KgaA, Darmstadt, Germany), MFC agar (Oxoid Ltd, Ba-

singstoke, Hampshire, England), Slatzet & Bartley medium (Oxoid Ltd, Basingstoke, Hampshire, England) and Pseudomonas CN (Oxoid Ltd, Basingstoke, Hampshire, England) agar for the detection and enumeration of total coliforms, faecal coliforms, enterococci and Pseudomonas aeruginosa respectively. Afterwards, the dishes were incubated properly. Characteristic colonies were counted and confirmatory biochemical tests were performed to verify the presence of the bacteria indicators. Generally, all stages and confirmatory tests were performed according to the technical specifications described by the respective standard methods [20]. More specifically, confirmation of total and faecal coliforms was based on subculturing at least five selected colonies in Lauryl Sulfate broth — Brilliant Green Bile broth and EC-MUG respectively; confirmation of intestinal enterococci was taken place by transferring the membrane, with all the colonies, onto Bile Aesculin Azide agar; confirmation of Pseudomonas aeruginosa species was occasionally based on the production of blue/green (pyocyanin) colonies; alternatively, if pyocyanin producing colonies were not present, confirmation was based on subculturing fluorescent colonies (not producing blue/green colour) in Acetamide broth and reddish-brown colonies in Acetamide broth, King's B medium and by performing oxidase test. From the number of characteristic colonies counted on the membranes, and taking account of the proportion of confirmatory tests performed, we calculated the number of confirmed bacteria and the results expressed as colony-forming units in a specific volume of sample (cfu/100 ml). In the Yeast Extract agar cultures no confirmatory tests were performed, since all grown microorganisms were calculated.

Statistical analyses. We investigated for potential associations between microbial count and MTBE concentration calculating the Pearson correlation coefficient

Table

The multiple linear regression analysis of MTBE influence on TC count

Model Unstandardized Coefficients Standardized Coefficients t Sig. 95% Confidence Interval for b Collinearity Statistics

b SE(b) Beta Lower Bound Upper Bound Tolerance VIF

(Constant) 35.221 5.374 6.554 0.000 24.342 46.100

AirTemp -1.111 0.674 -0.351 -1.648 0.108 -2.476 0.253 0.299 3.343

WaterTemp -0.256 0.748 -0.073 -0.343 0.734 -1.770 0.257 0.299 3.343

MTBE Concentration -0.113 0.023 -0.559 -4.800 0.000 -0.160 -0.065 1.000 1.000

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ПОВЕДЕНИЕ МЕТИЛ ТРЕТ-БУТИЛОВОГО ЭФИРА В МОРСКОЙ ВОДЕ ПО ОТНОШЕНИЮ К ОТДЕЛЬНЫМ ВИДАМ БАКТЕРИЙ Гаркавый С.С., Папапрепонис П., Каландзис Т.

Метил трет-бутиловый эфир (МТБЭ) (С5Н12О) является современным оксигенатором бензина (ОБ), который повышает его октановое число и уменьшает загрязнение атмосферного воздуха, снижая количество вредных выбросов автомобильных двигателей. Объемы производства и потребления МТБЭ в современных условиях составляют около 13,5 млн. тонн ежегодно, а его действие на окружающую среду и здоровье человека является недостаточно изученным. Целью работы было установить влияние МТБЭ на отдельные виды бактерий морской воды. Большинство известных микробиологических исследований посвящено выделению специфических микроорганизмов, способных к биодеструкции МТБЭ, или токсикологическим исследованиям (тест Эймса). Вместе с тем данных о влиянии МТБЭ на санитарно-показательную бактериальную флору объектов окружающей среды, в частности водоемов, опубликовано мало. В предыдущей работе нами была предпринята попытка установить влияние МТБЭ на санитарно-показательные микроорганизмы морской воды, однако полученные результаты оказались противоречивыми. В настоящей работе представлены результаты дополнительных исследований поведения возрастающих концентраций МТБЭ (от 0,015 до 150 мг/л) в морской воде по отношению к отдельным видам бактерий. Эксперимент проводился в микробиологической

лаборатории кафедры гигиены Университета Аристотеля города Салоники (Греция) в рамках выполнения проекта Европейского Союза Эрасмус Мундус. Объектом исследования была морская вода, отобранная из припортовой зоны термального залива Эгейского моря. После приготовления 10% разведений с использованием пептонной воды пробы морской воды смешивались с МТБЭ и микробиологически исследовались согласно стандартным методам — ISO 6222 (определение общего количества аэробных мезофильных микроорганизмов); APHA 9222 (определение бактерий группы кишечной палочки (БГКП) и их фекальных форм), ISO 7899-2 (определение энтерококков) и ISO 16266 (определение Pseudomonas aerugi-nosa). Полученные результаты показали, что среди исследуемых микроорганизмов наиболее чувствительными к МТБЭ оказались БГКП. Данные корреляционного анализа и анализа множественной линейной регрессии свидетельствовали о статистически достоверном уменьшении численности БГКП с увеличением концентрации МТБЭ. Колебания температуры воды и воздуха при отборе проб не оказывали существенного влияния на полученные результаты. Механизм действия МТБЭ на бактериальные клетки нами не изучался, однако есть предположение, что кроме МТБЭ бактерицидное действие проявляют продукты его распада — трет-бутиловый спирт (ТБС), трет-бутиловый формиат (ТБФ) и формальдегид.

Ключевые слова: оксигенаторы бензина, МТБЭ, вода, загрязнение, бактерии.

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(r); the non-parametric Spearman's correlation coefficient (rs); and by applying multiple linear regression analysis. In the regression analysis, air temperature and water temperature were considered as covariates in the model.

Results and discussion. The Spearman correlation test found a statistically significant result (rs=-0.386, p = 0.012) between total coliform (TC) count and MTBE. Although the strength of this association does not appear to be very high (absolute rs = 0.386, i.e. quite smaller than 1), this result suggests that the microbial count tends generally to decrease as the concentration of the chemical increases. The Pearson correlation test also gave a negative and even stronger association (r = -0.475, p = 0.001) for TCs, but the latter result cannot be evaluated, because none of the correlated variables was normally distributed; violations from normality is an issue of concern when interpreting Pearson's r.

The results of the multiple linear regression analysis are displayed in

table. The dependent variable in this analysis was the square root of TC count (sqrtTC); this transformation was applied in order to enable a normal distribution of the dependent variable, which enhances the validity of the significance test. The regression analysis revealed a highly significant (p<0.001) negative association between sqrtTC and concentration (b=-0.113). Water temperature and air temperature did not give significant b-coeffici-ents and, therefore, they do not seem to be predictors of sqrtTC.

The slope coefficient of concentration variable denotes that for every increase in the concentration of MTBE by 1 mg/l, the square root of TC is decreased by 0.113 units, which gives about 0.013 bacterial cells (i.e. the result transformed back to ordinary count). This suggests a very small effect of the chemical, which is statistically significant though. The adjusted >R2 is >0.444; this means that 44.4% of the variation of sqrtTC may be explained by variations in the concentration of the chemical. This value of 44.4% is

not large enough to suggest a strong linear relationship between the associated variables; consequently, the value of the slope coefficient (b) may not be considered appropriate for predictions of the number of TCs. Overall, the fact that the b-coefficient for concentration is highly significant provides evidence that there is a negative relationship between TC count and MTBE concentration and this result is adjusted for temperature variations in the seawa-ter and in the atmosphere.

The analyses for the other investigated bacteria — total microbial count, fecal coliforms, ente-rococci and pseudomonas species — gave no statistically significant results. There was no evidence of any association of the number of the above species with the concentration of MTBE.

Discussion. Studies on MTBE behavior in the environment are essential in assessing general potential hazards of this exogenous chemical. There have been a series of experiments on deriving specific bacteria from MTBE polluted sites

33 Environment & Health № 3 2011

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able to decompose this chemical, the results of which could be applied as an alternative treatment methodology. Still, to our knowledge, there is lack of information about the exact interaction of MTBE with the microbial flora of water and soil. Coliform bacteria are one of the most representative indicators of sanitary quality of water and their variability may be quite informative in evaluating environment pollution. In the present study we attempted to investigate a correlation between levels of MTBE pollution in sea water and the numbers of bacterial cells of certain sanitary indicator species. Our results have shown that only the total coliform bacteria have statistical response to MTBE. We have found dose-dependent changes in number of total coliforms bacteria — when concentration of MTBE is being increased, number of total coli-form bacteria are being decreased. The mode of this action needs to be clarified by biochemical and molecular studies which will aim to determine the molecular mechanisms of MTBE action on bacterial metabolism. We hypothesize that not only MTBE itself does it cause bacteria growth suppression but also its by-products, tert-butyl alcohol (TBA), tert-butyl formate (TBF) and formaldehyde (listed by EPA as potential human carcinogens), contribute to bactericidal action. We expected to find positive trend in growth of Pseudomonas species in high MTBE concentrations as there is the data about that P. aeruginosa can survive in fuel organics and even uses it as nutrients for growth. But our study did not clearly justify this assertion. Practically, our results may be applied in evaluating MTBE toxicity in water microorganisms. We have observed in our study that MTBE behaves in living microorganisms diversely, causing inhibition of some species. Therefore, we conclude that MTBE has to be routinely monitored in environmental media. Since the amounts of its production and usage in the world are still high and count around 13,5 Mt annually [21] there is a concern about its releases into environment. In spite of active promotion of biofuels containing bioethanol and its product bio-ethyl tert-butyl ether (ETBE), MTBE is, so far, the fuel oxygenate of choice in countries of the Eastern Europe, Middle East and Asia. Ukraine consumes nearly 200 thousand tons of MTBE yearly and this consumption is expected to raise in the near future, as the country is about to revise the regu-

latory base for fuel quality in order to meet the European Euro 2 and Euro 4 standards [22].

Conclusions. The results of the present study suggest that MTBE may be cause inhibition of total coliform bacteria in sea water. Taking into account our previous findings showing mixed results, current data point that of MTBE in ranges from 0,015 to 150 mg/L, impacted the TC growth. These results should enable the prospect studies on MTBE and water microbiology as part of studies for understanding the mechanisms by which MTBE can interact with some bacterial cells making them able to survive in hostile environments or to cause the inhibition of others. It needs to be pointed out that our results are applied on salt sea water and their relevance for fresh water still remains not evident. We believe that MTBE may be a quite suspicious volatile organic compound, soluble and stable in water, especially in ground water. Known cases of ground water pollution by MTBE in the USA and detection of MTBE in water of some countries of Europe suggest that this chemical deserves to be thoroughly studied for a better understanding of its behavior in the ambient media, so that enabling protection of the environment and public health.

Acknowledgments. Authors are thankful to EU Erasmus Mun-dus External Cooperation Window Lot 6 for their essential support. We are also grateful to the following people: professor Malama-tenia Arvanitidou, chief Laboratory of Hygiene, and professor Alexis Benos for their support in organization of the study; Dr. Vasileios Paraschos for his valuable guidance concerning the application of international microbiological analysis methods; and the technical staff of the Laboratory of Hygiene for their support during the experiments.

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Надiйшла до редакцИ20.12.2010.

HYGIENICAL QUESTIONS OF NHE MODERN STATE OF DRINKING WATER IN THE PASSENGER TRAINS

Tsurkan V., Dayduyn V., Shkuro V., Parats A.

ПГ1БН1ЧН1 ПИТАННЯ СУЧАСНОГО СТАНУ ПИТНОГО ВОДОПОСТАЧАННЯ ПАСАЖИРСЬКИХ ПОТЯГ1В

однi ресурси, якi використову-ються для пиття i господарсько-побутових цтей, е одним з го-ловних факторiв ризику, що мае суттевий вплив на рiвень здо-ров'я населення та потребуе ви-ршення тако! глобально! транс-кордонно! проблеми, як забез-печення доступу населення до питно! води гарантовано! якоcтi [6, 8, 11]. Залiзничний транспорт е значним споживачем питно! води. Оcтаннiми роками спостер^аеться тенденцiя до збiльшення використання води залiзницею, що пов'язано передуем зi збiльшенням обсягу пе-ревезень. Одним з основних на-прямюв дiяльноcтi державно! саштарно-етдемюлопчно! служби на залiзничному транс-портi е оргашза^я i здiйcнення державного саштарно-епщемю-лопчного нагляду за джерелами i водопостачанням об'екпв, якi обслуговують паcажирcькi пере-везення. Екcтериторiальний характер дiяльноcтi закладiв державно! саштарно-епщемюлопч-но! служби на залiзничному транcпортi, який сполучаеться з

ГИГИЕНИЧЕСКИЕ ВОПРОСЫ СОВРЕМЕННОГО СОСТОЯНИЯ ПИТЬЕВОГО ВОДОСНАБЖЕНИЯ ПАССАЖИРСКИХ ПОЕЗДОВ Цуркан В.Г., Дядюн В.Н., Шкуро В.В., Парац А.Н.

В статье рассматриваются вопросы современного состояния питьевого обеспечения пассажирских перевозок на железнодорожном транспорте Украины и пути его оптимизации. На основании ретроспективного анализа данных лабораторных исследований образцов воды систем водообеспечения пассажирских вагонов Донецкой железной дороги за последние 3 года делается вывод о снижении доли проб, которые не соответствуют нормативным требованиям. Описывается алгоритм контроля качества питьевой воды пассажирских вагонов, которого придерживаются специалисты Донецких линейных санэпидстанций. Отмечены существенные конструктивные несовершенства вагонных систем водообеспечения, которые эксплуатируются сегодня в Украине, что значительно увеличивает риск возникновения инфекционных болезней у пассажиров железной дороги. Подчеркивается необходимость незамедлительного внесения соответствующих конструктивных изменений в системы питьевого водообеспечения пассажирских вагонов с целью повышения их санитарно-эпидемиологической безопасности.

© Цуркан В.Г., Дядюн В.М., Шкуро В.В., Парац А.М.

СТАТТЯ, 2011.

ЦУРКАН В.Г., ДЯДЮН В.М., ШКУРО В.В., ПАРАЦ А.М.

Державний заклад "Саштарно-епщемюлопчна стан^я на Донецьюй залiзницi" МОЗ Укра!ни, м. Донецьк

Державна установа "1нститут тени та медично! екологи iм. О.М. Марзеева НАМН Укра!ни", м. Ки!в

УДК 613.31:543.39 - 002.1

35 Environment & Health № 3 2011

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