Determination of Phenols in River Waters: A Case Study for Keritis River at Northwestern Crete.

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1 Determination of Phenols in River Waters: A Case Study for Keritis River at Northwestern Crete. NIKOS LYDAKIS SIMANTIRIS, MICHALIS ANASTOPOULOS, EVAGELOS TERZIS, AND KONSTANTINOS VOZINAKIS Laboratory of Environmental Chemistry and of Biochemical Processes, Department of Natural Resources and the Environment, Technological and Educational Institution of Crete, Romanou, Halepa, Chania, 7 Crete, GREECE Abstract: The -aminoantipyrine colorimetric method was employed for the examination of the waters of Keritis River, which flows west of the city of Chania, Crete. Along the banks of Keritis River there are several olive mills. Olive oil production wastes contain considerable amounts of phenols which constitute a potential threat for the environment, especially in the case that these wastes are not properly deposited. In our study we collected water samples from six sites of the river, most of them next to olive mills. The frequency of sampling was approximately once a month and the sampling period was between October and April. Analyses of these samples for phenols showed a small increase of phenol concentration in samples collected from certain sites, mainly during the peak of olive harvest and, accordingly, olive oil production season. However, the levels of phenols concentration did not exceed the upper acceptable limits, set by the directory /EU for drinking water. Key words: Phenols, phenolic extraction, -aminoantipyrine colorimetry, drinking water. Introduction The release of phenol and its derivatives into the environment is of great concern. A considerable number of organic pollutants, widely distributed throughout the environment, have a phenolic structure. Phenolic compounds are a class of polluting chemicals, easily absorbed by animals and humans and their toxicity is directed towards a great variety of organs and tissues. The most important effects reported in short term animal studies were neurotoxicity, liver and kidney damage, respiratory effects, dermal corrosion and necrosis, and growth retardation []. Phenolic compounds have also been accused of carcinogenicity [],[, and references therein]. Many phenols, especially chlorophenols, are known for their persistency in the environment and propensity for bioconcentration []. Olive oil production generates a large amount of solid and liquid wastes. The liquid wastes especially, contain high concentrations of phenolic compounds, as high as 8 g/l []. Usually, the olive mills liquid wastes are transferred to specially designed open tanks, so they can undergo sedimentation, flocculation, and evaporation of their water content. At the end of the olive harvesting season, the solid material from these tanks is collected and, depending on the case, is either processed or properly deposited. Impermeability of these tanks is of crucial importance for preventing leaking of the wastes to the ground, and, subsequently, possibly to the water resources. Escape of the wastes to the environment can also happen by overflow of the liquid due to excessive deposition and / or rainfalls. In the study presented here, we applied the - aminoantipyrine colorimetric method [] in our laboratory, in order to monitor the levels of phenolic compounds in the waters of Keritis River, which flows west of the city of Chania, throughout the olive harvesting season of the winter of. Keritis River flows through an area covered mainly by olive trees. Several olive mills operate along the river every winter, and monitoring the levels of phenolic compounds in river waters is of Laboratory of Environmental Chemistry and of Biochemical Processes, and Department of Land Reclamation, Prefecture of Chania, Crete.

2 great importance, since water from the river is used widely by the inhabitants of the area. We note that although the phenolic compounds determined in the samples examined in this study are characterized as "total phenols", the - aminoantipyrine colorimetric method cannot detect all the phenolic compounds possibly present in water and wastewater samples. More specifically, this method is capable for detection of o- and m- substituted phenols and from p- substituted ones, only those with carboxylic, halogen, methoxy or sulphonic substitution []. Thus, the levels of the phenols detected with this method are considered as the low limits of the real concentration of the total phenolic compounds in the samples.. Materials and Methods The -aminoantipyrine colorimetric method was used, as it is described in [], with minor modifications in order to adjust it to our laboratory instrumentation. More specifically, instead of using sample volumes of ml for phenol extraction, we used samples of ml. The amounts of all the reagents and solvents used for the extraction were adjusted accordingly. For optical spectroscopy we used. cm cuvettes. The method was tested for its performance in our laboratory using the statistical analysis method Student's t-test []. Samples were collected from six preselected sites, next to olive mills. Care was taken to collect samples from the center of the flow, not close to the river banks. The samples were transferred to the laboratory and analyzed immediately, or acidified with conc. H SO (. % v/v), and kept at ºC until analysis. Analysis was performed usually within the next two or three days, except for the experiment where the decay of the phenols concentration vs. time was determined (see below). Sample distillation. About ml of sample were adjusted to ph. with H PO :9 (v/v) when samples were treated immediately after sampling, or with NaOH. N, when samples were acidified and refrigerated prior to use. Then, three portions of ml each were distilled separately in a ml distillation apparatus. When ml of sample were distilled, the apparatus was dismantled, ml of deionized water were added and the distillation was continued to a final volume of ml. For the construction of a calibration curve (see below), the same procedure was repeated for a blank (deionized water) and a series of standards containing,,,, 8 and ppb of pure phenol. Extraction of phenols. In each distillate. ml of NH OH. N were added and the ph was adjusted to ph 7,9 by phosphate buffer..9 ml of -aminoantipyrine solution % (w/v) and.9 ml of K Fe(CN) solution 8 % (w/v) were then added and the distillates were left to stand for min for color development. Extraction of phenols was carried out with 7. ml of CHCl. After the extraction, trichloromethane was passed through a conical paper filter which contained approximately g of Na SO as a drying agent. Absorption measurements. Absorbance of the samples and the standards was measured at nm. Absorbance values from standards were used to construct a calibration curve. The equation of the best fit was used to calculate the phenol concentration of the samples.. Results and Discussion Figure presents a hydrological map of the area west of the city of Chania, in Chania prefecture in the island of Crete, where Keritis river flows. The springs of Keritis are at an altitude of m, and the direction of the river is roughly S N. The sampling sites are shown on the map with numbers, which correspond to the numbering used in the Tables of the results. Sample was collected right from the springs of the river, whereas sites,,, and were chosen for their proximity to olive mills. Although site is also close to an olive mill, waters from other resources of the area have already entered the river at that point. The first samples were collected by the end of October, before the beginning of the rain season and the oliveharvesting season. The last sampling was done by the end of April, when the olive harvest is over. Validation of the method. Student-t test [] was used in order to check the operation of the method in our hands and in our laboratory. For this, six sets of standards with concentrations of,,,,, 8 and ppb of pure phenol were prepared and analyzed according to the method described above. Before the measurements, the

3 spectrophotometer was zeroed to an empty holder (air), so the reproducibility for the absorption values of the ppb standard could also be checked. The results of this analysis are shown in Table and Figure. In table we show the absorbance values of the standards as well as the mean, the minimum and maximum values, the range and the standard deviation. For confidence level 9 %, the confidence limits are also shown. Figure presents the mean of the absorbance values at nm as a function of the concentration of the standards, along with the confidence limits and their range for confidence level 9 %. As one can realize from Table and figure, the phenol determination in water samples with the -aminoantipyrine method is reproducible and reliable, at least for the range of the concentrations examined in our laboratory. The slight modifications we introduced to the method did not affect its performance.,,,9 Absorbance,8,7,,, Figure. Hydrological map of the area west of the city of Chania. Keritis River is shown from its springs to its estuary. Figure. The mean of the absorbance values for the six set of standards presented in Table as a function of the concentration. The 9 % confidence limits are also shown. The effects of sample storage on the phenolic content was also checked. One set of samples, collected from all the sites, was acidified and stored at ºC. Analysis of parts of these samples was performed the next day, and then once a week for the next four weeks. Table. Student-t test for six sets of standard phenol solutions analyzed by -aminoantipyrine colorimetric method Phenol concentration (ppb) 8,7,8,,8,7,877,9,7,9,7,,77,9,987,,8,97,77,799,89,9,,,9,,777,9,99,,,,,78,899, Absorbance,,,9,98,78,87,99 Mean,,,9,7,79,89,978 Max,7,8,9,98,799,9, Min,,,,,7,89,99 Range,,,8,7,,7, st. dev Conf. lim 9%

4 No significant alterations between the results acquired from the analysis performed immediately after sampling and those of the next analyses were observed (data not shown). Examination of samples from Keritis River for phenols contamination. Samples from the six sites shown in the map in figure were collected approximately once a month, from October through April. The samples were analyzed immediately, or treated and stored as described in Materials and Methods section. For the analysis of each set of samples, fresh standards were prepared and a new calibration curve was constructed. For all calibration curves, a R coefficient of was observed (data not shown). The results of the analyses of all the sets of the samples are presented in Table. The histograms of Figure present the levels of total phenols in the corresponding sites as they were determined in the samples collected between October and April. Oct- Dec- Jan- Feb- Apr- Concentration (ppb Oct- Dec- Jan- Feb- Apr- Oct- Dec- Jan- Feb- Apr- Oct- Dec- Jan- Feb- Apr- Oct- Dec- Jan- Feb- Apr-

5 Table. Total Phenol Sampling sites October December January February March April Oct- Dec- Jan- Feb- Apr- Figure. Histograms presenting the concentration levels of total phenols at the sites of Keritis River as they are marked in the map of Fig.. As it is shown in Figure, the total phenol concentration determined in all the samples examined in this study did not exceed the low limit of ppb set by [7], except one sample taken from site, in February of (shown as a lined bar in histogram and as a grey box in Table ). In all the other samples the total phenol concentration was considerably lower, or zero. The variation of the phenol concentration on each sampling date vs the sampling sites is shown in Figure. The lines which connect the data points represent the best fits of the variation. In general, February seems to be the month with the highest level of contamination at most of the sites of the river neighboring olive mills. Comparatively high concentrations of phenols appear at several sites in January as well. The only site which showed a considerable level of phenol concentration in December was site. Keritis River was literally free of phenols in March and April, at least at the sites we examined. Regarding the variation of the total phenol concentration at each site throughout the sampling period, figure shows an increase in the samples collected from the springs of the river especially in January (peak of the olive harvesting season), but also in October and in February. The rest of the samples collected from the springs showed very small, or zero concentration of phenols. The origin of the contamination is, at this point, unclear. One possibility is the appearing phenols to exist in the underground water which comes out directly in the springs, and, alternatively, to originate from anthropogenic activities above ground. The relief of the area around the springs is such that could rationalize this hypothesis. In any case, further examination is needed in order to make safe conclusions. 7 Sampling sites Figure. Total phenol concentration vs sampling sites. October, December, January, February, March, April. Samples from site show an increase in phenol concentration mainly in December and February, and very low or zero concentration in the other samples. Especially in February, the total phenol levels seem to exceed the limit of ppb [7]. Most likely, the appearance of phenols at this site originates from overflow of

6 liquid wastes from the olive mill close to the site, due to rainfalls. Leaking of liquid wastes from the olive mill sedimentation / evaporation tanks cannot be excluded, however, in that case one should expect to observe higher phenol concentrations in samples collected in January from this site. Site showed an increase in total phenol concentration in January and February. The interpretation of these results could be the same as of those for site, with the addition that, probably, the size of the tank and the waste production of the olive mill close to this site are such that an overflow of wastes due to rainfall was prevented in December. Site showed a considerable level of phenols in January and February which could be explained the same way as above. Sites and showed very low concentrations of phenols throughout the sampling period, with the exception of site in February, which showed a rather small increase.. Conclusions The -aminoantipyrine colorimetric method was adopted and slightly modified in our laboratory in order to monitor the total phenol levels of the waters of Keritis River. This river could be considered as a subject of potential pollution due to the olive mills which they operate along its banks every winter. The liquid wastes of these olive mills are transferred to special open tanks for sedimentation and evaporation before subsequent treatment. There have been cases where these tanks were not impermeable, so leaking of the olive mills liquid wastes to the environment has been observed. Additionally, overflow of the wastes due to rainfalls (the olive harvesting period coincides with the raining period in Crete) is also a strong possibility for waste leaking. Due to their phenolic content, liquid wastes from olive mills are considered serious pollutants. In this study we show that there is a correlation of the total phenol concentration in samples collected from sites next to olive mills, with waste production: the highest concentrations of phenols appeared in the samples collected at the peak of the harvesting season. Even though in this study the levels of the total phenol concentration, as it is determined by - aminoantipyrine method, did not exceed the limits set by [7], care must be taken in order to prevent any escape of liquid wastes to the environment. Especially for Keritis River, the waters of which are used widely, a further study regarding the presence of phenols is underway in our laboratory. Acknowledgments. The authors thank Dr. Nikos Xekoukoulotakis for useful discussions and suggestions. The hydrological map of fig. was kindly provided by Dr. P. Soupios (Dept. of Natural Resources and the Environment, TEI of Crete). References: [] International Programme on Chemical Safety, c/ehc.htm [] Salaman, M. H., and Glendenning, O. M., Tumor Promotion in mouse skin by sclerosing agents, Br. J. Cancer,,, 97. [] Smith, C. J., Perfetti, T. A., Morton, M. J., Rodgman, A., Garg, R., Selassie, C. D., and Hansch, C., The Relative Toxicity of Substituted Phenols Reported in Cigarette Mainstream Smoke, Toxicological Sciences, 9, 78,. [] Lopez, R., Land Treatment of liquid wastes from the olive oil industry (Alpechin). Fresenius Envir Bull, 9, 99. [] American Public Health Association, Standard Methods for the Examination of Water and Wastewater, method, th edition, Washington D.C., 999. [] Skoog, D. A., West, D. M., Holler, F. J., and Crouch, S. R., in "Analytical Chemistry: An Introduction", 7 th ed. Harcourt Inc. NY,. [7] Directory / EU.