Evaluation of the actual standard procedures for analysis of total extractable hydrocarbons in environmental water matrices

Desalination 273 (2011) 308–315

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Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l

Evaluation of the actual standard procedures for analysis of total extractable hydrocarbons in environmental water matrices Carina Pinho a,⁎, Catarina Mansilha b,c, Paula Gameiro a a b c

Requimte, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre 4169-007 Porto, Portugal Departamento de Saúde Ambiental, Instituto Nacional de Saúde Doutor Ricardo Jorge, Rua Alexandre Herculano, 321, 4000-055 Porto, Portugal Requimte, Universidade do Porto, Portugal

a r t i c l e

i n f o

Article history: Received 21 September 2010 Received in revised form 7 January 2011 Accepted 17 January 2011 Keywords: Total extractable hydrocarbon Liquid–liquid extraction Solid phase extraction Microwave assisted extraction FTIR

a b s t r a c t Hundreds of different hydrocarbon compounds derived from agricultural, industrial and domestic practices are released daily into aquatic systems with adverse effects in the desirable potable characteristics, in aquatic fauna and flora, and on tourism, recreation and aesthetics of the impacted areas. Total petroleum hydrocarbon concentration data cannot be used to quantitatively estimate human health risk, but can be used as an important tool for three purposes: verifying if there is a problem, assessing the severity of the contamination and following the progress of a remediation effort. The objective of this work was the optimization of the experimental conditions for the determination of total hydrocarbons in water by liquid–liquid extraction coupled to infrared spectrophotometry, with the goal of improving the qualitative information provided by the spectra. The method was validated with satisfactory detection and quantification limits and demonstrated acceptable levels of precision, accuracy and analyte recoveries. Solid phase extraction and microwave assisted extraction procedures were tested and compared to the liquid extraction. Additionally, a spectra library was prepared with different natural and synthetic oils. The developed method was applied to the analysis of Portuguese inland and coastal bathing waters and wastewaters and their compliance with national and European legislation was assessed. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Aquatic pollution is an enormous global problem once water plays an essential role in sanitation and public health [1]. Among the pollutants, hydrocarbons, generally called Total Extractable Hydrocarbons (TEH) or Oils and Greases (OG), remain an important index of water quality, attracting the attention of environmental researchers owing to its adverse impact on human health, aquatic life and deleterious aesthetical effect [2–4]. The Environmental Protection Agency (EPA) and the American Petroleum Institute (API) United States, the International Maritime Organization (IMO), the World Health Organization (WHO), the Environment Canada and the Ministry of Water Resources in China are examples of government and private entities that are active in the participation and support of standards and methods for monitoring hydrocarbons in water [4–8]. The term “Total Hydrocarbons” encompasses a broad family of chemical compounds such as fatty material of biogenic origin or mineral hydrocarbon constituents [9]. They are widely used in agriculture, industrial and domestic activities and can reach the

⁎ Corresponding author. Tel.: +351 220402000; fax: +351 220402009. E-mail address: [email protected] (C. Pinho). 0011-9164/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2011.01.047

environment through storm water run-off, spills from road asphalts, fuelling depots, transportation and haulage, cooling water systems, refining processes of crude oil, manufacturing facilities such as automotive, plastics and steel production, accidents and wood distillation industries. “Total Petroleum Hydrocarbons” (TPH) is a term used to describe a group of several hundred chemical compounds that originally come from crude oil and are considered the most deleterious for human health. As a gross parameter, TPH shares with TEH the property of lacking any consistent relationship to a biological hazard, as very little is known about the toxicity of many compounds and recognized toxic chemical components vary with the hydrocarbon source. Therefore, no general statements based on biological effects can be made regarding “safe” levels of TEH or TPH [10]. In environmental waters it is scientifically proven that hydrocarbon effects include changes in the respiratory system of fish by oil adhesion to the gills, adhesion and destruction of algae and plankton, changes in feeding and reproduction of water life (plant, insect, and fish) and aesthetics effects [6,7,11,12]. Regarding human health, various TPH, such as benzene, toluene, xylene and n-hexane can cause fatigue, headache, nausea, drowsiness, and affect the human central nervous system, immune system, liver, spleen, kidneys and lungs [13]. They are classified as carcinogens by the International Agency for Research on Cancer [14] and WHO [4].

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With the aim of protecting public health and environment, discharges of hydrocarbons in the sewer systems are regulated by both European (Directive 76/160/CEE) and Portuguese National legislation (Decreto Lei 236/98, 1 August), with a limit of 15 mg/L for total OG. For drinking water, a strict limit of 0.10 μg/L for polycyclic aromatic hydrocarbons (Decreto Lei 306/2007, 27 August) is established. Regarding bathing waters, compliance with the new Portuguese Decreto Lei 135/2009 (that is the transposition of the European Bathing Water Directive 2006/7/EC [15] to the National law) only requires a visual inspection for chemical pollution in replacement of the previous 0.3 mg/L for OG (Portuguese Decreto Lei 236/98 and the European Directive 76/160/CEE), despite the actual tendency to increase the water quality assumptions to allow a tighter control of the environmental pollution. As Portugal has a large coast and pleasant weather, motorized water sports have gained popularity in the last years and there are increasingly more boats and personal water crafts for tourism, recreation and sport use that, fuelled by gasoline and diesel, are important sources of pollution. Some concern has been expressed about the risk to bathers from the presence of chemicals in recreational waters but very few studies have been found in the literature that equate a hazard to the health of swimmers [16,17]. Despite the lack of a toxicological basis, gross hydrocarbon parameters can still be a very useful tool for three purposes: 1) as a screening tool at contaminated site assessment; 2) as investigative surrogates on a site-specific basis; and 3) as remediation criteria when toxicological, organoleptic and water protection concerns have previously been addressed [18]. Thus, although restrictive, hydrocarbon contamination investigations should require a combination of gross parameter measurements with subsequent specific analyses for the expected contaminants. As TEH represent a group of substances which have similar physical characteristics defined by their common solubility in the organic solvents, the standard methods are based on an extraction followed by a gravimetric or infrared (IR) analytical methodology [19–22]. Although gravimetric-based methods are simple, quick and inexpensive, they present disadvantages such as low sensitivity and specificity, loss of volatile compounds and restriction to compounds NC10 [9,10,18,23]. IR-based methods measure the absorbance of the C―H bond and are more sensitive. The measurement of the stretching of aliphatic CH2 groups is at 2930 cm− 1. For CH3 groups measurement is at 2960 cm− 1 and for aromatic C―H bonds it is at 3010–3100 cm− 1, allowing a quantitative and a qualitative assessment of the analytes [9,18]. The aim of the present work was the development of a liquid– liquid extraction (LLE) procedure coupled to a Fourier Transformed Infrared (FTIR) methodology based on Standard Methods [22] and EPA's methods [20,21] for detection and quantification of total and petroleum hydrocarbons in water with an improvement on the qualitative information provided by the spectra. A library of FTIR spectra was made using several animal, vegetable and mineral oils in order to enable the identification of the type of hydrocarbons that may affect watercourses. 2. Experimental 2.1. Chemicals Carbon tetrachloride (99.8%) was purchased from Prolabo (Briare, France) and 1,1,2–Trichlorotrifluoroethane (freon, ≥ 99.9%) from Merck (Darmstadt, Germany). Analytical-reagent grade n-hexadecane was obtained from J.T. Baker (Deventer, Holland) and isooctane (≥ 99.8%) and benzene (≥ 99.8%) were obtained from Merck (Darmstadt, Germany). Stock standard solution, also referred as reference oil, was prepared by mixing 37.5% (v/v) n-hexadecane, 37.5% (v/v) isooctane and 25% (v/v)

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benzene. Calibration standards were prepared in 10 mL volumetric flasks by proper dilution of the stock standard solution with carbon tetrachloride (CCl4). Chloroform (99%), methanol (for GC SupraSolv®), acetone (≥ 99.8%), isopropanol (99.7%), ethyl acetate (for GC SupraSolv®) and silica gel 60 (0.2–0.5 mm) were purchased from Merck (Darmstadt, Germany). Acetonitrile (99.9%) was purchased from Panreac (Barcelona, Spain), HCl 37% from Prolabo (Briare, France) and anhydrous granulated sodium sulphate (99.0%) from J.T. Baker (Deventer, Holland). Ultra-pure water (0.054 μS/cm) was obtained by using a Milli-Q system from Millipore (Milford, MA, USA). 2.2. Apparatus A Shimadzu IR Affinity-1 system, equipped with deuterated L-alanine doped triglycine sulfate (DLATGS) detector with a KBr beamsplitter, was used for collection of FTIR interferograms. The spectrometer was employed with a resolution of 4 cm− 1, accumulating 45 scans per sample and using a quartz cell of 10 mm pathlength. Absorbance measurements were carried out between 3040 and 2930 cm− 1 with a baseline established between 3200 and 2600 cm− 1 (and were done against a pure solvent of CCl4 as background spectra). The equipment carried the IR Solution software for the acquisition and processing of the FTIR absorbance data. Solid phase extraction was conducted in a SPE vacuum manifold system from Phenomenex (Torrance, California, United States). A microwave oven MARS-X 1500 W (Microwave Accelerated Reaction System for Extraction and Digestion, CEM, Mathews, NC, USA) configured with a 14 position carousel was employed for microwaveassisted extraction using laboratory made 50 mL internal volume PTFE reactors. During operation, both temperature and pressure were monitored in a single vessel. 2.3. Sampling procedure Wastewaters were collected in influx and efflux systems of several sewage treatment plants (domestic and industrial) in North of Portugal, and treatment efficiency was evaluated. Samples of coastal and inland bathing waters were collected in Douro and Algarve. Water samples were collected in amber glass bottles, 1 L capacity, and acidified to a pH lower than two with 1.2 M HCl to avoid the loss of the target compounds due to microorganisms' metabolism and reactions of hydrolysis and precipitation. Samples were stored in a refrigerator at 4 °C and processed in a maximum of 48 h after their harvest [1,20,21]. 2.4. Spectra library A library of FTIR spectra was prepared using several animal (lard and tallow), vegetable (olive oil, sunflower oil, almond oil and cedar oil) and mineral oils (gasoline and unleaded 95, ultimate diesel, AC Delco DEXRON III oil, Porsche DOT-4 oil, SHE Mobil 10W/40 Super Turbo Diesel, Mobil Penetrating Oil Aerosol, antifreeze, waste engine oil, CHF steering oil, brake oil and valvoline oil). Samples were diluted in CC14 and IR spectra were obtained according to the equipment conditions specified at 2.2. 2.5. Liquid–liquid extraction (LLE) According to the EPA 413.2 [20] sample volume used in the extraction of TEH should be 1 L. However, in order to optimize LLE procedure, especially to reduce the volume of the organic solvent used for extraction, different sample volumes (1000 mL, 500 mL and 100 mL) were tested for extraction efficacy maintaining a proportion of 10% of the extraction solvent.

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LLE optimized conditions were as follows: 100 mL water sample, previously acidified with 1.2 M HCl solution, to a pH lower than two, were extracted with 3 × 3 mL of CCl4 in separatory funnels (250 mL) by shaking vigorously for 2 min. The filtration of the organic extracts was done through glass microfiber filters (Whatman Nº1 ∅ 9 cm) with anhydrous sodium sulphate and diluted with CCl4 to 10 mL in volumetric flasks. After that, FTIR measurements were carried out. 2.6. Solid phase extraction (SPE) In order to optimize the extraction efficiency of the procedure, different cartridges and conditioning and elution solvents were tested. TEH were extracted from water samples (100 mL of water previously acidified pH lower than two with 1.2 M HCl solution) using different SPE cartridges: Grace Pure™ Fast (C18) and Grace Pure™ Low (C18) from Grace (Deerfield, United States), Strata™ X (C18) and Strata™ SDB-L (styrene-divinylbenzene) from Phenomenex (Torrance, California, United States) and LiChrolut® EN/PR-18 (C18) from Merck (Darmstadt, Germany). Columns were previously activated and conditioned with a mixture of acetone, isopropanol and water (20:15:10 v:v:v) or methanol and water (5:5 v:v). After application of the samples (flow rate 1–3 mL/min), the columns were washed with 10 mL of water and dried for 50 min under vacuum (not exceeding a pressure of 20 mm Hg). Several elution solvents were tested, including: CCl4, freon, chloroform, methanol, acetonitrile and ethyl acetate. After elution, the extracts of chloroform, methanol, acetonitrile and ethyl acetate were evaporated until dryness in a rotative evaporator (Buchi/Brinkman Rotavapor RE-111 and Water Bath B-461) and then re-suspended with CCl4 to a final volume of 10 mL. CCl4 and Freon extracts were directly analyzed by FTIR at 2930 cm− 1. 2.7. Microwave-assisted solvent extraction (MASE) The procedure was based on a quantitative microwave-assisted solvent extraction of TEH from water samples using CCl4 as solvent. Factors such as temperature (90 °C and 110 °C) and extraction time (10 min and 15 min) were considered to optimize the microwave extraction process. An exit power of 300 W and 150 psi were employed. MASE optimized conditions were as follows: 25 mL water sample, previously acidified to pH lower than two with 1.2 M HCl solution, were introduced in PTFE reactors with 10 mL of CC14. Reactors were hermetically closed and introduced into the microwave. After irradiation at 90 °C for 10 min reactors were cooled and opened. The organic phases were quantitatively removed, filtered through glass microfiber filters (Whatman N°1 ∅ 9 cm) containing anhydrous sodium sulphate and then FTIR measurements were carried out.

Following the pioneering study of Simard [28], Standard Methods [22] and EPA [20,21] a mixture of n-hexadecane, isooctane and benzene (37.5% + 37.5% + 25% v:v:v) was employed as calibration standard for the analysis of hydrocarbons in polluted waters by IR spectrometry. Procedure was validated for TEH in terms of calibration range, linearity, detection and quantification limits, precision, accuracy and sensitivity of the method. Linearity was tested using five calibration standards, in levels from 2.99 to 49.89 mg/L, prepared from the stock standard solution. Analytical method was performed using ten independent replicates analyzed at 2930, 2960 and 3040 cm− 1. As the magnitude of the coefficients of determination is a poor indicator of linearity [29–31], the assumption of homoscedasticity, or homogeneity of variances, was tested by applying the F-test in accordance with the following statistics [25,32,33]. The limits of detection (LOD) and quantification (LOQ) were calculated from the first calibration standard, i.e., the lowest possible limit of the working calibration range. Precision was determined in terms of intra-day precision and inter-day test and sensitivity was studied in terms of the slopes of the calibration function within the working range.

3. Results and discussion 3.1. Appreciation of the actual Standard Methods procedures The determination of total OG and TPH in waters was performed mainly in accordance with EPA 413.2 [20] and EPA 418.1 [21] methods, respectively, and in accordance with Standard Methods [22]. The reference calibration solution was composed of a ternary mixture of n-hexadecane, isooctane and benzene, with characteristic absorbance bands in IR (Fig. 1). As can be seen, there are two well established absorbance maxima at 2930 cm− 1 and 2960 cm− 1, the first corresponding to the stretching of aliphatic –CH2– and the second to the stretching of aliphatic –CH3– chemical bonds, characteristics of n-hexadecane and isooctane, respectively. The –CH– absorbance bands corresponding to the stretching of aromatic groups are located at wavenumbers higher than 3000 cm− 1 and are characteristic of benzene. Although Standard Methods and EPA propose a mixed standard calibration solution with three distinct absorbance spectral regions they fixed the IR absorbance measurements at 2930 cm− 1 for TEH and TPH analysis as this band is common to many organic compounds. Nevertheless, this can lead to incorrect or false negative results regarding compounds with

2.8. Total petroleum hydrocarbons (TPH) analysis TPH were quantified after removal of polar compounds from the organic extracts by adding approximately 3 g silica gel. Solutions were stirred for a minimum of 5 min on a magnetic stirrer. Silica gel has the ability to adsorb polar components such as fatty acids that are removed selectively from solution. Then, the solution was filtered through glass microfiber filter (Whatman N°1 ∅ 9 cm) and FTIR measurements were carried out [21]. 2.9. Method validation Analytical tools require that the methods used are fit for their purpose and the instruments are operating correctly [24], so the analytical performance characteristics of the optimized method were studied and validated according to an International Standard Organization (ISO) method [25], the International Union of Pure and Applied Chemistry (IUPAC) [26] and Guia Relacre requirements [27].

Fig. 1. FTIR spectrum of n-hexadecane (2 2), isooctane (- - -), benzene (…) and a 37.5:37.5:25 (v/v) standard ternary mixture (2) of these three compounds diluted with CCl4. Spectra was obtained from the accumulation of 45 scans, working with a nominal resolution of 4 cm− 1.

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different absorption characteristics like isooctane and benzene. Thus, our method was developed and validated based on the experimental results obtained using absorbance peak height values at 2930, 2960 and 3040 cm− 1 which allow an improvement in the quantitative and qualitative data assessment. 3.2. Spectra library A library of FTIR spectra using several animal, vegetable and mineral oils was elaborated. Some of the oils analyzed are shown in Fig. 2. All animal and vegetable oils analyzed presented a characteristic maximum absorption band at 2930 cm− 1. However, some petroleum sub-products showed a significant absorbance band at 2960 cm− 1, namely gasoline. This evidence can easily demonstrate that Standard Methods and EPA's measuring condition could be inefficient to assess water quality. The same is valid for aromatic compounds with spectra similar to benzene. The importance of tracing these spectra allowed the knowledge of different spectral patterns of widely used oils and greases. On the other hand, it enables a first qualitative assessment of the profile of the TEH in the analyzed samples.

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Table 1 Comparison of calibration curve results obtained for n-hexadecane, isooctane and benzene in CCl4. Parameters

n-Hexadecane at 2930 cm− 1

Isooctane at 2960 cm− 1

Benzene at 3040 cm− 1

Regression line (mean values) R2 (mean values) Fexp n LOD (mg/L) LOQ (mg/L)

y = 0.001x − 4 × 10− 04 0.9999 4.62 10 0.09 0.3

y = 0.001x − 2 × 10− 04 0.9997 3.81

y = 0.0002x − 7 × 10− 05 0.9998 5.30

y: absorbance; x: concentration; R2: coefficient of determination; Fexp: testing value for
homoscedasticity F exp = s22 = s21 with s22 〉s21 ; n: number of replicates of the calibration lines.

Results of sensitivity were 8.2% for n-hexadecane, 7.9% for isooctane and 7.8% for benzene.

3.4. Extraction procedures 3.4.1. Liquid–liquid extraction

3.3. Performance of the analytical method — validation of the FTIR methodology for TEH The LLE-FTIR method was validated and correlated with extractive alternatives based on SPE and MASE [9,23,34–36]. Studies of linearity of the method showed a linear relationship between the analytical signal (y) and the concentration (x), with a correlation coefficient greater than 0.999. Results are shown in Table 1. No significant differences between variances were obtained since the testing values (Fexp) were lower than the critical value of the F-distribution (Ftab = 5.35) at a confidence level of 99% for 9 degrees of freedom. Good sensitivity (LOD), 0.09 mg/L, was found under the optimized experimental conditions and LOQ was equal to 0.3 mg/L. Precision, expressed as relative standard deviation (%RSD), was established in terms of repeatability and intermediate precision and accuracy was described in the sense of bias [30]. They were obtained comparing the observed concentrations (Cobs), that represents the average of the results obtained through the regression model equation of the calibration curves (x), and the theoretical or nominal concentrations (Cnom) [37] (Table 2). Results demonstrated acceptable levels of precision (%RSD ≤ 10%) and accuracy (%Bias ≤ 10%), as defined by acceptance criteria [26,27].

3.4.1.1. Effect of sample volume. The effect of sample volume was studied in a total of sixteen experiments using ultra-pure water and real samples of waste, drinking and bathing waters. By application of the statistical t-test it was found that there were no statistical differences (P N 0.05) between the quantification at 2930 and 3040 cm− 1, over the course of the study. However, at 2960 cm− 1 it was found that the sample volume affects the extraction efficiency (P b 0.05). Fig. 3 shows the spectra of two extracts obtained from LLE of different volumes (100 mL and 500 mL) of the same water sample. As it can be seen, the absorption band at 2960 cm− 1 has a better resolution when using a lower sample volume which may influence the accuracy of the water quality results.

3.4.1.2. LLE-FTIR recovery studies. To confirm the efficiency of the developed procedure water samples (100 mL) were spiked with the stock standard solution at five level concentrations. Quantification was performed using the calibration curves aforementioned at 2930, 2960 and 3040 cm− 1. Measurements were carried out against unspiked waters from the same source as background spectrum. Although tests were performed in order to avoid possible losses, mean recoveries were higher for n-hexadecane (99 ± 11%, n = 47) when compared to isooctane (66 ± 11%, n = 32) and benzene (59±10%, n = 41). This may be due to the increased volatility of benzene (bp: 80.1 °C) when compared to isooctane (bp: 99.3 °C) and n-hexadecane (bp: 287 °C). In addition, bands of benzene are less intense, so they could be more susceptible to interferences from the sample matrices. Similar results were obtained by Daghbouche et al. and Farmaki et al. [9,35].

Table 2 Intra-day (n = 2) and inter-day (n = 10) precision (% RSD) and accuracy (% bias). TEH

Precision (% RSD) Inter-day

Fig. 2. FTIR spectra examples of mineral (gasoline and diesel ultimate), animal (lard) and vegetable (olive oil) oils and greases. Oils and greases analyzed were diluted in CCl4.

Nominal conc. (mg/L) n-Hexadecane at 2930 cm−1 Isooctane at 2960 cm−1 Benzene at 3040 cm−1

Accuracy (% bias) Intra-day

Inter-day

2.99 29.93 49.89 2.99 29.93 49.89 2.99 29.93 49.89 9.0

3.0

0.8

2.0

2.0

0.6

6.8

0.1

0.1

6.0

1.0

0.8

1.0

2.0

0.6

7.2

0.8

0.1

7.0

3.0

0.8

4.0

5.0

1.6

6.1

0.4

0.1

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Additionally some recovery studies were also carried out, using drinking water samples spiked with different CCl4 solutions of gasoline (16.73 mg/L), box car oil (21.07 mg/L) and olive oil (19.84 mg/L). For gasoline, a mean recovery of 71% was obtained; for box car oil and olive oil, mean recoveries of 51% and 64% were obtained, respectively. More accurate results could probably be obtained by using calibration standard solutions prepared with the same compounds as the samples instead of the stock standard solutions [34]. There are thousands of different TEH and as the environmental sample composition is almost totally unknown, this procedure in practice is quite difficult to perform. 3.4.1.2.1. Matrix effects. From the standpoint of validation methods, requirements set by regulatory agencies are basically the same and some considerations could be made related to the matrix effect, since this may cause a withdrawal or increased extraction efficiency. A statistical analysis of the recoveries was made differentiating each type of water: waste drinking and bathing water. It was obtained a better recovery value for drinking and bathing waters, than for wastewaters (Table 3). These results were expected since wastewaters are more heterogeneous, making the extraction a difficult process due to the formation of emulsions and a consequently substantial loss of the analytes. In these cases, submit separatory funnel to sonication for several minutes can help the procedure of phase separation. 3.4.2. Solid-phase extraction Recoveries of TEH were determined by spiking 100 mL ultra-pure water with 38.38 mg/L of the stock standard solution. Regardless to the cartridge used, recoveries were very low (between 3–32%) when compared with those obtained by LLE. Two factors may have contributed to these results. First, analytes may have been retained in the solid phase since the solvent chosen as eluent could have been inappropriate. In this case, other elution solvents were tested such as Freon, chloroform, methanol, acetonitrile and ethyl acetate, but no improvements were observed. A second factor may be the reduced affinity of the analytes to the solid phases. To test this hypothesis, samples were collected after their passage through the cartridges and processed by LLE. Tests with gasoline (peak height measurement at 2960 cm− 1), box car oil and olive oil standards were also conducted. The SPE experiments were done using Grace Pure™ Low cartridges and conditioning was performed with methanol and water. 32% of recovery for box car oil and 39% for olive oil were obtained. Recovery of gasoline was near 0%. Samples were then collected after their passage through the cartridge and were processed by LLE. In this case, we obtained recoveries of 100% for gasoline, 32% for box car oil and 66% for olive oil. Contrary to some scientific publications on the subject, SPE recovery results showed to be much lower than those obtained by manually shaken LLE [35,38].

Table 3 Matrix effect on recovery experiments. Standard

n-Hexadecane Isooctane Benzene

Extraction yield ± S.D.(%) n

Drinking water and bathing water

n

Wastewater

40 24 34

99 ± 11 66 ± 12 60 ± 9

7 8 7

96 ± 7 65 ± 7 51 ± 6

S.D. — standard deviation; n — number of independent trials.

3.4.3. Microwave assisted solvent extraction Some MASE experiments were performed using different water matrices and oil standards. Water samples were spiked at different concentration levels (23.03 to 29.93 mg/L). The results are presented in Table 4. The recoveries of the stock standard solution, independently of the water matrices, were higher (89 to 121%) compared with gasoline, box car oil and olive oil (67 to 80%) and comparable to those obtained by LLE. These results indicate that this method could be an alternative to LLE and could also avoid solvent losses by using closed systems. 3.5. Determination of TEH and TPH in environmental water samples Analysis of water samples were performed by the optimized and validated LLE-FTIR procedure. Despite the benefits of the microwave extraction, it was decided to process water samples by LLE as it is a less expensive methodology, uses the same volume of organic solvent and provided very similar recoveries. 3.5.1. Bathing waters analysis Analyses of TEH were performed in fifty-three Portuguese inland and coastal bathing waters from twenty-one different sampling points. The percentage distributions of total hydrocarbons found in the environmental samples are shown in Fig. 4. As can be seen, 47% of the samples quantified at 2930 cm− 1 and 57% at 2960 cm− 1 had values of TEH equal or above 0.3 mg/L. Moreover, the results evidenced that the methodologies currently used with analysis only at 2930 cm− 1 do not adequately assess OG in water matrices [20,22]. Besides, 30% of the bathing waters studied shows a maximum absorption band at 2960 cm − 1, which is characteristic of gasoline (Fig. 2). Analyses of TPH were carried out in seven of the bathing waters (inland and coastal) with TEH above 0.3 mg/L. As shown in Fig. 5, the large majority of TEH found in water samples corresponded to mineral oils (75.7% at 2930 cm− 1). At 2960 cm− 1, 53.3% are probably gasoline or gasoline derivatives (except sample 5). Considering the new EU Bathing Water Directive [15], although films have neither been detected nor reported throughout the bathing season, a significant number of samples showed TEH above the old recommended guideline value of 0.3 mg/L. To verify if the new Directive safeguards bathers against this possible hazard exposition, results were correlated with the quality parameters established by the new EU Bathing Water Directive [15] — Escherichia Table 4 Recoveries of various oils in different water matrices processed by MASE-FTIR.

Fig. 3. FTIR spectra of two extracts obtained from LLE of different volumes, 100 mL (2) and 500 mL (…), of the same water sample with an absorbance band at 2960 cm− 1.

Samples

Oil added

Extraction yield ± S.D. (%)

Drinking water

Box car (n = 2) Olive (n = 2) Gasoline (n = 3) Reference oil (n = 4) Reference oil (n = 3) Reference oil (n = 1)

70 ± 4 67 ± 0 80 ± 8 111 ± 17 89 ± 40 121

Drinking water Wastewater Bathing water

S.D. — Standard deviation; n — number of independent trials.

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Fig. 4. Percentage distribution of the samples with TEH concentration lower, equal or above 0.3 mg/L. All the samples were quantified at 2930, 2960 and 3040 cm− 1.

Fig. 6. Percentage distribution of microbiological indicators in waters with TEH b 0.3 mg/L and TEH ≥ 0.3 mg/L.

coli, in accordance with ISO 9308-3, and intestinal enterococci, in accordance with ISO 7899-2, (Fig. 6). These results were gently provided by the microbiology laboratory of INSA, I.P. According to the results present in Fig. 6, 32% of the samples with TEH concentration below 0.3 mg/L and 40% of the samples with TEH concentration equal or above 0.3 mg/L presented microbiological indicators above the guide values established by the new EU Directive. Statistical comparison between two microbiological parameters (E. coli and intestinal enterococci) and samples with and without TEH was examined by carrying out an unpaired sample t-test. Results showed that there were no statistically significant differences (P N 0.05) in TEH-positive and TEH-negative samples. These results lead us to conclude that TEH are independent from microbiological evaluation, demonstrating that the new Directive does not efficiently safeguard public health and environment regarding petroleum hydrocarbon exposure.

sewage waters and the other thirteen were the correspondent treated waters. As can be seen, for influent sewage waters TEH quantified at 2930 cm− 1 and at 2960 cm− 1 correspond to 69% and 8%, respectively. These results indicate levels of TEH greater than those established by Decreto Lei 236/98 (15 mg/L). In treated waters this percentage was substantially reduced to 15%, at 2930 cm− 1, but was maintained on 8% at 2960 cm− 1, revealing the partial efficiency of the treatments. Samples complied with the recommended guideline value when analyzed at 3040 cm− 1. Studies of TPH were carried out using eighteen wastewater samples. Results were compared with those of TEH (Fig. 8). According to the Fig. 8, water samples are mostly composed by nonpolar hydrocarbons (28.5% at 2930 cm− 1, 11.5% at 2960 cm− 1 and 72.6% at 3040 cm− 1). To assess the effectiveness of treatments, samples were analyzed as two independent groups: influent and effluent systems, provided from nine sewage water treatment plants. Results consider influent waters as 100% of total TPH concentration (Fig. 9). As we can see in Fig. 9, after treatment there is a significantly decrease of TPH concentration, (P b 0.05) regarding compounds quantified at 2930 cm− 1 and at 3040 cm− 1. Compounds measured at 2960 cm− 1 remained in the water. Consequently, compounds like gasoline are inefficiently removed during treatments. Results from wastewater samples demonstrated that several waters discharged in the sewer systems returned to the aquatic environment with TEH concentrations exceeding 15 mg/L, including

3.5.2. Wastewaters analysis The method developed was applied to the analysis of twenty six water samples from thirteen sewage water treatment plants of domestic and industrial effluents (Fig. 7): thirteen were influent

Fig. 5. Analysis of TEH and TPH in seven bathing waters. Results obtained using absorbance peak height values at 2930 cm− 1 (a) and 2960 cm−1 (b).

Fig. 7. Levels of TEH in wastewaters. (a) Influent sewage waters; (b) effluents of sewage water treatment plants.

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Fig. 9. Total concentration percentages of TPH calculated from nine influent sewage samples, considered as 100%, and nine effluent samples collected at the same treatment plants. Results obtained using absorbance peak height values at 2930 cm− 1, 2960 cm− 1 and 3040 cm−1.

than only one providing more qualitative information and reducing the likelihood of false negative results in samples. A spectra library was also prepared by using different animal, vegetable and mineral oils before method validation and three extractive procedures were tested. Recovery results suggested that LLE and MASE were much more efficient than SPE. The method was validated and considered appropriate for the monitoring of environmental waters being applied to the study of several bathing waters and wastewaters. Studies conducted in bathing waters showed that the new EU Bathing Water Directive only requires a visual inspection for chemical pollution in replacement of the previous 0.3 mg/L for TEH established by the old Directive 76/160/CEE. Our results showed that although oil films were not visible our bathing waters presented values for TEH above the limit of 0.3 mg/L. We conclude that the new Directive does not properly protect bathers against possible exposure to hydrocarbons. Regarding wastewaters, results of TEH from samples collected in influent and effluent systems revealed that contamination decreased significantly after treatment. However, several waters were still discharged in the sewer systems with large amounts of TEH and TPH compounds that may suggest a hazard, requiring more specific analyses.

Acknowledgements The authors are grateful to Dra. Cristina Pizarro, Dra. Carla Coelho and Dra. Ana Heitor of the laboratories of UAS of INSA-Porto and to Doutora Simone from ISEP for the use of the microwave oven.

References Fig. 8. Analysis of TEH and TPH in eighteen wastewaters. Results obtained using absorbance peak height values at 2930 cm− 1 (a), 2960 cm− 1 (b) and 3040 cm− 1 (c).

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