QUALITATIVE STUDY OF ORGANIC COMPOUNDS IN

Environmental Monitoring and Assessment (2006) 116: 103–110 DOI: 10.1007/s10661-006-7230-4

c Springer 2006

QUALITATIVE STUDY OF ORGANIC COMPOUNDS IN WASTEWATERS OF A SWINE SLAUGHTERHOUSE CAMILA PEDOTT AGUILAR1 , MARCIELI PERUZZOLO1 , MARCO DI LUCCIO2,∗ , ´ ´ DO NASCIMENTO FILHO1 ROGERIO MARCOS DALLAGO1 and IRAJA 1

Chemistry Department; 2 Food Engineering Department, Universidade Regional Integrada do Alto Uruguai e das Missões, Campus de Erechim, Av. Sete de Setembro, 1621, 99700-000, Erechim-RS, Brazil (∗ author for correspondence, e-mail: [email protected])

(Received 31 August 2004; accepted 12 May 2005)

Abstract. The main purpose of this work was the preliminary qualitative study of organic compounds in wastewaters of swine slaughterhouses. The samples were collected in a local abattoir and submitted to Liquid-Liquid Extraction (LLE) and Solid-phase Extraction (SPE) with XAD-4TM resin as stationary phase. The instrumental analysis was performed by Gas Chromatography with Mass Spectrometer Detector (GC/MSD). The compounds present in the LLE and SPE extracts were identified by the GC/MSD library (Wiley). The results pointed out that SPE and LLE can extract practically the same classes of compounds at the same amounts. LLE works well for the extraction of polar organic compounds, with acidified samples, while SPE presents a better performance for the extraction of less polar organic compounds. Aldehydes were main class of the compounds extracted by SPE and LLE and decenal was the major aldehyde identified. Fatty alcohols and carboxylic acids were also identified but in minor proportions. Keywords: GC/MSD, LLE, organic compounds, slaughterhouse, SPE

1. Introduction Meat industries are known to produce the highest loads of waste in the food industry. The wastewater of slaughterhouses and meat processing plants has a very complex composition and is very harmful to the environment (Polprasert et al., 1992). Thassitou and Arvanitoyannis (2001) have reported that such wastewaters usually show high amounts of blood, fats, intestine residues and other wastes. Several organic compounds resulting from the degradation of these residues can be found in the effluent of these industries, such as aldehydes, carboxylic acids, ketones, among others. Compounds present in stabilizers, antioxidants, dyes and disinfectants, which are widely used in the manufacture of meat products and cleaning and disinfection of plant installations, may also be found in smaller amounts. When at high concentrations in wastewaters, these compounds can represent a serious hazard for the human health and the environment, mainly by the possibility of contamination of superficial and groundwaters. The presence of organic pollutants in these samples is of great concern because slaughterhouse wastewaters

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are commonly used as natural organic fertilizers. Even at trace level, some organic compounds can cause injury to aquatic organisms and bioaccumulation in the food chain (Galceran and Jáuregui, 1995; Buchholz and Pawlliszyn, 1993; Heberer and Stan, 1997). Although carboxylic acids are not classified as important pollutants, from the environmental point of view, they can contribute to the leaching and consequent bioavailability of toxic compounds like polychlorinated biphenyls (PCBs) and heavy metals present in the soil, by changes in their solubilities in water or organic matter (Möder et al., 1998; You et al., 1996). It is also of concern the presence of nitrogen compounds in the environment, since these compounds can be hazardous for all life forms. Tests with rats pointed out that the exposition to tetranitromethane in a concentration range from 2 to 5 mg/L−1 can induce the development of neoplasias in the lung alveolus and bronchi. The majority of the animals with bronchi-alveolar neoplasm had neoplasias detected as carcinomas and these neoplasma frequently disseminate to other organs. On the other hand, if recovered, many of these compounds can return to the production lines as raw materials with a consequent reduction of costs for the industry. In this way, the development of a study where the main objective is the screening of these compounds in slaughterhouse wastewaters will contribute to solve the problems of environmental preservation and reduction of operational costs. Few reports dealing with chemical characterization of meat industry wastewater using SPE or LLE can be found. Most studies are focused in volatile compounds (Yo, 1999), estrogens and steroids present in wastewater from swine farms and dairy plants (Fine et al., 2003; Rajraman et al., 2004; Kolodziej et al., 2004). In this work, samples of swine slaughterhouse wastewaters were submitted to LLE and SPE for the investigation of organic pollutants. The instrumental analysis was made by Gas Chromatography with Mass Spectrometry Detection (GC/MSD). The individual compounds were identified by the GC/MSD library (Wiley). A semiquantitative approach was performed taking total peak areas of the Total Ion Current (TIC) chromatograms.

2. Materials and Methods 2.1. S AMPLES

AND SOLVENTS

From August, 2002 to January, 2004 wastewater samples (6 L) were collected monthly from a local swine slaughterhouse in glass bottles, from 8:00 am to 8:00 pm, every 2 h. All the samples were mixed and 1 L sample was collected from the mixture (composite sample). All reagents and solvents were of analytical grade and distilled twice in glass apparatus, when necessary. All glass material was washed with n-hexane, acetone and dichloromethane (DCM) and dried at 120 ◦ C for 4 h. Plastic or rubber materials were not used to avoid contact with samples or solvents.

WASTEWATERS OF SWINE SLAUGHTERHOUSE

2.2. L IQUID–LIQUID

EXTRACTION

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(LLE)

For the LLE experiments three organic solvents were tested: dichloromethane, nhexane and acetone. The dichloromethane shows the following advantages over the other solvents: shorter time of evaporation and better observation of the cut line between the aqueous and organic phases. In this way the LLE with dichloromethane was performed as follows: a) 100 ml of sample was acidified (until pH ∼ 2) and placed in a separation funnel; b) 20 ml of dichloromethane (DCM) were added; c) The sample was shaken for 20 s and allowed to rest for other 20 s. These procedures were repeated three times. The first organic phase was then extracted; d) The steps described in (b) and (c) were repeated twice and three organic phases were obtained; e) The organic phases were mixed in a glass flask. The LLE procedures were performed with sample triplicate. A sample aliquot was acidified samples (pH ∼2) to retain the polar compounds in their neutral forms and, in this way, improve their migration to the organic phase. The acidified samples were submitted to those same LLE extraction procedures. The volume of the LLE organic phases for both neutral and acidified samples was reduced to 10 ml under vacuum. For removal of the residual water, the organic phases were percolated through a glass column packed with 2 g of anhydrous sodium sulfate. The organic phases were dried until constant weight under a gentle flux of ultrapure nitrogen. The average mass of the dry extracts was 0.28 g. 2.3. S OLID- PHASE

EXTRACTION

(SPE)

For the SPE experiments, the XAD-4, and activated carbon stationary phases were tested. The activated carbon shows problems of total column plugging after the percolation of approximately 10 ml of the samples. The XAD-4 resin does not show this problem and, in this way, was chosen for SPE experiments. The XAD˚ was 4 resin (20–50 mesh, active surface: 750 m2 /g, average pore diameter: 50 A) submitted to Soxhlet extraction with n-hexane (4 h), DCM (5 h) and acetone (4 h) for removal of impurities and water traces. The glass wool used in the extractions was also Soxhlet extracted with DCM (4 h). The column overload was avoided using an optimum sample volume/stationary phase mass rate, as described in previous works (Filho et al., 2003, 2004). The SPE procedures were as follows: a) A glass column (15 cm × 11 mm i.d.) was dry packed with the XAD-4 resin (1 g) previously treated as described above; b) The column was washed with 20 ml of DCM; c) 100 ml of the composite sample was percolated through the column;

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d) After sample percolation, the column was vacuum dried by 10 min; e) The analytes were eluted with 15 ml of DCM. This procedure was performed in triplicate. After elimination of residual water by percolation through an anhydrous sodium sulfate column, the organic extracts were dried until constant mass in a fume hood overnight. The average mass of the dry extracts was 0.24 g. Commercial SPE cartridges were not used in this work to avoid the samples contamination with plasticizers compounds like phthalate esters. 2.4. GC/MSD

ANALYSIS

A GC/MSD, Shimadzu QP5050A was used in the instrumental analysis. A 30 m × 0.25 mm fused silica capillary column DB-5 (0.25 μm film thickness) was used for the GC separation using the following oven temperature program: 65 ◦ C heating to 200 ◦ C at 2 ◦ C/min and heating to 300 ◦ C at 5 ◦ C/min (5 min hold). The temperature and injector temperatures were 280 ◦ C and 300 ◦ C, respectively. 3. Results and Discussions Figure 1 shows the Total Ion Current (TIC) chromatograms of the extracts obtained by SPE (A) and LLE of the neutral samples (B). Table I shows the identification (GC/MSD library) of the numbered peaks in Figure 1. No relevant differences were observed between the chromatograms of the LLE extracts for both neutral and acidified samples, indicating a low content of polar compounds in the samples.

Figure 1. TICs of the organic extracts: SPE (A) and LLE (B).


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TABLE I Identification (GC/MSD system library) of the numbered peaks in Figure 1. Relative areas and concentration of the compounds extracted by SPE and LLE #Peak SPE 1 2 3 4 5 6 7 8 9 10 11 LLE 12 13 14 15 16 17 18 19 20

Compound

MQ (%)a

Area %b

Heptenal Benzoic acid Octenal Nonanal Decenal Decadienol Undecenal Octadecanal Dodecanol Dioctylphthalatec Cholesterol

92 93 89 91 91 91 91 89 84 93 80

1.24 1.39 0.55 1.38 13.91 3.22 2.80 1.11 1.02 8.60 20.11

Hexanoic acid Propenyl aziridine Nonanal Methyl butanol Decenal Undecene Dioctylphthalate Ursene Methyl comate

87 80 89 83 94 87 94 68 80

2.65 0.99 2.51 1.08 19.59 3.45 23.20 1.81 1.61

a

Match quality (GC/MSD library). Percent of the total chromatogram area. c Sample contaminant. b

Aldehydes and carboxylic acids (mainly benzoic acid) were the extracted compounds with higher match quality (over 90%) and the discussions were then focused on these compounds. Although the qualitative analysis is the focus of this work, the peak area summations of the TIC chromatograms can be a good approach for quantitative inferences and to analyze the hazardous and economic potentials of slaughterhouse wastewaters. In this way, Table I shows that aldehydes were main class of the compounds extracted by SPE and LLE (20.99% and 22.10% of the total chromatograms areas, respectively). Decenal was the major extracted aldehyde (13.91% for SPE and 19.59 for LLE). The SPE allows the preferential extraction of a range of aldehydes from medium to high molecular weight (C7 to C18 ) while in the LLE, the aldehydes were of medium molecular weight (C9 and C10 ). Such

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aldehydes are possibly products of fermentation of organic acids present in the wastewater (Weinlig, 1973). These results can be explained by the fact that the carbon chain extension exerts a strong influence on the polarity of the compound. Thus, compounds with larger carbon chain are less polar and can be better extracted by SPE, when an apolar stationary phase is employed. On the other hand, compounds with shorter carbon chain are more polar. In this way, acidification of the samples increases the dieletric constant and the more polar compounds remain in the organic fraction. The benzoic acid is a well know raw material for food preservative, plastics, and insect repellents manufacture. This compound has already been identified in leachate samples of sanitary landfill that contain a great amount of food (Filho et al., 2003). In this way, it is not surprising to find such compound in slaughterhouse wastewaters. However, the presence of benzoic acid in any kind of environmental sample is of concern because this compound is very slightly soluble in water and highly soluble in organic matter. High bioaccumulation can be expected for benzoic acid (Filho et al., 2003). Although some authors have highlighted LLE deficiencies (Zhang and Fei, 1995; Borra et al., 1986) this work shows clearly that this method works well for the extraction of polar organic compounds, in this kind of samples. Even considering the difficulties and limitations of LLE, this method is currently widely employed in the enrichment of organic compounds, even in environmental samples. The methods 604, 625 and 8270 of the EPA are based in LLE (Borra et al., 1986).

4. Conclusions For the studied samples, the conclusions are the following: a) The choice of the extraction method (SPE or LLE) is directly related with the chemical characteristics of the target compounds. The LLE works well for the extraction of polar organic compounds, while the SPE with non-polar resins as XAD-4TM is a better choice for the retention of less polar compounds; b) Classes of organic compounds of environmental interest, namely: carboxylic acids aldehydes and alcohols were identified in both SPE and LLE fractions, suggesting that these wastewaters show a high toxicological potential; c) Aldehydes and alcohols with carbon chains ranging from C7 to C18 and C5 to C12 , respectively, are the main contaminants in this kind of sample, possibly resulting from fermentation of other compounds by microorganisms present in the wastewater; d) In spite of the low match quality (80%), the presence of propenyl aziridine (suspect of carcinogenic action) in this kind of sample is of great concern and must be carefully evaluated.


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However, these results are preliminary and must be corroborated by the analysis of standard compounds. The presence of potentially toxic compounds, even after wastewater treatment, justifies the concern about this kind of pollutant and claims for more accurate research for the reduction or elimination of such compounds from slaughterhouse wastewaters. Acknowledgment Authors would like to acknowledge FAPERGS for financial support. References Borra, C., Corcia, A. D., Marchetti, M. and Samperi R.: 1986, ‘Evaluation of graphitized carbon black cartridges for rapid organic trace enrichment from water: Application to priority pollutant phenols’, Anal. Chem. 58, 2048–2052. Buchholz, K. D. and Pawlliszyn, J.: 1993, ‘Determination of phenols by solid-phase microextraction and gas chromatographic analysis’, Environ. Sci. Technol. 27, 2844–2848. Filho, I. N., Mühlen, C., Schossler, P. and Caramão, E. B.: 2003, ‘Identification of some plasticizers compounds in landfill leachate’, Chemosphere 50, 657–663. Filho, I. N., Schossler, P., Freitas, L. S., Melecchi, M. I. S., Vale, M. G. R. and Caramão, E. B.: 2004, ‘Selective extraction of benzoic acid from landfill leachate by solid-phase extraction and ion-exchange chromatography’, J. Chromatogr. A 1027(1/2), 167–170. Filho, I. N., Schossler, P., Freitas, L. S., Melecchi, M. I. S., Vale, M. G. R. and Caramão, E. B.: 2002, ‘Chemical composition of Hibiscus tiliaceus L. flowers: A study of extraction methods’, J. Sep. Sci. 25, 1–6. Fine, D. D., Breidenbach, G. P., Price, T. L. and Hutchins, S. R.: 2003, ‘Quantitation of estrogens in ground water and swine lagoon samples using solid-phase extraction, pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography–negative ion chemical ionization tandem mass spectrometry’, J. Chromatogr. A 1017(1/2), 167–185. Galceran, M. T. and Jáuregui, O.: 1995, ‘Determination of phenols in sea water by liquid chromatography with electrochemical detection after enrichment by using solid-phase extraction cartridges and disks’, Anal. Chim. Acta 304, 75–84. Heberer, T. and Stan, H. J.: 1997, ‘Detection of more than 50 substituted phenols as their t-butyldimethylsilyl derivatives using gas chromatography-mass spectrometry’, Anal. Chim. Acta 341, 21–34. Kolodziej, E. P., Harter, T. and Sedlak, D. L.: 2004, ‘Dairy wastewater, aquaculture and spawning fish as sources of steroid hormones in the aquatic environment’, Environ. Sci. Technol. 38, 6377–6384. Möder, M., Popp, P. and Pawliszyn, J.: 1998, ‘Characterization of water-soluble components of slurries using solid-phase microextraction coupled to liquid chromatography mass spectrometry’, J. Microcol. Sep. 10, 225–234. Polprasert, C., Kemmadamrong, P. and Tran, F. T.: 1992, ‘Anaerobic baffle reactor (ABR) process for treating slaughterhouse wastewater’, Environ. Technol. 13, 857–865. Rajraman, D., Williams, E. L., Layton, A. C., Burns, R. T., Easter, J. P., Daugherty, A. S., Mullen, M. D. and Sayler, G. S.: 2004, ‘Estrogen content of dairy and swine wastes’, Environ. Sci. Technol. 38, 3567–3573. Thassitou, P. K. and Arvanitoyannis, I. S.: 2001, ‘Bioremediation: A novel approach to food waste management’, Trends Food Sci. Technol. 12(5/6), 185–196.

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