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Ecotoxicology and Environmental Safety 61 (2005) 380391 www.elsevier.com/locate/ecoenv

Risk assessment of domestic and industrial efuents unloaded into a freshwater environment
enza, J. Alberdia, M. Tortorellia, Galassi Silvanac W.D. Di Marzioa,b,, M. Sa
a

n en Ecotoxicolog a, Departamento de Ciencias Ba sicas, Universidad Nacional de Luja n, C.C. 221, 6700 Luja n B, Argentina Programa de Investigacio b n Nacional de Investigaciones Cient cas y Te cnicas CONICET Comisio c ` degli Studi di Milano-Bicocca, Dip. di Biotecnologie e Bioscienze, Milano, Italia Universita Received 13 November 2003; received in revised form 14 September 2004; accepted 18 October 2004 Available online 26 January 2005

Abstract n River. It is an important freshwater An ecotoxicologic study was performed to assess the environmental status of the Luja system in the northeast of Buenos Aires Province, Argentina. Surface waters (SWs) and liquids efuents (LEs), before they reached the river, and sediments were assessed via acute toxicity screening using a battery of tests with native species. Additionally, the presence, in each LE and SW sample, of bioaccumulatable compounds was checked by SPME extraction and gas chromatographMS determination. An environmental risk assessment of each LE was carried out via toxic units and assessment factors approach and through extrapolation methods. Hazardous concentrations for each LE were compared with their river efuent concentrations. Ninety-one percent (91%) of the total toxic load of the river was due to 4 of 11 LEs (37%) evaluated. Although SW samples were not toxic, a real environmental risk was found for this freshwater environment. Sediment toxicity was found to be related to the proximity to pipe discharges. Bioaccumulatable compounds were found in SWs and in LEs. Esters of phthalic acids, morpholine, hydroquinone, and nonylphenol were found throughout the river at different sample sites and in different months during the 1-year sampling program. r 2004 Elsevier Inc. All rights reserved.
Keywords: Environmental risk assessment; River; Bioaccumulatable compounds; Acute toxicity; Sediments; Toxic units; Assessment factors; Extrapolation methods

1. Introduction The utility of a battery of biotests is well established for environmental hazard assessment of chemicals and chemical products, and biotests are used routinely to evaluate the toxicity of complex mixtures such as industrial wastewaters (Baun and Nyholm, 1996). A toxicity evaluation is an important parameter in wastewater quality monitoring, as it provides an overview of

Corresponding author. Programa de Investigacio n en Ecotoxico sicas, Universidad Nacional de log a, Departamento de Ciencias Ba n, C.C. 221, 6700 Luja n B, Argentina. Fax: +54 2323 423171x285. Luja E-mail addresses: dimarzio@speedy.com.ar, dimarzio@mail.unlu. edu.ar (W.D. Di Marzio).

the response of test organisms to all the compounds in the wastewater (Wang et al., 2003). n Rivers basin is situated in the northeast of The Luja Buenos Aires Province, Argentina. It emerges from the conuence of the Los Leones and El Durazno streams and runs 130 km through an area of 2300 km2. One million people are connected to this area. It receives the discharge of industrial and sewage efuents from its source to its outlet in the R o de la Plata River. n River runs through a region Furthermore, the Luja with typical agricultural and cattle activity. Its water quality had deteriorated throughout the three decades enz et al., 1996; Alberdi et al., prior to this study (Sa 1996). Sporadic sh die-offs had been observed throughout the river. As a result, the interaction of people with the river was reduced, since almost no recreational

0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.10.002

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activities on the river were possible. The people of the region feel that the river is polluted and that anything originating in its water is contaminated. No one seems to be responsible for this deterioration or for halting it. As in lotic systems, pollution seems to come from upstream; thus, each city and industry minimizes its responsibility. Environmental legislation does not include toxicity evaluation of liquid efuents (LEs) discharged into the n River. To assess the environmental hazard of the Luja discharges into the river, both the physico-chemical (PC) and toxic characteristics should be evaluated (Grothe and Reed-Judkins, 1996). An evaluation of the toxicity of the surface water (SW) and the toxic load that is being incorporated into the system should give us a picture of the rivers status. Studies of wastewater efuents indicate that toxicity tests have a viable role in water quality monitoring and control in rural areas. Sponza (2003) demonstrated that there is no single method that constitutes a comprehensive approach to aquatic life protection. For this reason, a spectrum of toxicity tests containing sensitive microorganisms should be applied to complement each other and chemical analyses. Wang et al. (2003) indicated that bioassays of SW and domestic efuent concentrates have considerable potential as a monitoring tool for organic contaminants in water. Bioassays of aquatic concentrates provide a direct functional response to the overall toxic properties of the mixtures of compounds present in a sample. Bioassays also are a cost-effective alternative to comprehensive chemical analysis. A toxicity identication approach allows connections to be drawn between the toxic effects observed with the compounds detected. This type of information can help one select appropriate treatments or source-reduction methodologies. We proposed a study to identify zones within the river that show acute toxicity to native organisms, to create toxic load proles for the more important industries and domestic efuents discharged, and to measure the environmental risk via a toxic-unit approach and by using extrapolation methods. Bioassays with native aquatic organisms were performed. We also conducted a qualitative survey of the bioaccumulatable compounds n River and in liquids found in water samples of the Luja efuents (LEs) prior to discharge.

follows: Site 1 (Los Leones), an unpolluted stream. Site 2 (El Durazno) is a stream that is inuenced by efuents from the milk industry. Site 3 (Suipacha) is where the n River rises from the conuence of the Los Leones Luja and El Durazno streams. Site 4 is where the river receives efuents from chemical industries and from the city of Mercedes, which had no wastewater treatment plant at the time of this study. At Site 5 (Las Tropas) the river receives efuents from the textile, meat, tannery, and enzyme purication industries. At Site 6 (La Loma) the river receives the efuents of the sewage plant of the n, of textile, chemical, and food industries. city of Luja At each point water samples were taken at 1 m below the surface by a horizontal water bottle made of polyvinyl chloride (Wildco Beta). The river ow measurement was obtained from an automatic ow meter placed downstream of the city of Mercedes by personnel of the Department of Hydrology of Buenos Aires Province. PC parameters of water samples were measured in situ by a water quality checker for simultaneous multiparameter measurement (HORIBA U10). These parameters were dissolved in oxygen concentration, turbidity, salinity, conductivity, temperature, and pH. We also evaluated the chemical oxygen demand (COD) and biochemical oxygen demand (BOD) of each water sample according to APHA-AWWAWPCF (1998). An Ekman dredge was used to take sediment samples. All sediment samples were composites of ve grab samples at each sampling point. They were characterized by pH, ammonia, sulde, and organic matter. Standard methods for these parameters were according to APHA-AWWA-WPCF (1998). 2.1. Toxicity tests Acute toxicity assessment of water samples was performed with the green alga Scenedesmus quadricauda and the following native organisms: the microcrustacean Daphnia spinulata, the amphipod Hyalella curvispina, and the poeciliid Cnesterodon decemmaculatum. Tests were performed following the United States Environmental Protection Agency (US EPA, 1991) and OECD (1981) guidelines for receiving waters and LEs. 2.1.1. Algal toxicity tests The strain S. quadricauda CCAP 276-21 was used in 96-h algal toxicity tests. Algal stock cultures were maintained in modied Detmers nutrient medium (pH 7.5) under controlled conditions in a climatized room at 2271 1C, 3000 lx/cm2 of continuous cool-white uorescent lighting, and 100 excursions/min on a shaker (Walsh, 1988). The inocula were prepared from these cultures to provide an initial cell density of 5 104 cell/ ml in treated and control asks. Test solutions consisted of enriched river water samples with algal medium nutrient solutions. Control cultures were incubated in

2. Materials and methods During 2003, water samples were taken from the n River at 2-month intervals. During surface of the Luja each sampling period samples were taken for 15 days at six sampling points. Fig. 1 shows the area under study and the sampling sites that cover the run of the river from sources to outlet. These sites are characterized as

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Argentine

N
Lujn river
6 5 4
Mercedes Lujn

Rio de la Plata

Buenos Aires city

1 2 3 Suipacha

sampling sites

20 km

n River basin). Numbers are the SW and sediment sampling sites. Fig. 1. Map of the study area (the Luja

the same medium. Control and treated cultures were grown under the same conditions of temperature, photoperiod, and shaking and were carried out in triplicate. Each toxicity test was conducted three times. Cell counts were correlated with absorbance (750 nm) on a Shimadzu MUV 240 spectrophotometer (Walsh, 1988). Denitive protocols for toxicity testing using this enz et al. (1996). species are described in Sa 2.1.2. Microcrustacean toxicity test Microcrustacean tests were performed with D. spinulata, a native Argentine cladoceran zooplankton that is widespread in freshwater environments of Buenos Aires Province. Acute toxicity tests under static conditions were carried out with D. spinulata o24 h old. Test conditions were static or without the renewal of medium, 48 h in duration, and conducted at 2071 1C in the dark. Organisms placed in articial pond water (pH, 7.870.2; total hardness, 95.876 mg CO3Ca/L; conductivity, 475.5746.3 mS/cm; and alkalinity, 189.3714.5 mg CO3Ca/L) served as controls. Denitive protocols for toxicity testing using these species are described in Alberdi et al. (1996). 2.1.3. Amphipod toxicity tests The native amphipod H. curvispina was selected as the test organism. H. curvispina were collected from a

population in an articial pond in a eld of Universidad n. They were placed in laboratory Nacional de Luja tanks of 500 L with a continuous ow of groundwater under natural conditions (water quality: hardness, 126 mg CO3Ca/L; conductivity, 800 mS/cm; pH, 8.26; alkalinity, 412 mg CO3Ca/L). Ten-day-old individuals were chosen because of there relative sensitivity [EC50, 96 h in 0.31 mg potassium dichromate/L (condence interval of 0.120.53 at Po0:05)] and size appropriate for test handling (Simionato et al., 1997). An age control was made using the equation T len 0:71 0:037t; where Tlen is the total length in millimeters and t is the time in days. The dilution water (DW) proposed for Borgmann (1996) was used in the experiments because it includes the bromide ion essential to Hyalella sp. The water quality for the DW was hardness, 82 mg CO3Ca/ L; conductivity, 500 mS/cm; pH, 8.3; and alkalinity, 212 mg CO3Ca/L. Other conditions were a temperature of 2171 1C and a 12-h dark/light photoperiod. SWs and LEs were assayed with 10-day-old and adult organisms. A 96-h static test was carried out in darkness with 40 amphipods of each age per sample. Denitive protocols are described in Di Marzio et al. (1999). 2.1.4. Fish toxicity tests Fish toxicity tests were performed with species C. decemmaculatum. It is a native member of the Family

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Poeciliidae that is widespread in the temperate water region throughout Buenos Aires Province. Its regional distribution reaches larger rivers in the Patagonia area, n River, which has colder waters. such as the Neuque Ninety-six-hour semistatic acute tests were conducted. Fish captured in an articial freshwater pond were acclimated in groundwater (pH, 7.9; conductivity, 400 mS/cm; dissolved oxygen (DO) concentration. 8.20 mg/L; hardness, 98 mg CaCO3/L) for 20 days under environmental test conditions. During this period they were fed daily with natural (algae+Daphnia sp.) and articial food, not exceeding 5% of their total individual weight. Tests were made using groundwater in glass aquaria at 2071 1C and a photoperiod of 12 h light/12 h dark. The nal volume was 2 L in order to get a loading rate of 0.5 g of sh/L. Samples and the control were quadruplicated. Eighty individuals of 1 month in age were used for each sample (40 males/40 females). Each aquarium was aerated to ensure a DO concentration greater than 5 mg/L. The solutions were renewed daily. Every 24 h the mortality was recorded. The denitive protocol is described in Di Marzio et al. (1996). 2.2. Sediment test Sediment toxicity was assessed with H. curvispina in a 10-day test. Sediment test conditions are summarized as follows: river sediment/control sediment; homogenized 50 g/250-mL DW; eight replicates; 1015-day-old H. curvispina. The overlying water was aerated and cleaned every 24 h. Mortality and growth were recorded at the end of exposure. Sediment of an unpolluted stream containing less than 3% organic matter was used as the control in the experiments. Sediment organic matter was measured according to APHA-AWWA-WPCF (1998). Further details of the protocol used are in US EPA (1998) and Di Marzio et al. (1999). 2.3. Analytical determination of bioaccumulatable compounds in surface water and liquid efuents Grab samples for analytical determinations were collected at each sampling point or efuent. They were refrigerated at 4 1C and kept in glass containers that were rinsed with tap water followed by high-purity water prior to the addition of the samples. They were ltered across a 0.45-m-ber glass lter and pH corrected at 7.5 according to Gert Jan de Maagd (2000). Samples were placed in 4-ml vials with a Teonfaced septum. A manual solid-phase microextraction holder was used with unbonded bers of polydimethylsiloxane (PDMS) at a stationary phase of 100- and 7-mm lm thickness and one of 85 mm of polyacrylate (PAC) (Supelco, Bellefonte, PA, USA). These were conditioned as follows: PDMS 100 at 250 1C for 1 h, PDMS 7 at

320 1C for 3 h, and PAC 85 at a 300 1C gas chromatograph (GC) injector temperature for 2 h. The bers were immersed into the sample, which was heated to 25 1C and agitated with a magnetic stirring bar at 560 rpm. The adsorption time was 1 h. The bers were then immediately inserted into the GC injector and the analysis was carried out. The desorption time was 4 min and the desorption temperature was set at 280 1C. A Shimadzu gas chromatograph 17A V 1.3 model with mass spectrometer QP 5050A and an MS Workstation Class 5000 (Shimadzu Corp., 1999) were used. Experimental conditions included PTE-5 fused-silica capillary column 30 m 0.25 mm 0.25-mm lm thickness (Supelco); a linear velocity of carrier Helio, 36.2 cm/s, splitless, with sampling time of 4 min and total ow of 11.7 mL/min; a temperature program of 100 1C for 2 min heated to a nal temperature of 280 1C at 10 1C/min and held at this temperature for 10 min; an injector temperature of 280 1C; and a capillary interface temperature of 280 1C. The mass spectrometer detector scan mode ranged from 50 to 350m/z. Computer searching of the acquired mass spectral data against libraries of reference mass spectra was used. The NIST (1998) and Wiley Library (1995) libraries of mass spectra were used. The NIST database reference mass spectra was of 142,341 compounds with 99% of molecular formula associated. Wiley had 229,119 compounds with more than 40% of the molecular structure associated. PBM o probability-based matching algorithm for calculating the similarity index (SI) between spectra was used. The SI quantitatively expresses the difference between the spectrum of an unknown sample and a spectrum registered in a library. In general, the differences between the respective intensities of the spectral peaks at a certain mass number are determined and the smaller those differences are, the greater the degree of similarity. The following equation was used: 2 3   6 Ium=z Itm=z 7 6 7 7 100: SI 6 61 m 7 =z P 4 5 Ium=z Itm=z
m=z

where Ium=z is the relative spectral intensity for mass number m=z of the mass spectrum of an unknown sample. Itm=z is the relative spectral intensity for mass number m=z of the mass spectrum registered in a library. If the patterns of the two mass spectra are identical the SI is 100, and, conversely, if they are completely different the SI will be 0. We take into account compounds with a SI greater than 70. The coefcient of partition octanol/water log P or log Kow was obtained for each compound by using the C log P program. Bioconcentration factors (BCF) were determined by

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Connells equation (Connell, 1990) as follows: log BCF 6:9 103 log K ow 4 1:85 101 log K ow 3 1:55log K ow 2 4:18 log K ow 4:79: 2.4. Physico-chemical characterization of liquid efuents The following parameters were used according to APHA-AWWA-WPCF (1998): COD, BOD, pH, alkalinity, sulde, sulfate, conductivity, free chlorine, ammonium, salinity, chloride, reactive phosphorous, nitrate, hardness, and residual solids after 10 and 120 min of settling. The majority of these are included in the ofcial regulations for LEs discharged into SW. 2.5. Statistical analysis A one-way statistical analysis of variance (ANOVA) (Po0:05) in conjunction with Dunnets test was used to compare responses with the control using the computer program Toxstat V 3.5 (WEST, Inc., 1996). Regression analysis in conjunction with ANOVA was made between mortality and organic matter data from sediment toxicity tests using the program Statistica for Windows.

LC50 or EC50 and their ducial limits at 95% were determined by a probit method using the US EPA program V5 1.4 (US EPA, 1991; Berthouex and Brown, 1994; Sparks, 2000). Efuent samples were analyzed by principal component analysis (PCA) to explore the degree of correlation in the toxicity tests and PC parameters. Two matrices were constructed, one using the PC parameters alone and the second considering the PC and the toxicity indices for all species assayed. The 11 LEs were the horizontal rows of the PCA data matrix. The PCA was performed using StatGraphics Plus software V 2.1.

3. Results 3.1. Water quality The range of in situ physicochemical parameters measured for 1 year is shown in Table 1. The arithmetic n River is 1.159 m3/s with a mean of the ow of the Luja standard deviation of 4.513 and a coefcient of variation of 389%. The COD and BOD values are plotted with DO concentrations in Fig. 2.

Table 1 n River The range of physico-chemical parameters measured in 150 water samples from the Luja Parameter Total ow (m /s) Dissolved oxygen (mg/L) Conductivity (mS/cm) Salinity (%)[AU: What unit intended here?] Turbidity (nephelometric units) Temperature (1C) PH
3

Summer 07.44 018.95 159011790 0.013.00 90 to 1000 1827.5 8.511.4

Autumn 1.312.3 1.946.76 12006500 0.011.90 50700 1219 89.2

Winter 110.4 2.597.34 10205000 0.011.79 50400 813 7.69

Spring 0.2314.69 0.5313.11 6005500 0.012.5 120 to 41000 1024 810.9

Fig. 2. The DO concentration, BOD, and COD averages of all sampling sites. Values are in mg/L.

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3.2. Surface water toxicity tests Nontoxicity effects were observed for the algal tests in the majority of the samples. On the contrary, a stimulation of algal growth ranging between 20% and 70% with respect to the control was observed for all samples taken in winter and spring. This could be related to the accumulation of nutrients or their improved bioavailability, which occurs during this period in aquatic environments (Margalef, 1983). Samples taken in the summer close to pipe discharges of chemical industries, in the cities of Mercedes and n, showed toxicity of about 60% inhibition, Luja conductivity values of 12,000 mS/cm, and a pH of 11 (Fig. 3). In the majority of experiments with D. spinulata, immobility was not observed in the organisms exposed or was lower than 15%; only in ve experiments was the immobility 100% after 48 h of exposure. Nontoxicity effects were observed in D. spinulata in the majority of samples, excepts in Sites 46 in the summer samples and Site 4 in the winter samples (signicant differences, ANOVADunnettest at Po0:05). These results coincided when high values of conductivity and salinity and low values of DO were recorded. Fish mortality was not recorded in any surface samples. 3.3. Efuent toxicity tests and physico-chemical parameters Physicochemical data and the results of toxicity tests for each LE are reported in Table 2. Fig. 4 shows the PCA results taking the PC data of each efuent. Three groups were obtained: Group a, Mme, MCMe, and MS formed for domestic liquids; Group b, TL1, TL2, TMCMe, FS, Cme, ML, and CL
200 Stimulation growth % 180

(this joined mainly chemical efuents or mixed with organics); and Group c, with only BL as a member (M, municipal; T, textile; C, chemical; F, food; B, biotech n; Me, city of Mercedes; S, city of nology; L, city of Luja Suipacha.; 1, component 1; 2, component 2). Group b was discriminated in PCA analysis according to specic PC values regarding pH, conductivity, sulfate, ammonium, BOD, and COD variables. However, PCA performed with all parameters, PC plus the toxicity index (Figs. 5 and 6), denes the following groups: Group a, MCMe, Mme, and MS; Group b, FS, TMCMe, and Cme; Group c, TL2, TL1, ML, and CL; and Group d, BL. The latter is the more toxic efuent assayed and between Groups a, b, and c it is possible to say that there are differences in both PC and toxicity values, dening the rank of toxicity as aoboc. 3.4. Sediment toxicity tests Sediment samples were toxic in sites close to industrial and municipal discharges (Sites 46). There was a relationship between organic matter concentration and individual mortality (Fig. 7). Sediment pH values ranged from 6 to 7.6; sulde concentrations were not detectable; ionized ammonia (NH+ 4 ) was 50600 mg/L. Higher values of NH+ were found next to higher organic 4 matter contents (OMC) and ranged between 350 and 600 mg/L for sampling Sites 46. 3.5. Bioaccumulatable compounds Table 3 indicates a qualitative screening of the bioaccumulatable compounds and suspected hormonal disruptors found.

sampling points order

160 140 120 100 80 60 40 20 -5 5 15 25
1 2 3 4 5 6

winter

spring

summer

autumn

Inhibition growth %
35 45

algal growth
55

Time relative scale (days)

n River samples. Control growth at 96 h is considered to be 100%. Growth lesser or greater than that of Fig. 3. Algae growth responses in the Luja the control is considered inhibition or stimulation, respectively. Growth o80 or 4120% was signicant at Po0:05 (ANOVADunnets test).

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386 W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391 Table 2 Physico-chemicals parameters of assayed efuents, all values are expressed in mg/L Parameter COD BOD SS 10 min SS 120 min pH Conductivity Salinity Free chlorine Sulde Ammonium Nitrate Reactive phosphorous Sulfate Alkalinity Chloride Hardness Algae Cladocerans Amphipod adult Amphipod young Fish TL 548 300 0 0 11.42 2900 2 0 0.13 5.4 4.8 0.14 103.7 170 15 0 17.32 16.52 17.32 3 6 TL 190 40 0 0 8.26 1200 1 0.06 0.08 5.4 3.5 1.49 147 65 35 17.1 100 100 70 4.24 100 BL 1754 890 0 0.1 7.6 8000 5.3 0.05 13.9 1214.2 11.4 54.13 2647.2 135 20 58.5 35 4.95 4.24 1.97 3 ML 468 140 0.6 0.9 7.82 1480 0.9 0.09 0.04 28.4 1.1 12.33 121 60 25 19.5 4.25 71 28.98 50.86 71 CL 240 50 0 0 8.1 12,000 5 0.04 0.2 100 3.5 0.1 40 400 2,000 130 71 17.32 70.7 17.32 35.35 T+M+CM 792 270 0 0 7.12 631 0.32 0 0.072 12.5 13.2 0.81 86 600 200 258.1 5.61 NT NT NT NT MM 216 110 4 4 6.84 210 0.09 0.06 0.052 12.9 10.5 6.53 90 580 35 325 100 17.32 30.33 11.77 2.22 M+CM 36 10 0 0.1 7.15 152 0.06 0.05 0.004 0.20 32.9 2.09 112 510 20 334.5 100 70.71 55.82 43.75 6.86 CM 71 20 0.25 0.25 7.91 158 0.07 0.09 0.009 0.25 13.7 0.68 25 470 22 152.9 4.3 NT NT NT NT MS 159 54 0 0 7.20 251 0.11 0 0.004 2.9 14.7 3.21 79 630 38 372.8 100 NT NT NT 71 FS 250 90 0 0 7.09 184 0.08 0.06 0.032 13.6 14.9 8.88 120 700 20 219.8 6.89 NT NT NT NT

Conductivity is expressed in mS/cm, salinity in %, acute toxicity endpoints as efuent percentage (%). SS are solids at 10 and 120 min of n; M, sedimentation. NT, not acute toxicity; T, textile; B, biotechnology; C, chemical; M, municipal; F, food efuents. Superscripts: L, city of Luja city of Mercedes; S, city of Suipacha.

2.2 MMe MCMe MS 1.2

2.2 MCMe MS MMe BL 0.2 FS TMCMe CMe TL2 TL1 ML 2 4 Component 1 6 8

Component 2 (17 %)

1.2 Component 2

0.2 BL TL1 -0.8 ML -1.8 -7 -5 -3 -1 TL2 TMCMe

FS CMe

-0.8

CL 1 3

-1.8 -2

CL 0

Component 1 (62 %)
Fig. 4. Two-dimensional scattergrams corresponding to the distribution of the PC parameters of each efuent. M, municipal; T, textile; C, n; Me, city of chemical; F, food; B, biotechnology; L, city of Luja Mercedes; and S, city of Suipacha. The two components explained 75% of total variance. Fig. 5. Two-dimensional scattergram corresponding to the distribution of the PC parameters of each efuent and the toxicity indices for all assayed species. M, municipal; T, textile; C, chemical; F, food; B, n; Me, city of Mercedes; and S, city of biotechnology; L, city of Luja Suipacha. The two components explained 75% of total variance.

4. Discussion Acute toxicity of SWs was infrequent for animals (sh and invertebrates) and related to special environmental conditions, such as samples with heavy conductivity and salinity. On the other hand, algal growth stimulation was observed in many of the assayed samples. It is possible that this stimulation is related to phosphorous (P) bioavailability, since it is a key factor that denes the

algal growth. Phosphorous is usually the limiting nutrient for algal growth in freshwater environments. In the regional legislation, before discharge an efuent must meet a limit concentration of total phosphorous (TP) of less than 10 mg/L. But TP alone is not the best criterion for eutrophication control measures (Ekholm and Krogerus, 1998). Fig. 3 shows that a stimulation of growth, with respect to the control, was largely observed, despite all controls and river samples being

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spiked with the same PO4-P concentrations. Algae are able to use several sources of P, such as reactive and nonreactive phosphorous and particulate P. In addition, increased bioavailability due to strongly alkaline conditions likely occurs during toxicity testing carried out with algae (Persson, 1990). Also, algal toxicity may be correlated signicantly with some components of SWs. Graff et al. (2003) found that parameters like COD, hardness, conductivity, total suspended solids, and dissolved organic carbon were signicant factors affecting toxicity from river waters. The importance of using a battery of species in the LEs evaluation is remarked upon in this study. PCA analysis was a good tool to express how PC parameters and TI can together give a complete understanding of each efuent. LEs could be grouped regarding their organic, industrial, or mixed nature based on their toxicity. Results from toxicity tests performed with aquatic invertebrates seem to be explained by some PC variables like COD or ammonium (Fig. 7). Fish and
2.2 NO3 Algae Fish 1.2 Component 2 ALK AnfA daph PO4 NH4 COD AnfC Cond

0.2

-0.8

-1.8 -2.1 -0.1 1.9 3.9 5.9 7.9 Component 1

Fig. 6. Biplot of two-dimensional scattergram corresponding to the distribution of PC parameters of each efuent and the toxicity indices for all assayed species. The partial correlation for each variable is shown. The two components explained 75% of total variance.
120

algae were not linked with any efuent-based PC measured. This would conrm the idea that an efuent might be within permitted PC limits yet still be toxic. The discharge of sewage into SWs represents a major source of pollutants globally (Walker et al., 1996). Wang et al. (2003) showed that municipal sewage treatment plants were contributing to the total biological toxicity emission of receiving water bodies. They remarked that the preservation of the ecology of rivers and lakes requires regulations that consider ecotoxicity. Domestic wastes are discharged mainly into sewage systems. Industrial wastes are discharged either into sewage systems or directly into SWs. Several treatments may be applied to these efuents to improve their quality before they are discharged. The more popular wastewater treatment plant consists of a suspended and aerated culture of microorganisms forming microstructures called ocs or activated sludges. Others include oxidation lagoons, biolters, and bed reactors. All of these treatments improve the water quality of the efuents in terms of COD, BOD, and suspended solids removal; further treatments will remove mainly phosphate and nitrate. Before efuents are discharged, they must meet the threshold values for these parameters as dened on environmental legislation. The aim of this regulation is to prevent oxygen depletion and eutrophication of water bodies. But it does not include limits on toxicity and the toxic load of efuents into the environment. Buenos Aires Province has no regulatory protocol to measure either acute or chronic ecotoxicity. In this work, we have shown that LEs being discharged n River are ecotoxic even when they meet into the Luja the legal requirements for an authorized discharge. The toxic unit (TU) measures the strength of each LE assessed. It is expressed as a fraction or proportion of its lethal or effective threshold concentration: actual percentage of the (LE)/L(E)C50. If this number is

100

M = 3.929 * OM - 10.85 Fisher's value = 36.06 p < 0.05

Mercedes S4 La Loma S6

80 Mortality M (%)

R = 0.905

Las Tropas S5

60

40

20 Los Leones S1 Suipacha S3 0 0 4 8 12 16 20 24 28 32 El durazno S2

Regression 95% confid.

Organic matter OM (%)

Fig. 7. Regression analysis (signicant at Po0:05) between OMC in sediments and mortality recorded for the amphipod H. curvispina. The F value was 14.11 at Po0:05; the condence interval of the slopes was 1.096.61 at Po0:05: S1 is Site 1, S2 is Site 2, etc.

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388 W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391 Table 3 Organic compounds found in surface water and liquid efuents Log Kow 4.51 2 NC 0.59 4.79 8.23 NC 3.53 5.29 3.80 4.98 3.42 5.19 1.08 4.79 3.02 Log BCF 3.35 1.26 NC 2.83 3.33 3.9 NC 2.28 4.07 2.58 3.81 2.17 4.32 NC 3.99 1.78 Compound 1,2-Dichloronaphtalene Phenol, 4-methyl Phenol, 2-chloro-5-methyl Hydroquinonea Phenanthrene p-Nonylphenola Nonylphenol diethoxylate 4-Heptanol Phenol, 4,40 methylethylidene,bis1-Butanol, 4-butoxy 3,3-Dimethyl, 14-heptanol 1,2-Benzenedicarboxylic acid di-n-Butyl phthalatea Morpholinea Diethyl phthalatea Furanone, 5-ethyldihydro-5-methyl Efuents TMCMe ML, CL CL TL1, TL2, ML Not found MS, FS, ML, TMCMe ML TL1, TL2 Not found TL1, TL2 Not found MS, ML, Cme Ml, CL, MS, MMe TL1, TL2, ML ML, TL1, TL2 Not found Sample Mercedes (4) La Loma (6) La Loma (6) La Loma (6), Las Tropas (5) La Loma (6) La Loma (6) Las Tropas (6) Las Tropas (6) Las Tropas (5) Las Tropas (5) Las Tropas (5) La Loma (6) Mercedes (4) Mercedes (4) Mercedes (4), La Loma (6) Las Tropas (5)

BCF, bioconcentration factor; Kow, coefcient of partition for octanol/water; NC, not calculated; M, municipal; T, textile; C, chemical; F, food; L, n; Me, city of Mercedes; S, city of Suipacha; 1, component 1; 2, component 2. In parentheses are the numbers of each site sample (refer city of Luja Fig. 1). a Detected throughout the sampling year.

greater than 1, more than half of a group of aquatic organisms will be killed or affected by the LE. TUs were used to calculate the toxic load of each efuent as ToxLoad UT FLE, where FLE is the ow of LE (see Table 4). A frequent approach to dening an environmental threshold of LE to avoid acute effects on native organisms is to consider that no effect occurs when the percentage of LE dilution in the river water is lower than 0.3 TUs (Grothe and Reed-Judkins, 1996). The toxicity index for the more sensitive species is taken and is compared with the expected LE dilution, taking into account its ow and the mean ow or some critical ow value for the river and to obtain a river concentration value (RCV). [The RCV could also be termed the predicted environmental concentration for each efuent.] The RCV in our study was calculated according to the following equation: RCV F LE =F LE F RM 100; where FLE is the ow of LE and FRM is the mean ow for the river. Table 4 shows the RCV and TU for each LE evaluated. Six of 11 of the LEs studied represent a hazard in terms of acute toxicity to aquatic organisms. The assessment factor (AF) may also be used to extrapolate from the lowest chronic no observed effect concentration (NOEC) to the eld situation, from short to long exposure time, and from the concentration with acute effects to the NOEC. For each extrapolation step a factor of 10 is suggested. Consequently, if a data set contains only one LC(E)50 for one species, the environmental concern level is estimated from LC(E)50/

10 10 10 (OECD, 1995). We use also this approach taking an AF of 100 since our scope was to dene an acute environmental threshold. We have taken for each LE the more sensitive species and divided it by 100 as the acute AF. Obtained data are shown in Table 3; according to the AF approach, we nd environmental risk only for one efuent, a chemical industry discharged near the city of Mercedes. Another approach to dening a hazardous concentration (HC) for the substances or LEs is through extrapolation methods. A common feature of these methods is that they use the toxicity data for all tested species instead of those for more sensitive species and derive a maximum tolerable concentration or a HC (OECD, 1995). The variability in the sensitivity of the test species is assumed to be representative of the variability of all species in the aquatic community. Table 4 indicates these HC values according to the models of Wagner and Lkke (1991) and Aldenberg and Slob (1993), both recommended methods by OECD (1995). In this case, all LEs will be hazardous for the aquatic n River, at organisms present in the waters of the Luja least with respect to their potential to exert acute toxicity. Although the SWs have not produced an acute toxicity it is probable that a real environmental risk exists in this freshwater environment. However, from an empirical point of view, we must also remark that the three approaches overestimated the environmental risk for the assayed LEs in the order TUoAFoHC. The breach between the LEs and SW toxicity could be explained by chelation and the distribution process. It is

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W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391 Table 4 Environmental risk assessment according to toxic unit and extrapolation methods for each assayed efuent Toxic load ML TL1 TL2 CL TMCMe FS MS MCMe MMe BL CMe 1180 1000 710 690 81.99 59.02 56.40 53.35 47.54 40 3.41 RCV in TU 2.52 2.24 1.58 1.29 0.21 1.27 0.12 0.13 1.11 0.10 0.01 RA 1 R R R R NR R NR NR R NR NR RCV in % 10.70 6.71 6.71 22.34 1.18 8.75 8.75 0.87 2.45 0.19 0.04 HC 5% AS 0.02 0.06 0.01 0.15 0.01 0.02 2.29 0.03 0.00 0.01 0.01 RA 2 R R R R R R R R R R R HC 5% WL 0.02 0.07 0.01 0.18 0.02 0.03 2.38 0.04 0.01 0.01 0.01 AF 0.042 0.03 0.042 0.17 0.056 0.069 0.71 0.069 0.022 0.02 0.04 RA 3 R R R R R R R R R R NR 389

R, acute risk; NR, no acute risk; RCV, river concentration value, obtained with mean river ow; RA 1, risk assessment for toxic unit (TU), if RCV40.3 risk; RA 2, risk assessment for extrapolation methods, if RCV in % 4HC (hazardous concentration) risk for 5% of species; AS, Aldenberg and Slob (1993); WL, Wagner and Lkke (1991); RA 3, risk assessment for assessment factor (AF) (OECD, 1995), if RCV in % 4AF risk. Toxic load is UT FLE.

known that humic and fulvic acids reduce the bioavailability of heavy metals. Organic compounds could reach bottom sediments by being adsorbed by suspended solids. Biodegradation of these compounds is also possible, but the results showed a permanent decit of oxygen in all sampling sites, indicating an oxygenstressed environment with a COD:BOD:DO relationship as high as 350:25:5 (see Fig. 2). In that way, aerobic processes could be reduced, resulting in an increasing environmental half-life for these organic chemicals. In this scenario, one would expect to nd toxicity in the sediments. This is related to interstitial water concentrations of chemicals and is a function of the concentrations sorbed to sediment organic carbon under equilibrium conditions. It has been reported that the toxicity of sediments is largely due to interstitial water (Swartz and Di Toro, 1997). Organic matter plays an important part in determining chemical bioavailability. Higher OMC increases chemical sorption, reducing the nal concentration in interstitial water (Power and Chapman, 1992; Swartz and Di Toro, 1997). The positive relationship between OMC and sediment toxicity on H. curvispina would be related to higher NH+ 4 concentrations, which were measured in sampling points with lesser DO concentrations. High values of NH+ 4 concentrations could be able to mask chronic effects due to other organic and inorganic chemicals. In these environments nitrication processes could be reduced. The results obtained with sediment toxicity tests can be an important tool for making decisions regarding the extent of remedial action needed for the Mercedes, Las Tropas, and La Loma contaminated sites. Lethal effects on sh were not found in any of the water samples assayed. Toxicity tests with different aquatic organisms indicated that zones with potential acute toxicity were near the points of discharge of LEs

n. This acute from the cities of Mercedes and Luja toxicity could be related to high conductivity, salinity, and pH and low DO concentrations. This study indicated that toxicity and possible eutrophication problems were mainly located in the lower river due to the discharge of both industrial and sewage efuents n. from the cities of Mercedes and Luja As was shown in Table 3, potential bioaccumulatable compounds were found. Ester of phthalic acids, morpholine, hydroquinone, and nonylphenol (NP) were found in all samples taken at Sites 46 and in many of the tested efuents. NP, an estrogen agonist, is the ultimate degradation product of nonylphenol polyethoxylate nonionic surfactants (NPE) and has been reported to be an endocrine disruptor. Long-chain NPE also breaks down into shorter-chain NPEs, with toxicity inversely related to ethoxyl chain length. It has been suggested that disruption of endocrine balance due to exposure to NP might compromise the reproductive tness and survival of shes (Purdom et al., 1994). NP reduced fecundity, disrupted testosterone metabolism, and increased production of larval storage protein in aquatic invertebrates (DeFur et al., 1999). Di-n-butyl phthalate (DBP) is another suspected compound that shows a weak estrogenic activity and can adversely affect the endocrine systems of animals (McNeal et al., 2000). DBP is used mainly as a specialty plasticizer for nitrocellulose polyvinyl acetate and polyvinyl chloride. It also is used in coatings, adhesives, lacquers, and paper coating (WHO (World Health Organization), 1997). DBP is biodegradable in natural SWs with an estimated half-life in the range of 114 days, although longer in anaerobic or anoxic conditions. The log Kow values of DBP and diethyl phthalate indicate that they potentially could be bioaccumulated by aquatic organisms. However, their accumulation is

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390 W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391

inuenced by the capability of an organism to metabolize them. Several authors have shown the ability of sh to metabolize DBP (WHO (World Health Organization), 1997). The laboratory BCF for Pimephales promelas ranged between 570 and 590 and for Cyprinodon variegatus to 11.7. The greater BCF recorded was 1400 for the amphipod Gammarus pseudolimnaeus (WHO (World Health Organization), 1996). The theoretical BCF for DBP from Table 2 is 20,890, which would conrm the aforementioned. Hydroquinone has a multitude of uses; it is used as a developer in black and white photography and related graphic arts such as lithography, rotogravure, and medical and industrial X-ray lms. It is also widely used in the manufacture of rubber antioxidants and antiozonants, monomer inhibitors, and food antioxidants to prevent deterioration in many oxidizable products. With a log Kow of 0.59 it can be considered that hydroquinone does not bioaccumulate. The log BCF found in the literature for aquatic organisms is between 1.60 and 2.94. Biodegradation of hydroquinone is closely related to many variables, such as pH, temperature, and whether conditions are aerobic or anaerobic. Its BOD/COD relationship is 0.53, indicating that it is readily biodegradable under aerobic conditions. It is a carcinogenic, neurotoxic, and nephrotoxic chemical. Morpholine is an extremely versatile chemical. It is most important as a chemical intermediate product, such as for the production of agrochemicals and performance polymers (WHO (World Health Organization), 1996). Morpholine can undergo a variety of reactions. It behaves chemically as a secondary amine. Under environmental and physiological conditions, the proven animal carcinogen N-nitrosomorpholine is formed by the reaction of solutions of nitrite or gaseous nitrogen oxides with dilute solutions of morpholine. Among the aquatic organisms tested, cyanobacteria and unicellular green algae appear to be the most sensitive taxa, with toxicity threshold values of 1.74.1 mg/L (WHO (World Health Organization), 1996). Because of its log Kow (1.08) it is not a bioaccumulatable chemical. Morpholine is biodegraded in aerobic conditions by a restricted range of microbes, which have a low growth rate and a lag period greater than 8 days. The presence of morpholine in the environment indicates the lack of treatment of industrial wastewater or bad operational conditions in an activated sludge plant.

incorporated into the river. The environmental risk for these efuents exists, but it was overestimated, although different risk evaluation methods were used. Even if SWs did not show acute toxicity, it is possible that aquatic organisms are bioaccumulating organic compounds. Besides, they are exposed to hormonally disrupting chemicals. The environmental half-life of these organic compounds could be increased since the permanent lack of DO slows all aerobic biodegradation process. It is also possible that the sediment remains a natural reservoir of chemicals. Although we recorded sediment toxicity, this would be an overestimate due to a high interstitial ammonium concentration.

Acknowledgments The authors thank Jean Franc - ois Masfaraud (ESE, de Metz, Metz, France) for his assistance in Universite PCA analysis. The research was supported by grants sicas and from the Departamento de Ciencias Ba n, Argentina, SECYT of Universidad Nacional de Luja and from the Agregadur a Cient ca de la Ambasciata dItalia en Buenos Aires, Argentina.

References
enz, M.E., Tortorelli, M.C., 1996. Alberdi, J.L., Di Marzio, W.D., Sa n de la toxicidad aguda con Daphnia Protocolo para evaluacio spinulata (Cladocera). Resu menes X Congreso de Toxicolog a, Buenos Aires. Aldenberg, T., Slob, W., 1993. Condence limits for hazardous concentrations based on logistically distributed NOEC toxicity data. Ecotoxicol. Environ. Safety 25, 4863. APHA-AWWA-WPCF, 1998. Method 2540. In: Franson, M. (Ed.), Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Association, American Water Works Association, Water Environment Federation, Washington, DC. Baun, A., Nyholm, N., 1996. Monitoring pesticides in surface water using bioassays on XAD-2 preconcentrated samples. Water Sci. Technol. 33 (6), 339347. Berthouex, P.M., Brown, L.C., 1994. Statictics for Environmental Engineers. Lewis, Boca Raton, FL, USA, p. 335. Borgmann, U., 1996. Systematic analysis of aqueous ion requirements of Hyalella azteca: a standard articial medium including the essential bromide ion. Arch. Environ. Contam. Toxicol. 30, 356363. Connell, D.W., 1990. Bioaccumulation of xenobiotic compounds. CRC Press, FL, USA, 219p. DeFur, P.L., Crane, M., Ingersoll, C., Tatterseld, L., 1999. Endocrine Disruption in Invertebrates: Endocrinology, Testing, and Assessment. SETAC, Pensacola, FL, USA. enz, M.E., Alberdi, J.L., Tortorelli, M.C., 1996. Di Marzio, W.D., Sa Protocolo para evaluar la toxicidad aguda sobre Cnesterodon decemmaculatus (Pisces, Poeciliidae). X Congreso de Toxicolog a, Buenos Aires. enz, M.E., Alberdi, J.L., Tortorelli, M.C., 1999. Di Marzio, W.D., Sa Assessment of the toxicity of stabilized sludges using Hyalella curvispina (Amphipod) bioassays. Bull. Environ. Contam. Toxicol. 63, 654659.

5. Conclusions A eld ecotoxicological study was performed to dene n River plain. The the environmental status of the Luja toxic load was quantied, and four industries of the 11 evaluated represent 91% of the total toxic load

ARTICLE IN PRESS
W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391 Ekholm, P., Krogerus, K., 1998. Bioavailavility of phosphorus in puried municipal wastewater. Water Res. 32, 343351. Gert Jan de Maagd, P., 2000. Bioaccumulation tests applied in the whole efuent assessment: a review. Environ. Toxicol. Chem. 19, 2535. Graff, L., Isnard, P., Cellier, P., Bastide, J., Cambon, J.P., Narbonne, J.F., Budzinski, H., Vasseur, P., 2003. Toxicity of chemicals to microalgae in river and in standard waters. Environ. Toxicol. Chem. 22, 13681379. Grothe, K.D., Reed-Judkins, D. (Eds.), 1996. Whole Efuent Toxicity Testing: An Evaluation of Methods and Prediction of Receiving System Impacts. SETAC, Pensacola, FL, USA, p. 340p. Margalef, R., 1983. Limnolog a. Omega Ediciones, Barcelona. McNeal, T.P., Biles, J.E., Begley, T.H., Craun, J.C., Hopper, M.L., Sack, C.A., 2000. Determination of suspected endocrine disruptors in foods and food packaging. In: Keith, L.H., Jones Lepp, T.L., Needham, L.L. (Eds.), Analysis of Environmental Endocrine Disruptors, American Chemical Society Symposium Series 747, Washington, DC, p. 173. NIST, 1998. National Institute of Standars and Technology/Environmental Protection Agency/National Institutes of Health Mass Spectral Database. NIST, Gaithersburg, MD, USA. OECD, 1981. Guidelines for Testing of Chemicals. OECD, Paris. OECD, 1995. Guidance Document for Aquatic Effects Assessment. OECD Publ. No. 92. OECD, Paris. Persson, P., 1990. Utilization of phosphorus in suspended particulate matter as tested by algal bioassay. Verh. Internat. Verein. Limnol. 24, 242246. Power, E., Chapman, P., 1992. Assessing sediment quality. In: Burton, Jr., G.A. (Ed.), Sediment Toxicity Assessment. Lewis, Boca Raton, FL, USA. Purdom, C.E., Hardiman, P.A., Bye, V.J., Eno, N.C., Tyler, C.R., Sumpter, J.P., 1994. Estrogenic effects of efuents from sewage treatment works. Chem. Ecol. 8, 275285. enz, M.E., Alberdi, J.L., Di Marzio, W.D., Tortorelli, M.C., 1996. Sa Protocolo estandarizado de ensayos de toxicidad con las algas de agua dulce Scenedesmus acutus y Scenedesmus quadricauda. X Congreso de Toxicolog a, Buenos Aires. Shimadzu Corp., 1999. MS Workstation Class 5000, Software Reference Guide and Users Manual. Analytical Instruments Division, Shimadzu Corp., Kyoto, Japan. enz, M.E., Tortorelli, M.C., Di Marzio, Simionato, E., Alberdi, J.L., Sa W.D., 1997. A native South American amphipod: Hyalella 391 curvispina used in ecotoxicological bioassays: growth and sensitivity. 18th Annual Meeting of Society of Environmental Toxicology and Chemistry, San Fransisco. Sparks, T. (Ed.), 2000, Statistics in Ecotoxicology. Wiley, Chichester, p. 320. Sponza, D.T., 2003. Application of toxicity tests into discharges of the pulp-paper industry in Turkey. Ecotoxicol. Environ. Safety 54, 7486. Swartz, R., Di Toro, D., 1997. Sediments as complex mixtures: an overview of methods to assess ecotoxicological signicance. In: Ingersoll, C., Dillon, T., Biddinger, G. (Eds.), Ecological Risk Assessment of Contaminated Sediments. SETAC, Pensacola, FL, USA. US EPA, 1991. Methods for Measuring the Acute Toxicity of Efuents and Receiving Waters to Freshwater and Marine Organisms, fourth ed. Publ. No. 600/4-90/027. US Environmental Protection Agency, Washington, DC. US EPA, 1998. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates, second ed. Draft 4/1/98. US Environmental Protection Agency, Duluth, MN. Wagner, C., Lkke, H., 1991. Estimation of ecotoxicological protection levels from NOEC toxicity data. Water Res. 25, 12371242. Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B., 1996. Principles of Ecotoxicology. Taylor & Francis, London. Walsh, G.E., 1988. Principles of toxicity testing with marine unicellular algae. Environ. Toxicol. Chem. 7, 979987. Wang, C., Wang, Y., Kiefer, F., Yediler, A., Wang, Z., Kettrup, A., 2003. Ecotoxicological and chemical characterization of selected treatment process efuents of municipal sewage treatment plant. Ecotoxicol. Environ. Safety 56, 211217. WEST Inc, 1996. TOXSTAT V3.5. Western EcoSystems Technology Inc., Cheyenne, WY, USA. WHO (World Health Organization), 1994. Hydroquinone. Environmental Health Criteria 157. WHO, Geneva. WHO (World Health Organization), 1996. Morpholine. Environmental Health Criteria 179. WHO, Geneva. WHO (World Health Organization), 1997. Di-n-butyl Phthalate. Environmental Health Criteria 189. WHO, Geneva. Wiley Library, 1995. Registry of Mass Spectral Data with Structure, sixth ed. Wiley (for Shimadzu Corp.), New York.