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Ecotoxicology and Environmental Safety 61 (2005) 380391
Risk assessment of domestic and industrial effluents unloaded
into a freshwater environment
W.D. Di Marzioa,b,, M. Sa enza, J. Alberdia, M. Tortorellia, Galassi Silvanac
aPrograma de Investigacion en Ecotoxicolog a, Departamento de Ciencias Basicas, Universidad Nacional de Lujan, C.C. 221, 6700 Lujan B, ArgentinabComision Nacional de Investigaciones Cient ficas y Tecnicas CONICET
cUniversita degli Studi di Milano-Bicocca, Dip. di Biotecnologie e Bioscienze, Milano, Italia
Received 13 November 2003; received in revised form 14 September 2004; accepted 18 October 2004
Available online 26 January 2005
Abstract
An ecotoxicologic study was performed to assess the environmental status of the Luja n River. It is an important freshwater
system in the northeast of Buenos Aires Province, Argentina. Surface waters (SWs) and liquids effluents (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 chromatograph-
MS 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 effluent 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). Atoxicity evaluation is an important parameter in waste-
water quality monitoring, as it provides an overview of
the response of test organisms to all the compounds in
the wastewater (Wang et al., 2003).
The Luja n Rivers basin is situated in the northeast of
Buenos Aires Province, Argentina. It emerges from the
confluence of the Los Leones and El Durazno streams
and runs 130 km through an area of 2300km2. One
million people are connected to this area. It receives thedischarge of industrial and sewage effluents from its
source to its outlet in the Ro de la Plata River.
Furthermore, the Luja n River runs through a region
with typical agricultural and cattle activity. Its water
quality had deteriorated throughout the three decades
prior to this study (Sa enz et al., 1996; Alberdi et al.,
1996). Sporadic fish die-offs had been observed through-
out the river. As a result, the interaction of people with
the river was reduced, since almost no recreational
ARTICLE IN PRESS
www.elsevier.com/locate/ecoenv
0147-6513/$- see front matter r 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2004.10.002
Corresponding author. Programa de Investigacio n en Ecotoxico-
loga, Departamento de Ciencias Ba sicas, Universidad Nacional de
Luja n, C.C. 221, 6700 Luja n B, Argentina. Fax: +54 2323 423171x285.
E-mail addresses: [email protected], [email protected].
edu.ar (W.D. Di Marzio).
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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 effluents (LEs) discharged into the
Luja n River. To assess the environmental hazard of the
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
effluents 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 effluent 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 identification approach allows connections tobe 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 profiles for the more important industries and
domestic effluents 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 compoundsfound in water samples of the Luja n River and in liquids
effluents (LEs) prior to discharge.
2. Materials and methods
During 2003, water samples were taken from the
surface of the Luja n River at 2-month intervals. During
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
follows: Site 1 (Los Leones), an unpolluted stream. Site
2 (El Durazno) is a stream that is influenced by effluents
from the milk industry. Site 3 (Suipacha) is where the
Luja n River rises from the confluence of the Los Leones
and El Durazno streams. Site 4 is where the river
receives effluents from chemical industries and from the
city of Mercedes, which had no wastewater treatmentplant at the time of this study. At Site 5 (Las Tropas) the
river receives effluents from the textile, meat, tannery,
and enzyme purification industries. At Site 6 (La Loma)
the river receives the effluents of the sewage plant of the
city of Luja n, of textile, chemical, and food industries.
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 flow
measurement was obtained from an automatic flow
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, tempera-
ture, and pH. We also evaluated the chemical oxygen
demand (COD) and biochemical oxygen demand (BOD)
of each water sample according to APHA-AWWA-
WPCF (1998). An Ekman dredge was used to take
sediment samples. All sediment samples were compo-
sites of five grab samples at each sampling point. They
were characterized by pH, ammonia, sulfide, 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 Environ-
mental 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 modified Detmers nutrient medium (pH
7.5) under controlled conditions in a climatized room at
2271 1C, 3000 lx/cm2 of continuous cool-white fluor-
escent 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 flasks. Test solutions consisted
of enriched river water samples with algal medium
nutrient solutions. Control cultures were incubated in
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the same medium. Control and treated cultures were
grown under the same conditions of temperature,
photoperiod, and shaking and were carried out intriplicate. Each toxicity test was conducted three times.
Cell counts were correlated with absorbance (750 nm) on
a Shimadzu MUV 240 spectrophotometer (Walsh,
1988). Definitive protocols for toxicity testing using this
species are described in Sa enz et al. (1996).
2.1.2. Microcrustacean toxicity test
Microcrustacean tests were performed with D. spinu-
lata, 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. spinulatao
24h old. Testconditions were static or without the renewal of
medium, 48 h in duration, and conducted at 2071 1C
in the dark. Organisms placed in artificial pond water
(pH, 7.870.2; total hardness, 95.876m g CO3Ca/L;
conductivity, 475.5746.3mS/cm; and alkalinity,
189.3714.5 mg CO3Ca/L) served as controls. Definitive
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 artificial pond in a field of Universidad
Nacional de Luja n. They were placed in laboratory
tanks of 500 L with a continuous flow of groundwaterunder natural conditions (water quality: hardness,
126mg CO3Ca/L; conductivity, 800mS/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 (confidence
interval of 0.120.53 at Po0:05)] and size appropriatefor test handling (Simionato et al., 1997). An age control
was made using the equation Tlen 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. Thewater quality for the DW was hardness, 82 mg CO3Ca/
L; conductivity, 500mS/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. Definitive 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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N
20 kmSuipacha
Mercedes
Lujn
Buenos Aires
city
Rio de la Plata
Lujn river
Argentine
4
1 2 3
56
sampling sites
Fig. 1. Map of the study area (the Luja n River basin). Numbers are the SW and sediment sampling sites.
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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,
such as the Neuque n River, which has colder waters.
Ninety-six-hour semistatic acute tests were conducted.
Fish captured in an artificial freshwater pond were
acclimated in groundwater (pH, 7.9; conductivity,400mS/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 artificial food, not exceeding 5% of their total
individual weight. Tests were made using ground-
water in glass aquaria at 2071 1C and a photoperiod
of 12 h light/12 h dark. The final volume was 2 L in order
to get a loading rate of 0.5 g of fish/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 24h the mortality was
recorded. The definitive 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 theend 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 effluents
Grab samples for analytical determinations were
collected at each sampling point or effluent. They wererefrigerated 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
filtered across a 0.45-m-fiber glass filter and pH
corrected at 7.5 according to Gert Jan de Maagd
(2000). Samples were placed in 4-ml vials with a Teflon-
faced septum. A manual solid-phase microextraction
holder was used with unbonded fibers of polydimethyl-
siloxane (PDMS) at a stationary phase of 100- and 7-mm
film thickness and one of 85mm of polyacrylate (PAC)
(Supelco, Bellefonte, PA, USA). These were conditioned
as follows: PDMS 100 at 250 1C for 1h, PDMS 7 at
320 1C for 3 h, and PAC 85 at a 3001C gas chromato-
graph (GC) injector temperature for 2 h. The fibers 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 fibers were then
immediately inserted into the GC injector and the
analysis was carried out. The desorption time was 4 minand the desorption temperature was set at 2801C. 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. Experi-
mental conditions included PTE-5 fused-silica capillary
column 30 m 0.25 mm 0.25-mm film thickness (Su-
pelco); a linear velocity of carrier Helio, 36.2 cm/s,
splitless, with sampling time of 4 min and total flow of
11.7 mL/min; a temperature program of 1001C for 2 min
heated to a final temperature of 280 1C at 10 1C/min and
held at this temperature for 10min; an injector
temperature of 2801C; and a capillary interface tem-
perature 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 anunknown 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:
SI 1 Ium=z Itm=z Pm=zm=z
Ium=z Itm=z
26666
4
37777
5
100:
where Ium=z is the relative spectral intensity for massnumber m=z of the mass spectrum of an unknownsample. Itm=z is the relative spectral intensity for massnumber 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 coefficient
of partition octanol/water log Por log Kow was obtained
for each compound by using the Clog P program.
Bioconcentration factors (BCF) were determined by
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Connells equation (Connell, 1990) as follows:
log BCF 6:9 103log Kow41:85 101log Kow
3
1:55log Kow2 4:18 log Kow 4:79:
2.4. Physico-chemical characterization of liquid effluents
The following parameters were used according to
APHA-AWWA-WPCF (1998): COD, BOD, pH, alka-
linity, sulfide, sulfate, conductivity, free chlorine, am-
monium, salinity, chloride, reactive phosphorous,
nitrate, hardness, and residual solids after 10 and
120 min of settling. The majority of these are included
in the official 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 tocompare 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 fiducial 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). Effluent 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 usingthe 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
mean of the flow of the Luja n River is 1.159 m3/s with a
standard deviation of 4.513 and a coefficient of variation
of 389%. The COD and BOD values are plotted with
DO concentrations in Fig. 2.
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Table 1
The range of physico-chemical parameters measured in 150 water samples from the Luja n River
Parameter Summer Autumn Winter Spring
Total flow (m3/s) 07.44 1.312.3 110.4 0.2314.69
Dissolved oxygen (mg/L) 018.95 1.946.76 2.597.34 0.5313.11
Conductivity (mS/cm) 159011790 12006500 10205000 6005500
Salinity (%)[AU: What unit intended here?] 0.013.00 0.011.90 0.011.79 0.012.5
Turbidity (nephelometric units) 90 to 1000 50700 50400 120 to 41000
Temperature (1C) 1827.5 1219 813 1024
PH 8.511.4 89.2 7.69 810.9
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 berelated 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
Luja n, showed toxicity of about 60% inhibition,
conductivity values of 12,000mS/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 five 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 (significant differences,
ANOVADunnettest at Po0:05). These results coin-cided 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. Effluent 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 effluent. Three groups were obtained: Group a,
Mme, MCMe, and MS formed for domestic liquids;
Group b, TL1, TL2, TMCMe, FS, Cme, ML, and CL
(this joined mainly chemical effluents or mixed with
organics); and Group c, with only BL as a member (M,
municipal; T, textile; C, chemical; F, food; B, biotech-
nology; L, city of Luja n; Me, city of Mercedes; S, city of
Suipacha.; 1, component 1; 2, component 2). Group b
was discriminated in PCA analysis according to specific
PC values regarding pH, conductivity, sulfate, ammo-nium, BOD, and COD variables.
However, PCA performed with all parameters, PC
plus the toxicity index (Figs. 5 and 6), defines 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 effluent assayed and between Groups a, b, and c it
is possible to say that there are differences in both PC
and toxicity values, defining 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; sulfide concentrations were not detectable;
ionized ammonia (NH4+) was 50600 mg/L. Higher
values of NH4+ were found next to higher organic
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.
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Time relative scale (days)
20
40
60
80
100
120
140
160
180
200
-5 5 15 25 35 45 5
algal growth
Stimulation growth %
Inhibition growth %
summerautumn
winter springsampling points order
12
3
4
56
5
Fig. 3. Algae growth responses in the Luja n River samples. Control growth at 96 h is considered to be 100%. Growth lesser or greater than that of
the control is considered inhibition or stimulation, respectively. Growth o80 or 4120% was significant at Po0:05 (ANOVADunnets test).
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4. Discussion
Acute toxicity of SWs was infrequent for animals (fish
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 defines the
algal growth. Phosphorous is usually the limiting
nutrient for algal growth in freshwater environments.
In the regional legislation, before discharge an effluent
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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Table 2
Physico-chemicals parameters of assayed effluents, all values are expressed in mg/L
Parameter TL TL BL ML CL T+M+CM MM M+CM CM MS FS
COD 548 190 1754 468 240 792 216 36 71 159 250
BOD 300 40 890 140 50 270 110 10 20 54 90
SS 10 min 0 0 0 0.6 0 0 4 0 0.25 0 0
SS 120 min 0 0 0.1 0.9 0 0 4 0.1 0.25 0 0pH 11.42 8.26 7.6 7.82 8.1 7.12 6.84 7.15 7.91 7.20 7.09
Conductivity 2900 1200 8000 1480 12,000 631 210 152 158 251 184
Salinity 2 1 5.3 0.9 5 0.32 0.09 0.06 0.07 0.11 0.08
Free chlorine 0 0.06 0.05 0.09 0.04 0 0.06 0.05 0.09 0 0.06
Sulfide 0.13 0.08 13.9 0.04 0.2 0.072 0.052 0.004 0.009 0.004 0.032
Ammonium 5.4 5.4 1214.2 28.4 100 12.5 12.9 0.20 0.25 2.9 13.6
Nitrate 4.8 3.5 11.4 1.1 3.5 13.2 10.5 32.9 13.7 14.7 14.9
Reactive phosphorous 0.14 1.49 54.13 12.33 0.1 0.81 6.53 2.09 0.68 3.21 8.88
Sulfate 103.7 147 2647.2 121 40 86 90 112 25 79 120
Alkalinity 170 65 135 60 400 600 580 510 470 630 700
Chloride 15 35 20 25 2,000 200 35 20 22 38 20
Hardness 0 17.1 58.5 19.5 130 258.1 325 334.5 152.9 372.8 219.8
Algae 17.32 100 35 4.25 71 5.61 100 100 4.3 100 6.89
Cladocerans 16.52 100 4.95 71 17.32 NT 17.32 70.71 NT NT NT
Amphipod adult 17.32 70 4.24 28.98 70.7 NT 30.33 55.82 NT NT NT
Amphipod young 3 4.24 1.97 50.86 17.32 NT 11.77 43.75 NT NT NT
Fish 6 100 3 71 35.35 NT 2.22 6.86 NT 71 NT
Conductivity is expressed in mS/cm, salinity in %, acute toxicity endpoints as effluent percentage (%). SS are solids at 10 and 120min of
sedimentation. NT, not acute toxicity; T, textile; B, biotechnology; C, chemical; M, municipal; F, food effluents. Superscripts: L, city of Luja n; M,
city of Mercedes; S, city of Suipacha.
Component 1 (62 %)
Component2(
17%)
BL
MMeMCMe
MS
TL1
ML
TMCMe
FS
CLTL2
-7 -5 -3 -1 1 3-1.8
-0.8
0.2
1.2
2.2
CMe
Fig. 4. Two-dimensional scattergrams corresponding to the distribu-
tion of the PC parameters of each effluent. M, municipal; T, textile; C,
chemical; F, food; B, biotechnology; L, city of Luja n; Me, city of
Mercedes; and S, city of Suipacha. The two components explained
75% of total variance.
Component 1
Component2
CL
TL2
ML
BL
CMe
TMCMe
MS
MCMe
TL1
MMe
FS
-2 0 2 4 6 8
-1.8
-0.8
0.2
1.2
2.2
Fig. 5. Two-dimensional scattergram corresponding to the distribu-
tion of the PC parameters of each effluent and the toxicity indices for
all assayed species. M, municipal; T, textile; C, chemical; F, food; B,
biotechnology; L, city of Luja n; Me, city of Mercedes; and S, city of
Suipacha. The two components explained 75% of total variance.
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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 condi-
tions likely occurs during toxicity testing carried out
with algae (Persson, 1990). Also, algal toxicity may be
correlated significantly with some components of SWs.Graff et al. (2003) found that parameters like COD,
hardness, conductivity, total suspended solids, and
dissolved organic carbon were significant factors affect-
ing 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 effluent. 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
algae were not linked with any effluent-based PC
measured. This would confirm the idea that an effluent
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 toxicityemission 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 effluents to improve their
quality before they are discharged. The more popular
wastewater treatment plant consists of a suspended and
aerated culture of microorganisms forming microstruc-
tures called flocs or activated sludges. Others include
oxidation lagoons, biofilters, and bed reactors. All of
these treatments improve the water quality of the
effluents in terms of COD, BOD, and suspended solids
removal; further treatments will remove mainly phos-
phate and nitrate. Before effluents are discharged, they
must meet the threshold values for these parameters as
defined on environmental legislation. The aim of this
regulation is to prevent oxygen depletion and eutrophi-
cation of water bodies. But it does not include limits on
toxicity and the toxic load of effluents 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
into the Luja n River are ecotoxic even when they meetthe 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
ARTICLE IN PRESS
Component 1
Component2
COD
Algae
ALK
NH4
Cond
NO3
AnfA
AnfC
daph
Fish
PO4
-2.1 -0.1 1.9 3.9 5.9 7.9
-1.8
-0.8
0.2
1.2
2.2
Fig. 6. Biplot of two-dimensional scattergram corresponding to the
distribution of PC parameters of each effluent and the toxicity indices
for all assayed species. The partial correlation for each variable is
shown. The two components explained 75% of total variance.
Organic matter OM (%)
MortalityM
(%)
0
20
40
60
80
100
120
0 4 8 12 16 20 24 28 32
Regression95% confid.
R2
= 0.905
M = 3.929 * OM - 10.85Fisher's value = 36.06 p < 0.05
Los Leones S1
Suipacha S3 El durazno S2
Las Tropas S5
Mercedes S4 La Loma S6
Fig. 7. Regression analysis (significant at Po0:05) between OMC in sediments and mortality recorded for the amphipod H. curvispina. The Fvaluewas 14.11 at Po0:05; the confidence interval of the slopes was 1.096.61 at Po0:05: S1 is Site 1, S2 is Site 2, etc.
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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 effluent as
ToxLoad UT FLE, where FLE is the flow of LE (see
Table 4).
A frequent approach to defining an environmental
threshold of LE to avoid acute effects on native
organisms is to consider that no effect occurs when thepercentage 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 flow and the mean flow or some critical flow
value for the river and to obtain a river concentration
value (RCV). [The RCV could also be termed the
predicted environmental concentration for each efflu-
ent.] The RCV in our study was calculated according to
the following equation:
RCV FLE=FLE FRM100;
where FLE is the flow of LE and FRM is the mean flow
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 field 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 environ-
mental 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 define 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 find environmental
risk only for one effluent, a chemical industry discharged
near the city of Mercedes.Another approach to defining a hazardous concentra-
tion (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
organisms present in the waters of the Luja n River, at
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
ARTICLE IN PRESS
Table 3
Organic compounds found in surface water and liquid effluents
Log Kow Log BCF Compound Effluents Sample
4.51 3.35 1,2-Dichloronaphtalene TMCMe Mercedes (4)
2 1.26 Phenol, 4-methyl ML, CL La Loma (6)
NC NC Phenol, 2-chloro-5-methyl CL La Loma (6)
0.59 2.83 Hydroquinonea TL1, TL2, ML La Loma (6), Las Tropas (5)4.79 3.33 Phenanthrene Not found La Loma (6)
8.23 3.9 p-Nonylphenola MS, FS, ML, TMCMe La Loma (6)
NC NC Nonylphenol diethoxylate ML Las Tropas (6)
3.53 2.28 4-Heptanol TL1, TL2 Las Tropas (6)
5.29 4.07 Phenol, 4,40methylethylidene,bis- Not found Las Tropas (5)
3.80 2.58 1-Butanol, 4-butoxy TL1, TL2 Las Tropas (5)
4.98 3.81 3,3-Dimethyl, 14-heptanol Not found Las Tropas (5)
3.42 2.17 1,2-Benzenedicarboxylic acid MS, ML, Cme La Loma (6)
5.19 4.32 di-n-Butyl phthalatea Ml, CL, MS, MMe Mercedes (4)
1.08 NC Morpholinea TL1, TL2, ML Mercedes (4)
4.79 3.99 Diethyl phthalatea ML, TL1, TL2 Mercedes (4), La Loma (6)
3.02 1.78 Furanone, 5-ethyldihydro-5-methyl Not found Las Tropas (5)
BCF, bioconcentration factor; Kow, coefficient of partition for octanol/water; NC, not calculated; M, municipal; T, textile; C, chemical; F, food; L,
city of Luja n; Me, city of Mercedes; S, city of Suipacha; 1, component 1; 2, component 2. In parentheses are the numbers of each site sample (referFig. 1).
aDetected throughout the sampling year.
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known that humic and fulvic acids reduce the bioavail-
ability 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 deficit of
oxygen in all sampling sites, indicating an oxygen-
stressed environment with a COD:BOD:DO relation-
ship 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 find toxicity in
the sediments. This is related to interstitial waterconcentrations 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
final 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
NH4+
concentrations, which were measured in samplingpoints with lesser DO concentrations. High values of
NH4+ concentrations could be able to mask chronic
effects due to other organic and inorganic chemicals. In
these environments nitrification 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 fish 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
from the cities of Mercedes and Luja n. This acute
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 effluents
from the cities of Mercedes and Luja n.
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 effluents. NP, an estrogen agonist, is theultimate degradation product of nonylphenol poly-
ethoxylate 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
fitness and survival of fishes (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 suspectedcompound 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
ARTICLE IN PRESS
Table 4
Environmental risk assessment according to toxic unit and extrapolation methods for each assayed effluent
Toxic load RCV in TU RA 1 RCV in % HC 5% AS RA 2 HC 5% WL AF RA 3
ML 1180 2.52 R 10.70 0.02 R 0.02 0.042 R
TL1 1000 2.24 R 6.71 0.06 R 0.07 0.03 R
TL2 710 1.58 R 6.71 0.01 R 0.01 0.042 R
CL 690 1.29 R 22.34 0.15 R 0.18 0.17 RTMCMe 81.99 0.21 NR 1.18 0.01 R 0.02 0.056 R
FS 59.02 1.27 R 8.75 0.02 R 0.03 0.069 R
MS 56.40 0.12 NR 8.75 2.29 R 2.38 0.71 R
MCMe 53.35 0.13 NR 0.87 0.03 R 0.04 0.069 R
MMe 47.54 1.11 R 2.45 0.00 R 0.01 0.022 R
BL 40 0.10 NR 0.19 0.01 R 0.01 0.02 R
CMe 3.41 0.01 NR 0.04 0.01 R 0.01 0.04 NR
R, acute risk; NR, no acute risk; RCV, river concentration value, obtained with mean river flow; 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.
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influenced by the capability of an organism to metabo-
lize them. Several authors have shown the ability of fish
to metabolize DBP (WHO (World Health Organiza-
tion), 1997). The laboratory BCF for Pimephales
promelas ranged between 570 and 590 and for Cyprino-
don variegatus to 11.7. The greater BCF recorded was
1400 for the amphipod Gammarus pseudolimnaeus(WHO (World Health Organization), 1996). The theo-
retical BCF for DBP from Table 2 is 20,890, which
would confirm 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 films. It is also widely
used in the manufacture of rubber antioxidants and
antiozonants, monomer inhibitors, and food antioxi-
dants to prevent deterioration in many oxidizable
products. With a log Kow of 0.59 it can be considered
that hydroquinone does not bioaccumulate. The
logBCF found in the literature for aquatic organisms
is between 1.60 and 2.94. Biodegradation of hydro-
quinone 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 Organiza-
tion), 1996). Morpholine can undergo a variety ofreactions. 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 condi-
tions by a restricted range of microbes, which have a lowgrowth 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.
5. Conclusions
A field ecotoxicological study was performed to define
the environmental status of the Luja n River plain. The
toxic load was quantified, and four industries of the 11
evaluated represent 91% of the total toxic load
incorporated into the river. The environmental risk for
these effluents 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 com-
pounds. Besides, they are exposed to hormonally
disrupting chemicals. The environmental half-life ofthese 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,
Universite de Metz, Metz, France) for his assistance inPCA analysis. The research was supported by grants
from the Departamento de Ciencias Ba sicas and
SECYT of Universidad Nacional de Luja n, Argentina,
and from the Agregadura Cientfica de la Ambasciata
dItalia en Buenos Aires, Argentina.
References
Alberdi, J.L., Di Marzio, W.D., Sa enz, M.E., Tortorelli, M.C., 1996.
Protocolo para evaluacion de la toxicidad aguda con Daphnia
spinulata (Cladocera). Resumenes X Congreso de Toxicologa,
Buenos Aires.Aldenberg, T., Slob, W., 1993. Confidence 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 artificial 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., Tattersfield, L., 1999. Endocrine
Disruption in Invertebrates: Endocrinology, Testing, and Assess-
ment. SETAC, Pensacola, FL, USA.
Di Marzio, W.D., Sa enz, M.E., Alberdi, J.L., Tortorelli, M.C., 1996.
Protocolo para evaluar la toxicidad aguda sobre Cnesterodon
decemmaculatus (Pisces, Poeciliidae). X Congreso de Toxicologa,
Buenos Aires.
Di Marzio, W.D., Sa enz, M.E., Alberdi, J.L., Tortorelli, M.C., 1999.
Assessment of the toxicity of stabilized sludges using Hyalella
curvispina (Amphipod) bioassays. Bull. Environ. Contam. Toxicol.
63, 654659.
ARTICLE IN PRESS
W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391390
7/27/2019 Riskassessment of Domestic and Industrial Effluents Unloaded
12/12
Ekholm, P., Krogerus, K., 1998. Bioavailavility of phosphorus in
purified municipal wastewater. Water Res. 32, 343351.
Gert Jan de Maagd, P., 2000. Bioaccumulation tests applied in the whole
effluent 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 Effluent Toxicity
Testing: An Evaluation of Methods and Prediction of Receiving
System Impacts. SETAC, Pensacola, FL, USA, p. 340p.
Margalef, R., 1983. Limnologa. 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/Environ-
mental 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 effluents from sewage
treatment works. Chem. Ecol. 8, 275285.
Sa enz, M.E., Alberdi, J.L., Di Marzio, W.D., Tortorelli, M.C., 1996.
Protocolo estandarizado de ensayos de toxicidad con las algas de
agua dulce Scenedesmus acutus y Scenedesmus quadricauda. X
Congreso de Toxicologa, Buenos Aires.
Shimadzu Corp., 1999. MS Workstation Class 5000, SoftwareReference Guide and Users Manual. Analytical Instruments
Division, Shimadzu Corp., Kyoto, Japan.
Simionato, E., Alberdi, J.L., Sa enz, M.E., Tortorelli, M.C., Di Marzio,
W.D., 1997. A native South American amphipod: Hyalella
curvispina used in ecotoxicological bioassays: growth and sensitiv-
ity. 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 significance. 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 Effluents
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 Bioaccumu-
lation of Sediment-Associated Contaminants with Freshwater
Invertebrates, second ed. Draft 4/1/98. US Environmental Protec-
tion 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 effluents 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. Environ-
mental Health Criteria 157. WHO, Geneva.
WHO (World Health Organization), 1996. Morpholine. Environmen-
tal 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.
ARTICLE IN PRESS
W.D. Di Marzio et al. / Ecotoxicology and Environmental Safety 61 (2005) 380391 391