Implementing Lecane quadridentata acute toxicity tests to assess the toxic effects of selected...

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Implementing Lecane quadridentata acute toxicity tests to assess the toxiceffects of selected metals (Al, Fe and Zn),$,$$

Felix Torres Guzman a,n, Francisco Javier Avelar Gonzalez b, Roberto Rico Martınez c

a Universidad Autonoma de Guerrero. Departamento de Quımica. Avenida Lazaro Cardenas s/n, ciudad universitaria. Chilpancingo 39660, Gro. Mexicob Universidad Autonoma de Aguascalientes. Centro de Ciencias Basicas. Departamento de Fisiologıa y Farmacologıa. Edificio 52, Avenida Universidad 940, Aguascalientes,

Ags. Mexicoc Universidad Autonoma de Aguascalientes. Centro de Ciencias Basicas. Departamento de Quımica. Edificio 60. Avenida Universidad 940. Aguascalientes, Ags. Mexico

a r t i c l e i n f o

Article history:

Received 18 April 2008

Received in revised form

14 October 2009

Accepted 18 October 2009Available online 14 November 2009

Keywords:

Aluminum

Iron

Zinc

Metal toxicity

Rotifer

LC50

San Pedro River

Mexico

a b s t r a c t

An environmental study revealed that three metals (Al, Fe and Zn) are common in the San Pedro River

(SPR) (Aguascalientes, Mexico). Regrettably, in many samples the concentrations of these metals

exceeded the maximum allowed toxicant concentrations levels as defined in by Mexican legislation.

The highest concentrations of the three metals were found during the 2005 dry season, with elevated Al

concentrations present along the entire river. Not surprisingly, the highest concentrations for all three

metals came from locations adjacent to industrial areas. Estimates of the contribution of these metals to

total toxicity revealed that these three metals are important contaminants of the river and responsible

for most of the lethal toxicity found in environmental samples. To assess the importance of these

reports, we conducted acute toxicity tests to determine LC50 for Al, Fe and Zn on the freshwater rotifer

Lecane quadridentata. This permitted us to estimate the contribution of these metals to total toxicity

during 2005–2006. Based on LC50 values, all three metals should be considered very toxic, with the zinc

LC50 value (0.12 mg L�1) making it the most toxic metal for L. quadridentata. This approach can be

applied to other sites with similar concentrations of these metals.

& 2009 Elsevier Inc. All rights reserved.

1. Introduction

Contamination of aquatic systems by metals is increasingglobally and is of critical environmental concern. Thus, knowledgeof the effects of metals on aquatic organisms is important for theestablishment of water quality criteria. Of the metals normallyencountered in aquatic systems, aluminum, iron and zinc arecommonly present in polluted aquatic ecosystems (Shaw et al.,2006).

Zinc (Zn) is ubiquitous in the environment, constituting20–200 ppm (by weight) of the Earth’s crust. Zn is released tothe environment from both natural and anthropogenic sources;however, releases from anthropogenic sources are greater thanthose from natural sources (ATSDR, 2005). The Zn background

concentration in surface waters is usually o0.05 mg L�1, but canrange from 0.002 to 50 mg L�1 (ATSDR, 2005). Zn is an essentialnutrient for most animals and it is necessary for the function of alarge number of metalloenzymes. It does not volatilize from waterand is deposited primarily in sediments through adsorption andprecipitation. Severe Zn contamination tends to be confined toareas near emission sources (ATSDR, 2005). In water, Zn occurs inthe environment primarily in the 2+ oxidation state. It dissolvesin acids to form hydrated Zn2 + cations and in strong bases to formzincate anions, which are hydroxo complexes, e.g. (Zn[OH]3)� ,(Zn[OH]4)2� and (Zn[OH]4[H2O]2)2� (O’Neil et al., 2001). In mostwaters, Zn exists primarily as the hydrated form of the divalentcation. However, the metal often forms complexes with a manyinorganic and organic ligands (ATSDR, 2005). Potential environ-mental risks of elemental Zn and Zn compounds may occur atlocal and regional scales in surface water (Bodar et al., 2005).

Aluminum (Al) may influence the cycling of importantelements like Phosphorus, organic carbon and trace metals. Al ispotentially toxic to aquatic organisms, especially to fish when Alconcentration is higher than 8 mg L�1 (ppb) (Driscoll, 1985). Al isubiquitous; the third most common element of the earth’s crust(Staley and Haupin, 1992; ATSDR, 2006). In the environment, Alexists in only one oxidation state (3+), and can react with othermatter in the environment forming several complexes (Snoeyinkand Jenkins, 1980). Al can dissolve in lakes, streams and rivers

ARTICLE IN PRESS

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/ecoenv

Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2009.10.006

$FUNDING SOURCES: The present contribution is part of the Project: ‘‘Estudio

sobre los agentes, cargas contaminantes y toxicidad que afectan la cuenca del Rıo

San Pedro en el Estado de Aguascalientes’’. Proyecto SEMARNAT-CONACyT. Code

Number of the Project: FOSEMARNAT-2004-01-78F.T. is currently supported by

the Graduate CONACyT Scholarship 198929.$$GUIDELINES FOR THE PROTECTION OF AQUATIC ANIMALS: As we were using

freshwater microinvertebrates the guidelines of our institution for protection of

animals do not apply. In any case, all freshwater microinvertebrates were treated

with care.n Corresponding author.

E-mail address: [email protected] (F. Torres Guzman).

Ecotoxicology and Environmental Safety 73 (2010) 287–295

Author's personal copyARTICLE IN PRESS

depending on the quality of the water. In surface water Alconcentrations are usually very low (o0.1 mg L�1) (ATSDR, 2006;Brusewitz, 1984; Martell and Motekaitis, 1989; Miller et al., 1984;Sorenson et al., 1974; Taylor and Symons, 1984). However, evenat neutral pH levels, higher Al concentrations have been foundin lakes with a high humic acid content (Brusewitz, 1984). Alpolymerization is affected by presence of dissolved silica; whenenough silica is present, Al is precipitated as crystallized claymineral species (Bodek et al., 1988). Its concentration in soilsvaries widely, ranging from about 7 to over 100 g kg�1 (ATSDR,2006). Soils rich in organic matter had the greatest solubility andmobility of aluminum when the pH is low (Cotton et al., 1999).

Exposure to low levels of aluminum is not harmful, butexposure to high levels cause death. Effects on immunological,neurological and reproductive systems have been reported(ATSDR, 2006).

Iron (Fe) is an essential nutrient that is vital to the processes bywhich cells transform energy. Fe modifies the expression of thecitric acid cycle enzyme aconitase via a mechanism involving ironregulatory proteins. Fe exposure influences the citric acid cycleactivity, which results in a complex interaction between Fehomeostasis, oxygen supply and cellular energy metabolism, ofwhich the consequences are not yet understood (Oexle et al.,1999; Strydom et al., 2006). The form of Fe frequently found insolution in groundwater is the ferrous (Fe2 +) ion, which isbelieved to be toxic. The availability of Fe in aqueous solution isaffected by environmental conditions, particularly changes in thedegree of oxidation or reduction (Hem, 1989), which is dependentprimarily on pH, redox potential and temperature (Wetzel, 1983).Fe is readily oxidized by dissolved oxygen to the ferric form in theneutral to slightly acidic pH range, while the ferrous ion is stableunder these conditions (Forstner and Wittman, 1979). Fe ispresent in organic wastes, plant debris in soils and the activitieswithin the biosphere may strongly influence the occurrence ofFe in water (Hem, 1989; Avenant-Oldewage and Marx, 2000).The main source of increased Fe concentrations in the aquaticenvironment is acid mine drainage (AMD), mineral and steelprocessing and industrial runoff (Forstner and Wittman, 1979;Cruywagen et al., 1981). Acid mine drainage releases ferrous andferric sulphates into the aquatic environment, while coal minewaste dumps and lagoons leach iron into the water (Ellis, 1989;Department of Water Affairs, 1992). Acid mine drainage occurswhen minerals containing reduced forms of sulfur (pyrites,sulfides) are oxidized upon exposure to water and oxygen. Theoxidation process results in production of strong acids, whichmobilize acid-soluble metal. The mixture of acid and dissolvedmetals creates a severe environment for aquatic organisms,including reduced taxon richness and abundance and shifts frompollution-sensitive to pollution-tolerant species (Soucek et al.,2001). Fe itself can be toxic to aquatic plants when present in highconcentrations. High (free) Fe in pore water have been shown tocause necrotic leaf spot development and increased iron plaqueformation on roots of Glyceria fluitans (Van Der Well et al., 2006).

The state of Aguascalientes is located in Lerma-Santiagohydrological region in Mexico. With a surface of 5658.70 km2,the San Pedro River (SPR) is the most important basin in the state.It represents the main collector of the pluvial precipitation,wastewater and treated water. To this river, 70 million m3 ofresidual and treated waters coming from industrial, agriculturaland domestic sources are discharged annually (Ruiz et al., 2000;INEGI, 1993). Along the river are 56 communities, includingseveral that comprise the city of Aguascalientes (CNA, 2003).

The metal contamination of the SPR around the city ofAguascalientes began in 1895 with the inauguration of the GreatCentral Mexican Smelting, a subsidiary of the GuggenheimSmelting Company, that received all rights to use the water of

the SPR (Gomez, 1982). This smelting industry operated in the cityof Aguascalientes between 1896 and 1930, and during this time, itwas among the most important producers of copper and lead inMexico, and in the world (Gomez, 1982). Lead was extracted fromgalena mineral whose impurities include among others iron(Gomez, 1982). As a result of poor environmental managementthe smelting industry established in Aguascalientes produced agreat accumulation of tailings rich in Fe that were colloquiallycalled ‘‘the mountain of fat’’ (Gomez, 1982). In 1990, eight metal-mechanic factories were established in Aguascalientes operatingwith wastewater treatment plants, nonetheless in El Niagarareservoir and in several stations along the SPR Fe were found inhigh concentrations (SEDUE, 1990). Castillo (2003) found that 95%of the wells in the municipality of Aguascalientes showed valuesof Fe above the 0.30 mg L�1 concentration allowed by theMexican Norm. In recent studies carried out in the 2003–2006period, several authors (Ramırez-Lopez, 2005; Torres, 2009), cameto the conclusion that although the presence of lead (Pb) andmercury (Hg) could be important in particular areas aroundthe SPR, the SPR is contaminated in most of its extension with themetals: Al, Fe and Zn, and that other metals are present in suchlow concentrations that do not represent a primary concern to theriver (Ramırez-Lopez, 2005; Torres, 2009). Presence of such highconcentrations of Al, Fe and Zn represents a clear threat to thebiota of the river and, of course, to human health. Moreover,Santos-Medrano et al. (2007) reports that in almost all samplingstations monitored the water is acutely toxic. The goal of ourstudy was to utilize the 48 h acute toxicity test with the rotiferLecane quadridentata (Perez-Legaspi and Rico-Martınez, 2001) toestimate metal toxicity at selected sampling stations along theSan Pedro River in Aguascalientes, Mexico. We first determinedthe responsiveness of L. quadridentata to Al, Fe and Zn toxicity incontrolled laboratory tests to estimate metal LC50, NOEC andLOEC values. Then, we calculated the expected toxicity due to theconcentration of these three metals in the environmental samplesusing the equations obtained from the toxicity tests.

2. Materials and methods

2.1. Sampling of the San Pedro River

Monitoring of the SPR was carried out during the dry season (February–March)

and rainy season (November) of 2005–2006. Thirty eight sites were sampled along

the basin (Table 1). The main body of the SPR (SP stations), as well as four

tributaries; Chicalote River (CH stations), Morcinique River (MOR stations), Arroyo

San Francisco (ASFA stations) and Arroyo La Escondida (AE stations) were included

in the survey (Fig. 1). Sampling stations 0–1 through 0–5 correspond to treated

water of the San Pedro River (Table 1 and Fig. 1). Four liters of water were collected

for the Al, Fe and Zn determination, and for toxicity evaluation. Water was

collected in polypropylene bottles, previously rinsed with 10% HNO3 and

deionized water (Standard Methods, 2005).

2.2. 48 h acute toxicity tests using L. quadridentata

We used L. quadridentata originally collected from Chapala Lake, Mexico

(Perez-Legaspi and Rico-Martınez, 2001). L. quadridentata was continuously

cultured in EPA medium (USEPA, 1985) and fed Nannochloropsis oculata (UTEX

strain LB2194). Asexual eggs were collected and incubated at 25 1C in Petri dishes

with EPA medium (pH 7.4–7.8; hardness 80–100 mg L�1 CaCO3). Acute toxicity

tests were conducted in 24-well polystyrene plates (Costar Co., USA), following the

protocol of Perez-Legaspi and Rico-Martınez (2001). Toxicity range finding tests

were conducted previous to the definitive tests for all three metals. Briefly, 10

neonates were placed in each well containing a test volume of 1.0 mL EPA medium

and incubated for 48 h at 25 1C in darkness. For the definitive tests, besides a

negative control, we used eight concentrations for aluminum as AlCl3 (ranging

from 8�10�4 to 0.5 mg L�1), five concentrations for zinc as Zn(NO3)2 (ranging

from 0.031 to 2.0 mg L�1) and seven concentrations for iron as Fe(NO3)2 (1�10�4

to 2.0 mg L�1), each with five replicates. Actual concentrations of the three

toxicants were measured at start of test. The actual values were: 83.0075.48% for

Al, 71.6776.83 for Fe and 90.83710.36 for Zn, of their nominal values (n=6 for

F. Torres Guzman et al. / Ecotoxicology and Environmental Safety 73 (2010) 287–295288


each metal). The test was concluded after 48 h, after that time the number of

immobilized rotifers was recorded and the LC50 (median lethal concentration), the

no-observed-effect concentration (NOEC), and the lowest-observed-effect con-

centration (LOEC) were determined.

2.3. Statistical analysis for the 48 h acute toxicity test

Data were analyzed by means of the software LD50 to obtain LC20, LC50 and

LC90 probit values. Statistica 6.0 (Stat-Soft Inc., 1993) was used to determine

NOEC, LOEC and r2 values. Tests were considered valid only if mortality was less

than 10% in the control.

2.4. Determination of hardness and pH

To determine hardness we used the Hardness Test kit HI 3812 (Hanna

Instruments) following the recommendations of Standard Methods (2005). To

determine pH we used a pH/mV/Ion Meter UltraBasic (Denver Instruments Inc.)

with capacity to measure the whole pH interval from 0 to 14 with an electrode

according to Standard Methods (2005).

2.5. Determination of the concentration of Al, Fe and Zn in the samples of the San

Pedro River

To determine the concentration of the three metals in the environmental

samples we used atomic absorption (AA) methods. The aluminum, iron and zinc

were determined by AA, modality of flame air–acetylene (method 3111B for Fe

and Zn) and nitrous oxide–acetylene (method 3111D for Al), using a model

Perking-Elmer AAnalyst 100, according to Standard Methods (2005). The detection

limits for Al, Fe and Zn were 0.130, 0.025 and 0.005 mg L�1, respectively. Samples

were collected in clean polypropylene bottles rinsed with deionizated water and

30% HNO3 Instra quality. A concentrated HNO3 Instra quality acid solution was

also used as the stopper solution (0.1%).

2.6. Quality assurance

Standard NIST (US National Institute for Standardization and Technology)

reference material 1643d (trace elements in water) was used for water. The

percent recovery rate was 93%. Each sample was analyzed in duplicate. Deionized

water was used for all analytical work, and glassware and all material used for

metal analysis were soaked in 10% nitric acid, rinsed with deionized water and

dried before use. As quality control, the correlation coefficient of the standard

curve was higher than 0.98 in each group of samples analyzed for these metals.

The percentages of recovery of an internal standard and of spiked samples were

between 80% and 110%. The coefficient of variation of duplicated samples was

always smaller than 15%. J.T. Baker standards were used for atomic absorption. The

analyses of all the standard AA solutions are verified against identifiable solutions

according to the NIST. The reference standard solutions (SRM) were: Al (6440-04),

Fe (6453-04) and Zn (6474-04). Each sample was analyzed by triplicate.

Coefficient of variation was always less than 15%.

3. Results

Toxicity of Al, Fe and Zn was determined for the freshwaterrotifer L. quadridentata by 48 h LC50 acute tests (Figs. 2–4). Zincwas the most toxic of the three compounds tested (see LC50 andLOEC values in Fig. 4), followed by Al and Fe (although the Al LOECvalue is lower than that of Zn). All values were in mg L�1.

Table 1Geographical localization of the discharges to the San Pedro River.

Key of the sampling place Name Coordinate

Geographic coordinates Utm units

X Y

CH-1 Descarga Gomez Portugal-Cremerıa 21159024.400N–102117015.400W 780,071 2,434,217

CH-2 Efluente laguna de estabilizacion Gomez Portugal 21159040.30N–10211709.300W 780,353 2,434,217

CH-5 Descarga directa de Loretito al rıo 22100018.400N–102114023.900W 791,526 2,435,848

CH-7 Descarga Jaltomate 22100049.500N–10210905.800W 794,072 2,437,089

CH-10 Confluencia-PIVA-Chicalote-Gomez Portugal 2115804900N–102117032.700W 779,581 2,433,102

CH-12 Primera descarga La Florida 21158045.800N–102118015.500W 778,368 2,432,998

CH-13 Segunda descarga La Florida 21158045.700N–102118021.400W 778,200 2,432,990

CH-15 Rancho Agropecuario El Becerro 22100010.600N–102110035.300W 791,526 2,435,848

SP-2 Descarga Canada Honda al lado del parque de beisbol 22100016.800N–102111057.900W 789,151 2,435,990

SP-3 Descarga municipal de Canada Honda 22100015.500N–10211201.400W 789,057 2,435,949

SP-4 Union rıo Chicalote y San Pedro 21157056.600N–102119015.700W 776,669 2,431,451

SP-8 Compuerta de San Miguelito-Presa de Jesus Marıa 21157016.300N–10212005.400W 775,259 2,430,188

SP-11 Descarga Mpl. tubo roto conducida al rıo Jesus Marıa 2115701.900N–10212006.400W 775,238 2,429,742

SP-12 Descarga Pocitos-Los Arquitos 21155027.100N–102119039.100W 776,074 2,426,840

SP-13 Descarga por debajo del Rıo San Pedro 21154022.600N–10211908.400W 776,991 2,424,873

SP-20 Descarga Canteras de San Jose 21151030.800N–102119041.200W 776,141 2,419,572

SP-21 Rıo San Francisco 2115100.900N–102119041.900W 776,136 2,418,649

SP-23 Union Rıo San Francisco y San Pedro 2115100.900N–102119043.200W 776,097 2,418,651

SP-25 Rastro municipal Jesus Marıa 21158027.400N–102119034.100W 776,125 2,432,394

SP-26 Descarga Los Ramırez y Los Vazquez 21156013.2600N–102119051.800W 775,722 2,432,017

SP-29 Descarga San Antonio de los Horcones 21159030.200N–102119020.900W 776,466 2,434,329

SP-33 Descarga FREASA rastro Mpl. Sn. Fco. de los Romo 22103052.500N–10211700.200W 778,491 2,439,012

SP-34 Primera descarga municipal de Cd. Cuauhtemoc, Zac. 22126033.3400N–102120041.0700 773,289 2,484,236

SP-35 Segunda descarga municipal de Cd. Cuauhtemoc, Zac. 2212604100N–10212004600W 773,143 2,484,471

SP-39 Estacion de Adame 22124042.100N–102116031.900W 780,478 2,480,941

SP-42 Descarga San Jacinto I 22120056.500N–102114019.800W 784,411 2,473,132

SP-44 Descarga El Chayote 2211508.500N–10211708.500W 783,645 2,467,035

SP-47 Descarga La Soledad 22125046.200N–102118013.400W 777,541 2,482,861

SP-49 Las Animas 22105017.600N–10211603000W 781,176 2,445,107

MOR-1 Los Arquitos 21155032.600N–102122057.600W 770,378 2,426,637

MOR-2 Los Negritos 21152026.200N–102120038.300W 774,474 2,421,261

ASFA-1 Puente de Bonaterra 21149028.700N–102117013.700W 780,443 2,415,885

AE-2 Descarga Fatima II 21146048.200N–102120029.100W 774,914 2,410,851

0–1 Descarga Nissan, Los Arellano 21148036.500N–102116036.800W 781,531 2,414,299

0–2 Cascada Sabinal 21145022.500N–102122015.600W 771,900 2,408,162

0–3 Sabinal 2114503800N–10212204100W 771,747 2,408,034

0–4 Presa Niagara 2114504800N–10212201.100W 772,272 2,410,801

0–5 Niagara despues del puente 21146042.900N–102122027.600W 771,516 2,410,628

F. Torres Guzman et al. / Ecotoxicology and Environmental Safety 73 (2010) 287–295 289


0-50-40-30-2

0-1AE-2

SP-8SP-4

SP-3SP-2CH-7CH-5

CH-2

MOR-2

MOR-1

SP-49

SP-47

SP-44

SP-42

SP-39

SP-35

SP-34

SP-33

SP-29

SP-26

SP-23SP-21SP-20

SP-13SP-12

720000

720000

740000

740000

760000

760000

780000

780000

800000

800000

820000

820000

840000

840000

2400000

2400000

2420000

2420000

2440000

2440000

2460000

2460000

2480000

2480000

0 5,500Meters

N

11,000 22,000 33,000 44,000

Fig. 1. Sampling sites along the San Pedro River, Aguascalientes, Mexico.

Fig. 2. Acute toxicity test for iron on L. quadridentata.



All LC50 values were well below the highest environmentalconcentrations for each toxicant found in the San Pedro River(SPR). The results for Al, Fe and Zn concentrations determined insamples collected along the SPR are shown in Table 2. Values forAl ranged from 0.06 to 62.6 in the 2005 dry season, from 0.10 to5.85 in the 2005 rainy season, from 0.18 to 9.05 in the 2006 dryseason and from 0.16 to 7.858 in the 2006 rainy season. Thecorresponding values for Fe were 0.16–22.6, 0.10–5.59, 0.07–9.42

and 0.10–7.65, respectively. Similarly for Zn the values were0.01–10.3, 0.03–0.46, 0.01–0.34 and 0.01–4.30, respectively.

After our initial analysis of samples, nine sampling sites wereselected (due to their high concentrations of Al, Fe and Zn), toestimate the contribution of these three metals to total toxicityand the effects of key environmental factors (pH and hardness).Table 3 shows the results of the determination of pH andhardness, as well as expected toxicity calculated from Figs. 2–4.

Fig. 3. Acute toxicity test for aluminum on L. quadridentata.

y = 42.34 + 33.16x; r2 = 0.58CL50 = 0.1231; NOEC = 0.001; LOEC = 0.01

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

2.00

3.00

Zn mg/L

-20

0

20

40

60

80

100

120

% M

orta

lity

Fig. 4. Acute toxicity test for zinc on L. quadridentata.



The LC50 data for each of these nine sampling sites werecalculated from toxicity tests on the samples obtained bySantos-Medrano et al. (2007).

4. Discussion

4.1. General assessment of contaminants

Several values of the environmental samples collected alongthe SPR are above the LC50 values found for the three metals(see Figs. 2–4, and more important, they are above the maximumallowed toxicant concentrations (MATC) of Mexican legislation(Table 4). What is the contribution of this high metalconcentration to the total toxicity of the samples? The answer isnot easy, since environmental samples are composed of a cocktailof multiple contaminants, but an approach that considers theexpected toxicity calculated from the equation of the regressioncurve (Figs. 2–4) suggests that an important contribution tomortality of this population comes from these three metals

(Table 3). This suggestion becomes more relevant when weconsider two important facts. (1) The concentrations of othermetals (e.g., As, Cd, Cu, Hg and Mn) and some organic compoundsalong the SPR are much lower than MATC values (Torres, 2009).(2) The environmental samples selected (Table 3) are very similarto the conditions of the EPA medium (pH=7.5, hardness=80–120 mg L�1). These high percentages of expected mortality forL. quadridentata might be unrealistic in some cases (the 3787.6%and 1672.1% values for Al in Table 4), because we need to considerdifferent species of the metals and their bioavailability. A betterapproach to consider bioavailability is presented in Table 5 forthree selected sampling stations corresponding to the SPR and itstwo main tributaries (Chicalote and Morcinique Rivers). Table 5shows the expected mortality calculated using the concentrationof dissolved metals instead of total metal as in Table 3. In thesethree sampling stations, the percentages of expected mortality aremore realistic and lower, especially for Fe. Nonetheless, thecontribution to expected toxicity derived from the dissolved metalconcentration of these three metals is still relevant (Table 5).Furthermore, the LC50 values obtained from environmental

Table 2Al, Fe and Zn levels in 38 sampling stations around the San Pedro River, Aguascalientes, Mexico.

Sampling sites codes Dry 05 Rain 05 Dry 06 Rain 06 Dry 05 Rain 05 Dry 06 Rain 06 Dry 05 Rain 05 Dry 06 Rain 06

Al Al Al Al Fe Fe Fe Fe Zn Zn Zn Zn

CH-1 0.55 0.74 0.83 0.35 0.71 1.05 0.64 0.38 0.12 0.30 0.03 0.05

CH-2 0.06 ND 0.43 2.21 1.60 0.15 0.22 2.13 0.04 0.07 0.02 0.11

CH-5 1.06 1.98 0.49 0.47 0.61 1.83 0.26 0.35 0.13 0.20 0.12 0.04

CH-7 1.69 ND 0.32 0.16 0.97 ND 0.16 0.16 0.11 ND 0.03 0.01

CH-10 19.40 5.85 0.85 7.86 22.60 4.81 0.48 1.85 10.3 0.37 0.02 0.19

CH-12 0.59 ND 1.33 0.34 0.45 ND 1.97 0.77 0.08 ND 0.10 0.05

CH-13 4.12 3.43 1.33 ND 2.66 3.47 1.95 0.36 0.21 0.24 0.08 0.03

CH-15 62.60 0.73 3.40 2.00 22.40 1.22 1.78 2.35 0.51 0.27 0.34 0.37

SP-2 4.86 1.26 0.32 ND 1.80 0.92 0.17 0.21 0.34 0.14 0.04 0.06

SP-3 4.74 0.63 0.18 0.54 2.88 0.56 0.12 0.36 0.29 0.09 0.01 0.07

SP-4 2.10 0.21 0.63 ND 0.92 0.41 0.60 0.29 0.86 0.05 0.04 0.04

SP-8 2.96 1.08 0.55 0.63 1.56 1.04 0.45 0.92 0.17 0.11 0.05 0.12

SP-11 1.63 1.22 0.58 0.42 0.96 0.50 0.48 0.66 0.12 0.06 0.06 0.09

SP-12 54.00 4.14 0.34 ND 18.60 2.19 0.35 0.24 3.35 0.07 0.02 0.02

SP-13 2.05 ND 0.75 2.08 1.05 ND 0.79 1.27 0.11 ND 0.08 0.07

SP-20 15.20 2.05 ND 0.60 7.64 1.83 0.13 0.83 0.12 0.17 0.02 0.02

SP-21 0.16 ND 0.46 0.67 0.18 ND 0.39 0.94 0.05 ND 0.09 0.09

SP-23 4.44 0.11 0.46 0.95 3.18 0.21 0.34 0.59 0.18 0.03 0.02 0.03

SP-25 4.42 0.10 0.42 ND 3.24 2.19 0.90 0.59 0.06 0.13 0.03 0.06

SP-26 3.60 ND 1.70 0.53 2.68 ND 1.79 1.06 0.19 ND 0.18 0.10

SP-29 0.77 ND 9.05 0.72 5.78 0.52 1.95 0.47 0.56 0.11 0.32 4.30

SP-33 4.01 ND 0.27 3.05 8.86 1.21 1.84 7.65 0.47 0.46 0.08 0.32

SP-34 0.38 ND 0.88 ND 0.53 ND 0.94 0.16 0.08 ND ND ND

SP-35 1.05 ND 0.36 0.54 1.79 ND 0.51 0.93 0.15 ND 0.02 ND

SP-36 ND ND ND ND 2.19 ND ND ND 0.10 ND ND ND

SP-39 2.23 ND ND ND 2.19 0.58 ND 0.10 0.39 0.09 ND 0.01

SP-42 2.20 ND ND 0.16 0.23 0.27 0.04 0.34 0.05 0.09 ND 0.02

SP-44 0.03 ND ND ND 0.73 ND 0.13 0.22 0.11 ND ND 0.03

SP-47 0.93 ND 0.13 ND 1.29 3.79 0.10 0.27 0.16 0.37 0.28 ND

SP-49 1.63 ND 0.59 ND 1.20 ND 0.28 0.27 0.08 ND ND 0.03

MOR-1 0.75 ND ND 2.33 0.48 ND 0.05 10.50 0.07 ND ND 0.04

MOR-2 0.26 ND 2.40 1.87 0.08 6.59 3.79 2.67 0.11 0.72 0.24 0.06

ASFA-1 0.87 ND ND 0.85 0.85 ND 0.40 0.98 0.17 ND 0.03 0.06

AE-2 0.73 ND 5.00 2.35 2.59 ND 9.42 1.90 0.14 ND 0.07 0.10

0–1 3.21 ND 0.78 1.01 0.16 0.20 0.76 0.50 0.02 0.03 0.02 0.02

0–2 ND ND ND 0.19 0.20 0.34 0.08 0.21 0.03 0.11 0.02 ND

0–3 ND ND 0.30 ND 0.18 ND 0.28 0.19 0.01 ND 0.02 ND

0–4 0.13 ND 2.96 0.16 0.07 ND 2.20 0.23 0.01 ND ND ND

0–5 ND ND ND 0.33 ND ND 0.07 0.16 ND ND ND ND

78 ND ND 0.18 ND ND ND 0.27 0.28 ND ND ND 0.03

MATC (mg/L) CE-CCA-001/89a 0.05 0.05 0.05 0.05 1 1 1 1 10b 10b 10b 10b

MATC (mg/L) CE-CCA-001/89c 5 5 5 5 5 5 5 5 10b 10b 10b 10b

All values are in mg L�1. AND=No detected. All the values exceeding the MATC are in bold.

a Mexican guideline for protection of aquatic life.b NOM-001-ECOL-1996.c Mexican guideline for cattle and agricultural use.



samples show that a dilution of 35.2% of river water can still killhalf the population of the rotifer in 48 h (LC50 column in Table 3).However, a detailed comparison of total versus dissolvedconcentration of each metal (and some selected organiccompounds) for each sampling station and perhaps furtherlaboratory experiments are necessary to fully associate toxicitywith a particular contaminant as it is done with the toxicityidentification evaluation.

The metals Al and Fe are considered non-priority pollutantsand they are usually associated to natural sediments. However,the presence of a very active smelting industry in the past andpresent in the city of Aguascalientes (Gomez, 1982; SEDUE, 1990)with its poor waste management (in the past wastewater wasdumped directly into the SPR) can be a part of the explanation forhigh concentrations of these metals. Our results from determina-tion of dissolved versus total metal, and the fact that the higherconcentrations of these three metals come from industriallocations, suggest that at least a fraction of them should beconsidered anthropogenic contaminants.

For the 2005 dry season, seven samples were close to theMATC for Al and three exceeded the 5 mg L�1 MATC for cattle andagricultural use (Table 2). However, comparing the MATC valuefor the protection of aquatic life, 89.5% of samples collected inthe 2005 dry season exceeded the Al MATC value (Table 4). The

corresponding percentages of samples exceeding the Al MATC forthe rain 2005, dry 2006 and rain 2006 seasons were 36.8%, 81.6%and 71.0%, respectively (Table 4).

Five samples exceeded the 5 mg L�1 MATC during Fe in the2005 dry season. During the 2006 dry season and the 2005 and2006 rainy seasons, Fe concentrations exceeded the MATC valuesin one sampling station per survey. For Zn only two samplesexceeded the 2 mg L�1 MATC value in the 2005 dry season andone exceeded the MATC during the 2006 rainy season.

4.2. Temporal variation

The 2005 dry season represents the time when the highestconcentrations of all three metals were detected. Al (mean=4.028,n=38) was the most abundant metal followed by Fe (mean=3.260,n=38) and Zn (mean=0.570, n=38). For Al there was an importantreduction in Al levels during the 2005 rainy season, but Al levelswere increased again during the 2006 dry season (with respectto the 2005 rainy season), although not to the same levels ofthe 2005 dry season. However, there was again an increase in theAl levels in the 2006 rainy season. Fe concentrations decreased(or remained almost constant) every year from the peakconcentration of the 2005 dry season. Zn concentrationsdecreased every year after the peak concentration of the 2005dry season.

4.3. Spatial variation

During the 2005 dry season nearly the entire river, as well asits main tributaries had high levels of Al (many of them above theMATC), with the highest value was recorded at CH-10. In factthroughout the 2005–2006 sampling period important Al inputcame from the Chicalote River (currently the main tributary ofthe San Pedro River). The sampling station CH-10 corresponds tothe discharges nearby the effluent of the Industrial Park of theAguascalientes Valley (PIVA) that has been identified as the mosttoxic discharge to the San Pedro River (Santos-Medrano et al.,2007). In the case of Fe also, the main inputs came from theChicalote River throughout the year. The source of this metal isalso CH-10 and CH-15. These sampling stations already receivedthe inputs from the PIVA and the Industrial Park of San Franciscode los Romo, sites where several metal-mechanic factories arelocated. By far, the highest input of Zn came from PIVA (CH-10).Fortunately the highest Zn concentration recorded in the 2005 dryseason was greatly reduced in all the following surveys and theimpact of this reduction in the total Zn concentrations in the restof the San Pedro River is evident by examining Table 2.

Table 3pH (n=4), hardness (n=2), CL50 (n=3) and expected percent mortality (n=4, calculated from slope of regression related to the total concentration of each of the three

metals evaluated) for selected sampling sites.

Sampling site code pH Hardness (mg L�1) CL50a Al expected Fe expected Zn expected

Mean71SD Mean71SD (%) Mean71SD (%) Mean71SD (%) Mean71SD (%) Mean71SD

CH-2 8.1870.71 150.0074.24 54.70722.90 (1.83 aTU) 162.027201.12 57.52744.53 44.3371.30

CH-10 6.7670.27 120.0074.24 82.907104.37 (1.21 aTU) 1672.0871516.28 344.687460.11 132.547167.64

CH-12 7.4470.62 183.0074.24 43.63714.89 (2.29 aTU) 140.757109.06 47.33737.77 44.2571.44

SP-8 7.3870.15 177.0074.24 71.37745.70 (1.40 aTU) 283.757217.90 50.69724.73 46.0771.63

SP-11 7.3870.25 117.0074.24 47.02710.20 2.13 aTU) 217.587108.87 48.24724.52 45.0870.95

SP-12 7.8270.32 108.0074.24 52.4978.15 (1.90 aTU) 3787.5775793.90 251.067397.91 71.02754.94

SP-13 7.5070.30 153.0070.00 35.20733.20 (2.84 aTU) 267.337197.61 46.43724.83 44.9671.54

SP-23 7.8470.28 138.0074.24 161.387160.85 (0.62 aTU) 503.707375.31 230.677175.85 53.2076.33

MOR-2 7.4770.32 120.0074.24 36.58719.57 (2.73 aTU) 323.367215.34 158.657120.84 51.72710.00

a Santos-Medrano et al., 2007.

Table 4Percentage of samples exceeding the Mexican MATC values for each metal.

Aluminum

Dry 2005 Rain 2005 Dry 2006 Rain 2006

89.5% 36.8% 81.6% 71.0%

Iron

Dry 2005 Rain 2005 Dry 2006 Rain 2006

55.3% 28.9% 23.7% 23.7%

Zinc

Dry 2005 Rain 2005 Dry 2006 Rain 2006

2.6% 0% 0% 0%

Table 5Expected percentage mortality calculated using dissolved concentrations (calcu-

lated from slope of regression related to each of the three metals evaluated) for

selected sampling sites.

Sampling

site code

Expected mortality

dissolved Al (%)

Expected mortality

dissolved Fe (%)

Expected mortality

dissolved Zn (%)

CH-10 59.44 39.60 43.83

SP-23 31.60 13.35 42.80

MOR-2 193.10 12.85 45.59



4.4. Acute toxicity tests

We successfully implemented acute toxicity tests to determinelethal concentrations for Al, Fe and Zn on neonates of thefreshwater rotifer L. quadridentata. The LC50 value that wedetermined for Zn (0.12 mg L�1) makes it the most toxic metalfor L. quadridentata neonates of those studied so far (Perez-Legaspi and Rico-Martınez, 2001). The Zn LC50 values are lowerthan titanium, chromium, lead, mercuric chloride and copper(8.52, 4.53, 3.78, 0.44 and 0.33 mg L�1, respectively) (Perez-Legaspi and Rico-Martınez, 2001). They also were lower than theLC50 for Al and Fe obtained in this work (Figs. 3 and 4). Accordingto our results, the LC50 values for Al, Fe, and Zn at o1.0 mg L�1

would be classified them as very toxic by FAO/UNESCO/WHOguidelines (Sprague, 1973). That differs with the results of Hirtand Domitrovic (2002) who found that for the fish Cichlasoma

dimerus Hg and Cu were very toxic (LC50 values rangedfrom 0.488 in Hg to 0.8 mg L�1 in Cu), and Al, Cd, Cr, Pb andZn were just toxic (LC50 values ranged from 6 in Al to 46 mg L�1

in Zn).When we compared the Zn LC50 values of L. quadridentata

with that of other species, we found that L. quadridentata is moresensitive than the freshwater rotifer Brachionus calyciflorus,LC50=1.3 mg L�1 (Snell et al., 1991) and Daphnia magna, LC50ranged from 0.57 mg L�1 (Burba, 1999) to 2.8 mg L�1 (PesticidesDatabase, 2006). For Fe, LC50 of D. magna is 23.78 mg L�1 (Burba,1999). For Al, B. calyciflorus LC5043.0 mg L�1 (Snell et al., 1991)and D. magna, whose LC50 ranges between 0.32 and 1.02 mg L�1

(Havas, 1985), are less sensitive that the corresponding LC50s forL. quadridentata. Therefore, our model organism is more sensitivefor two metals than B. calyciflorus and more sensitive for all threemetals than D. magna. This is an important comparison for Mexicobecause the official (and only) norm for freshwater acute test(NMX-AA-087-SCFI-1995) uses D. magna. However, Mexicanguidelines for protection of aquatic life are not particularly‘protective’ when we analyzed the LC50 data found in this workand also are less restrictive when compared to similar guidelinesfor other countries or international organizations (see Table 6).

Implementation of these acute tests is part of a multi-disciplinary project that is currently underway trying to establishthe basal level of several metal and organic toxicants, and thelevels of toxicity in water and sediments, in the SPR aroundthe city of Aguascalientes. The ultimate goal of the project is todevelop a plan for the bioremediation of the river. Acute toxicityhas been reported in the main creeks and sampling points aroundthe river (Santos-Medrano et al., 2007). Many sites heavilycontaminated with aluminum (Al), cadmium (Cd), chromium(Cr), iron (Fe), mercury (Hg), lead (Pb) and zinc (Zn) have beenreported in the margins of the river (Avelar & Llamas, 2000). Inthat context the implementation of Al, Fe and Zn acute toxicity

tests represents a preliminary step to assess the contribution ofthese metals to the total toxicity found in more than 38 samplingpoints in the river. The acute toxicity tests that we implementedcan be used to estimate the contribution of some of the metals tothe total toxicity of the sample. The determination of Al, Fe and Znconcentrations in the SPR and its main tributaries clearly showthe importance of these metals as contaminants of the river.

5. Conclusion

High concentrations of Al, Fe and Zn were found in the SPR andits main tributaries, in many cases exceeding Mexican MATCs. Allthree metals were found to be very toxic for L. quadridentata, withZn being the most toxic for this species. The highest concentra-tions for all three metals were found during the 2005 dry season,indicating a high degree of temporal variation, but strong spatialvariation along the SPR also exists, with the highest values for allthree metals present close to industrial areas. The high concen-trations of these three metals in the SPR make them importantcontaminants of the river and responsible for most of the acutetoxicity found in selected samples.

Unfortunately the Mexican MATCs values for protection ofaquatic life are set too low to be effective in protecting manyaquatic species: e.g., rotifers such as L. quadridentata. In ourassessment this species could not sustain a viable populationunder the current guidelines. And yet the toxicity for a singlespecies is not the real issue; L. quadridentata can be likened to a‘miner’s canary’—a sentential that reports of danger to others.

Because of its high sensitivity, application of the L.quadridentata acute toxicity test can be an important tool formonitoring metal toxicity worldwide.

Acknowledgments

We would like to thank Mexico’s Council for Science andTechnology (CONACYT) as well as Mexico’s Ministry of theEnvironment (SEMARNAP) for funding this investigation. Theauthors thank Dr. Terry W. Snell and Robert L. Wallace for criticalreview of the manuscript. Also Martha Evelia Perez Reyes, LauraYamamoto Flores, Jennifer Lopez Baltasar and Samanta RamosGomez, members of the Biotechnology and Environmental StudiesLaboratories of UAA, for their valuable help.

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Table 6Comparison between Mexican regulation values for Al, Fe and Zn, and correspondent values for selected countries.

Country and source Reference Al Fe Zn

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Mexico NOM-001-ECOL-1996 Agricultural and livestock use Secretarıa del Medio Ambiente y Recursos Naturales (1996) – – 10,000

Mexico CE-CCA-001/89.Protection of aquatic life Instituto Nacional de Ecologıa (1989) 50 1000 10,000

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United Nations global drinking water quality index United Nations (2007) 100 300 3000

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