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Ecotoxicological studies of environmental samples from Buenos Aires area using a standardized amphibian embryo toxicity test (AMPHITOX)
Environmental Pollution 116 (2002) 177–183 www.elsevier.com/locate/envpol
Ecotoxicological studies of environmental samples from Buenos Aires area using a standardized amphibian embryo toxicity test (AMPHITOX) Jorge Herkovitsa,*,1, Cristina Perez-Colla,1, Francisco D. Herkovitsa a
Programa de Seguridad Quı´mica, Instituto de Ciencias Ambientales y Salud (ICAS). Fundacio´n PROSAMA, Paysandu´ 752, (1405) Buenos Aires, Argentina. Received 28 December 2000; accepted 1 May 2001
‘‘Capsule’’: AMPHITOX was found to be useful in evaluating toxicity of a wide variety of samples. Abstract The toxicity of 34 environmental samples from potentially polluted and reference stations were evaluated by means of the AMPHITOX test from acute to chronic exposure according to the toxicity found in each sample. The samples were obtained from surface and ground water, leaches, industrial effluents and soils. The data, expressed in acute, short-term chronic and chronic Toxicity Units (TUa, TUstc and TUc) resulted in a maximal value of 1000 TUc, found in a leach, while the lower toxicity value was 1.4 TUa corresponding to two surface water samples. In five samples (four providing from reference places) no toxicity was detected. The results point out the possibility of evaluating the toxicity of a wide diversity of samples by means of AMPHITOX as a customized toxicity test. The fact that almost all samples with suspected toxicity in rivers and streams from the Metropolitan area of Buenos Aires city resulted toxic, indicates the need of enhanced stewardship of chemical substances for environmental and human health protection purposes. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: AMPHITOX; Toxicity test; Amphibian embryos; Environmental pollution; Buenos Aires Metropolitan area
1. Introduction Environmental contamination in general has increased in concordance with the growing pressures on the Earth resources and life support systems resulting from escalating human numbers and activities combined with inappropriate environmental management (Cairns, 1994; Strong, 1994; Herkovits et al., 1996). These facts are of major concern because ecosystem services, essential to the quality of human life, could be severely affected while and, on the other hand, there is an ethical responsibility to also preserve attributes important for species diversity conservation purposes (Black, 1994; Cairns and Niederlehner, 1994, Costanza et al., 1997; Herkovits, 1997).
* Corresponding author. Tel.: +54-11-4431-2445; fax: +54-114432-1111. E-mail address: [email protected] (J. Herkovits). 1 Members of the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina.
The aquatic environment is particularly important as a recipient, transporting medium and sink for the majority of xenobiotic chemicals. The studies on the fate and transport of pollutants in the environment point out the close-in problems for air, soil, sediments, surface and groundwater as interrelated compartments. In order to cope with such a wide range of environmental situations and to have the possibility to compare results from different matrixes, there is a need for more sensitive, cost-effective and short-term biological test methods. For water quality-based toxicity control purposes, the US EPA found a good predictive correlation between embryo-larval survival and independent ecological parameters, such as species richness and diversity (US EPA, 1991). Moreover, by focusing on the most sensitive life-cycle stages, the US EPA suggested that chronic toxicity tests could be shortened to 7-day exposure protocols for the evaluation of industrial effluents. A worldwide decline of amphibian populations linked to the increasing environmental degradation is widely documented (Simms, 1969; Cook, 1981; Baringa, 1990; Blaustein et al., 1994; Boyer and Grue, 1995; Burkhart
0269-7491/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00167-1
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J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183
et al., 1998) and adverse effects, such as malformations and disruption of metamorphosis, could be related to either to the presence of developmental toxicants or the absence of essential micronutrients (Dawson et al., 1985; Herkovits et al., 1989, Vismara et al., 1993; Fort et al., 1999, Burkhart et al., 2000; Tietge et al., 2000). Although aquatic toxicology has focused mainly on concerns for fishes, invertebrates and algae (e.g. US EPA, 1991), studies exploring the response of amphibians to environmental pollutants found that they are usually highly sensitive and appropriate for testing single chemicals
and complex mixtures (Pe´rez-Coll et al., 1988, Pe´rezColl and Herkovits, 1990; Herkovits and Pe´rez-Coll, 1990, 1991, 1993; Herkovits and Helguero, 1998; Herkovits et al., 1996, 1997, 2000; Burkhart et al., 2000; Tietge et al., 2000). AMPHITOX is a set of four standardized tests employing amphibian embryos that can be used to evaluate toxicity for acute (AMPHIACUT), shortterm chronic (AMPHISHORT), chronic (AMPHICHRO) and early life stage (AMPHIEMB) exposure to hazardous substances and samples (Herkovits and Pe´rez-Coll, 1999). In the present study, we report the
Fig. 1. Location of the samples obtained from rivers and streams in Buenos Aires neighborhood.
J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183
evaluation of toxicity of different environmental samples such as surface, groundwater, leaches, soils and industrial effluents by adjusting the AMPHITOX exposure period according to the toxicity of the sample.
2. Material and methods 2.1. Sample collection Environmental samples were obtained from the neighborhood of Buenos Aires City and were grouped in (1) surface water, (2) groundwater, (3) industrial effluents, (4) leaches (obtained from landfills and solid industrial wastes), and (5) soils. Samples of surface water were collected at the following stations far away from industrial or municipal discharge points (Fig 1): Table 1 Comparison of FETAX, artificial water and AMPHITOX solution water Ion
FETAX solution (mg/l)
Artificial watera (mg/l)
AMPHITOX solution (mg/l)
Na+ K+ Ca2+ Mg2+ Cl SO42 NO3 HCO3
272.0 15.7 19.4 15.2 403.0 93.3 0 69.7
0.72 0.72 8.48 1.57 0.66 26.5 0.04 1.88
14.75 0.26 0.36 0 22.71 0 0 1.45
Embryos/volume
25/10ml
a
From Tietge et al., 2000.
10/50ml
179
five samples from the Reconquista River (R1–R5) being the first of them (R1) a reference sample, five from Moron Stream (MS), a tributary of Reconquista River and nine from the Matanza-Riachuelo River and tributaries (MR1–MR9). Surface water samples were collected near the shore by means of a manual pump at a depth of 0.5 m. Groundwater samples were collected in two reference (G4 and G5) and three presumably polluted stations (G1, G2 and G3). Industrial effluents (E1–E4), leaches (L1–L4) and soil samples (S1, S2) were provided to our facility by a consultant firm in clean glass bottles maintained at 4 C. S1 was a reference sample and the industrial effluents were provided as blind samples from different activities. In the cases of soil and solid industrial wastes, a 24-h extraction procedure was applied (EPA, 1980) and toxicity test were performed with the processed material. The samples were obtained during 1994–1997 period and with the exception of surface water they were provided to the laboratory as coded samples. For each, 4 l or 1 kg (in the case of solids) were transported to the laboratory and maintained at 4 C; the toxicity tests were accomplished within the next 96 h. 2.2. Obtainment of Bufo arenarum (Hensel) embryos Bufo arenarum adult females weighing around 200– 250 g were collected in Lobos (Buenos Aires Province). Ovulation was induced by i.p injection of a suspension of one female homologous hypophysis in 2 ml of AMPHITOX’s solution, (AS), see Table 1. Oocytes were ‘‘in vitro’’ fertilized with a sperm suspension in AS. After fertilization, embryos were maintained in AS at
Fig. 2. Examples of toxicity values obtained from leaches and industrial effluents by means of AMPHITOX.
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J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183
20 1 C until the last stages of embryonic development (S. 23–25, Del Conte and Sirlin, 1951). 2.3. Toxicity tests The toxicity tests conducted to evaluate the environmental samples include three of the standardized conditions of the AMPHITOX tests: AMPHIACUT (96-h exposure), AMPHISHORT (7-day exposure) and AMPHICHRO (14 day-exposure) to evaluate acute, short-term chronic and chronic toxicity respectively (Herkovits and Perez-Coll, 1999). Batches of 10 embryos, in triplicate, were placed in 10-cm diameter glass petridishes containing 40 ml of medium and maintained at 20 1 C. Renewal of the solutions were done every 24 h. Embryonic survival was registered daily and dead embryos were removed. The exposure period was determined by the initial toxicity found in each sample. In the case of samples exerting a high toxicity, acute and short-term chronic data were reported. For samples with low toxicity, the exposure period was extended up to 14 days. Based on the preliminary toxicity results, tests employing dilutions of the samples were performed to determine the LC50 (acute) and NOEC values (short-term chronic and chronic tests). Solutions were prepared by diluting the samples with AS. Conductivity and pH were measured with a Luftman C400 conductimeter and a Luftman P300 pH meter, respectively. These measurements provided information about the water quality needed for control conditions of the toxicity bioassays. As an additional quality control for the toxicity test method, a reference
toxicant (cadmium) and the minimum criteria of acceptability including the percent coefficient of variation were evaluated. Data were statistically analyzed by means of PROBIT (US EPA, 1988) and transformed into acute Toxicity Units (TUa= 100/LC50) and short term-chronic or chronic Toxicity Units (TUstc or TUc=100/NOEC). For TUa and TUstc or TUc the maximal values recommended by USEPA for water quality control of industrial effluents are 0.3 and 1 respectively (US EPA, 1991).
3. Results and discussion Fig. 1 provides a view of the Metropolitan area of Buenos Aires city and the location of the 19 surface water samples obtained from rivers and streams in the region. The toxicity in all the samples evaluated ranged in a wide spectrum as illustrated in Figs. 2 and 3 which represent examples of the toxicity results obtained in this study. The highest toxicity was found in a sample of a leach (L2) that produced 100% lethality at a concentration of 0.9% within 24 h of exposure. The lowest toxicity (excluding no toxic samples) was registered in soil (S2), surface (R5) and groundwater (G2) samples, which exerted slight lethality only after 12 days of exposure. The coefficient of variation as a within laboratory precision data for acute, short-term chronic and chronic exposure conditions ranged between 16 and 48% that is within the accepted values for toxicity test purposes (US EPA, 1991).
Fig. 3. Examples of toxicity values obtained from surface and ground water samples by means of AMPHITOX.
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Because toxicity involves an inverse relationship to effective concentration (EC), it is helpful to view the magnitude of the hazard due to chemical stress by translating concentration-based toxicity measurements into toxicity units. In Table 2 the toxicity data for acute, short-term chronic and chronic exposure of embryos to the environmental samples evaluated are summarized. Taking into account the US EPA criteria for water quality-based toxicity control (US EPA, 1991), from a total of 34 samples studied, 19 exerted toxicity within the initial 96 h of exposure, while after 7 days of exposure the number of samples exerting lethal effects rose to 26. By expanding the exposure period to 14 days, only five samples did not exert toxicity. Among these samples with no toxicity detected, four belong to reference places (R1, G4 and G5 and S1) and therefore, 97% of the non-reference environmental samples had some measurable toxicity. The acute toxicity of the samples eval-
uated in most cases was around 2 TUa and represent 7 times over the maximal value recommended by US EPA for industrial effluents. For short-term chronic exposures in most cases the TUstc was between 1.2 and 20 times higher than that reference value for US EPA. For samples which exerted toxic effects only after chronic exposure, the toxicity ranged between 1.11 and 1.43 times over the maximal value recommended by US EPA for water quality-based toxic control in the case of chronic exposure. This result supports the suggestion from EPA that chronic toxicity test could be shortened to 7-day exposure protocols by focusing on the most sensitive life-stages (US EPA, 1991). As expected, the TUc/TUa ratios were 1 or higher, and the increase in this value could reflect a lower uptake rate of the pollutants, their delayed toxic effects on the embryos or both. The MR6 sample, a tributary of Matanza-Riachuelo River (Del Rey Stream), was not toxic although the
Table 2 Acute, short-term chronic and chronic toxicity data of different environmental samples obtained by means of AMPHITOX testa Sample (Group)
R1(a)b R2 (a) R3(a) R4(a) R5(a) MS1(a) MS2(a) MS3(a) MS4(a) MS5(a) MR1(a) MR2(a) MR3(a) MR4(a) MR5(a) MR6(a) MR7(a) MR8(a) MR9(a) G1(b) G2(b G3(b) G4(b)b G5(b)b E1 (c) E2 (c) E3(c) E4(c) L1(d) L2 (d) L3(d) L4(d) S1 (e)b S2 (e)
TUa (96 h) (Acute test)
TUstc (7 days) (Short-term chronic test)
TUc (14 days) (Chronic test)
No toxic No toxic No toxic No toxic No toxic 2.50 3.30 2.20 1.42 2.85 2.85 1.25 2 2.50 3.30 No toxic 1.42 1.33 1.67 No toxic No toxic No toxic No toxic No toxic 0.77 1.67 No toxic 5.88 10 200 No toxic 5.88 No toxic No toxic
No toxic 3.33 2 1.25 No toxic 14.29 20 14.29 8.33 16.67 5 5 5 2.5 10 No toxic 1.67 1.67 1.67 2 No toxic 1.33 No toxic No toxic 20 2.22 1.43 20 100 1000 1.11 10 No toxic No toxic
No toxic 10 5 2.5 1.25 Ne Ne Ne Ne Ne Ne Ne Ne Ne Ne No toxic Ne Ne Ne 5 1.43 2.5 No toxic No toxic Ne Ne 2 Ne Ne Ne 1.18 Ne No toxic 1.11
TUstc/TUa
– – – – – 5.72 6.06 6.50 5.87 5.85 1.75 4 2.5 1 3 – 1.18 1.26 1 – – – – – 25.97 1.33 4.77 3.40 10 5 – 1.72 – –
a Results are expressed as TUa, TUstc, TUc and TUstc/TUa ratios. (a) surface water; (b) groundwater; (c) industrial effluents; (d) leaches; (e) soil. Ne: No evaluated due to the toxicity found in acute and short-term chronic tests. b Reference samples.
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water of this place appeared with a non-pristine brown color. The results obtained conducting AMPHITOX as a customized test according to the toxicity of the sample points out the possibility to evaluate toxicity of samples with a wide range of different features. The toxicity of some of the samples (R1–R5) were also evaluated by means of a fish toxicity test employing Cnesterodon decemmaculatus, a sympatric specie of Bufo arenarum and in all cases the amphibian embryos were more sensitive at least by one order of magnitude (data not shown). Similar levels of higher susceptibility for amphibian embryos compared to fishes was found in studies conducted with single chemicals (Herkovits and Pe´rez-Coll, 1998). Based on stage-dependent susceptibility criteria, several studies have been performed to find out the most susceptible developmental stages to xenobiotics. Although, the last developmental stages of the embryos are not the most susceptible to xenobiotics (Pe´rez-Coll et al., 1988, Herkovits and Perez-Coll, 1993; Pe´rez-Coll and Herkovits, 1996), in our experience, they provide several advantages for routine toxicity screening purposes: (1) the relatively constant sensitivity to xenobiotics of embryos from stage 23 onwards which allows to conduct the preliminary and definitive test with embryos providing from the same couple (2) the very low mortality baseline (less than 10%) registered during all this period of about 20 days which facilitates to extend the toxicity assessment at least to 14 days of exposure, (3) the convenience of the biological material which allows register mortality with a limited expertise compared to teratological studies. In a recent study, Tietge et al. (2000) found out that inherent variability in water quality of field-collected samples could result in artifactual developmental effects when using FETAX. These artifacts were related to the low volume and high concentration of certain salts in the maintaining media of FETAX test, conditions which do not apply in the case of AMPHITOX (Table 1). The toxicity found in the samples from rivers and streams in the Metropolitan area of Buenos Aires city points out the need for enhanced stewardship of chemical substances for environment and human health protection in this urban ecosystem.
Acknowledgements The AMPHITOX test standardization was performed with a grant from the Pan American Health Organization and the Secretary of Science and Technology, Argentina. This study was financially supported by the Grupo de Investigaciones en Seguridad Quı´mica (CONICET) and Fundacio´n PROSAMA. The authors wish to thank Peter Daniel, Monica Stampacchio, Marcelo Merino and Miguel Angel Gomez Peral for their cooperation during the surface water sampling and
to Oscar Jimenez and Jorge Aragon from GEOCONSULTA S.A. for their contribution in the digitalization of the Buenos Aires metropolitan map. We also wish to thank the comments and editorial help provided by the reviewers. References Baringa, M., 1990. Where have all the froggies gone? Science 247, 1033–1034. Black, H., 1994. The price of progress: environmental health in Latin America. Environ. Health Perspect. 102, 1024–1028. Blaustein, A.R., Wake, D.B., Sousa, W.P., 1994. Amphibian declines: judging stability, persistence and susceptibility of populations to local and global extinctions. Conserv. Biol. 8, 60–71. Boyer, R., Grue, C.E., 1995. The need for water quality criteria for frogs. Environ. Health Perspect. 103, 352–355. Burkhart, J.G., Helgen, J.C., Fort, D., Gallagher, K., Bowers, D., Propst, T.L., Gemes, M., Magner, J., Shelby, M.D., Lucier, G., 1998. Induction of Xenopus laevis embryo mortality and malformation by water sources associated with field frog deformities. Environ. Health Perspect. 106, 841–848. Burkhart, J.G., Ankley, G., Bell, H., Carpenter, H., Fort, D., Gardiner, D., Gardner, H., Hale, R., Helgen, J.C., Jepson, P., Johnson, D., Lannoo, M., Lee, D., Lary, J., Levey, R., Magner, J., Meteyer, C., Shelby, M., Lucier, G., 2000. Strategies for assessing the implications of malformed frogs for environmental health. Environ. Health Perspect. 108 (1), 83–90. Cairns Jr., J., 1994. Third wave ecotoxicology. Ecotoxicology 3, 1–3. Cairns, J., Niederlehner, B.R., 1994. Predictive ecotoxicology. Handbook of Ecotoxicology. CRC Press, Lewis Publishers, Boca Raton, pp. 667–677. Cook, A.S., 1981. Tadpoles as indicators of harmful levels of pollution in the field. Environ. Poll. 25, 123–133. Costanza, R., d’Arge, R., de Groot, R., Farber, S., Grasso, M., Hannon, B., Limburg, K., Naeem, S., O’Neill, R., Paruelo, J., Raskion, R.G., Sutton, P., van der Bel, M., 1997. The value of the world ecosystem services and natural capital. Nature 387, 253–260. Dawson, D.A., McCornick, C.A., Bantle, J.A., 1985. Detection of teratogenic substances in acidic mine water samples using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). J. Appl. Toxicol. 5, 234–244. Del Conte, E., Sirlin, L., 1951. The first stages of Bufo arenarum development. Acta Zool. Lilloana 12, 495–499. EPA. SW 846 (1980) Test methods for evaluating solid wastes. Fort, D.J., Rogers, R.L., Copley, H.F., Bruning, L.A., Stover, E.L., Helgen, J.C., Burkhart, J.G., 1999. Progress toward identifying causes of maldevelopment induced in Xenopus by pond water and sediment extracts from Minnesota, USA. Env. Toxicol. Chem. 18 (10), 2316–2324. Herkovits, J., Pe´rez-Coll, C., Zeni, S., 1989. Protective effect of zinc against spontaneous malformations and lethality in Bufo arenarum embryos. Biol. Trace Elem. Res. 25, 247–251. Herkovits, J., Pe´rez-Coll, C.S., 1990. Zinc protection against delayed development produced by cadmium. Biol. Trace Elem. Res. 24, 217– 221. Herkovits, J., Pe´rez-Coll, C.S., 1991. Antagonism and synergism between lead and zinc in amphibian larvae. Environ. Poll. 69, 217– 221. Herkovits, J., Pe´rez-Coll, C.S., 1993. Stage-dependent susceptibility to cadmium on Bufo arenarum embryos. Bull. Environ. Contam. Toxicol. 50, 608–611. Herkovits, J., Pe´rez-Coll, C.S., Herkovits, F.D., 1996. Ecotoxicity in Reconquista River (Province of Buenos Aires): a preliminary study. Environ. Health Perspect 104 (2), 186–189.
J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183 Herkovits, J., 1997. Sustentabilidad: preservacio´n de los servicios del ecosistema. Gerencia Ambiental. 34, 274–276. and 343. Herkovits, J., Herkovits, F.D., Perez-Coll, C.S., 1997. Identification of aluminum hazard and aluminum-zinc antagonism in amphibian (Bufo arenarum) embryos. Environ. Sciences 5 (1), 57–64. Herkovits, J., Helguero, A.L., 1998. Copper toxicity and copperzinc interactions in amphibian embryos. Sci. Total Environ. 221, 1–10. Herkovits, J., Pe´rez-Coll, C.S., 1998. Toxicidad comparada de metales para Cnesterodon decemmaculatus., Abstract Book TR1O, 1st SETAC Meeting, Latinamerican Branch, Argentina Section, pp. 42. Herkovits, J., Pe´rez-Coll, C.S., 1999. Bioensayos para test de toxicidad con embriones de anfibio (‘‘ANFITOX’’). Ing. Sanitaria y Ambiental 42 (24–30) and 43 (50–55). Herkovits, J., Pe´rez-Coll, C.S., Herkovits, F.D., 2000. Evaluation of nickel-zinc interactions by means of bioassays with amphibian embryos. Ecotoxicol. Environ. Saf. 45, 266–273. Pe´rez-Coll, C.S., Herkovits, J., Salibia´n, A., 1988. Embryotoxicity of lead on Bufo arenarum. Bull. Environm. Contam. Toxicol. 41, 247– 252. Pe´rez-Coll, C.S., Herkovits, J., 1990. Stage-dependent susceptibility to lead in Bufo arenarum. embryos. Environ. Poll. 63, 239–245.
183
Pe´rez-Coll, C.S., Herkovits, J., 1996. Stage-dependent uptake of cadmium in Bufo arenarum embryos. Bull. Environ. Contam. Toxicol. 56 (4), 663–669. Simms, C., 1969. Indications of the decline of breeding amphibians at an isolated pond in marginal land, 1954–1967. Br. J. Herpetol. 4, 93–96. Strong M.F.(1994). Science and a sustainable earth. Professor C.G. Bernhard Lecture, Royal Swedish Academy of Science. Tietge, J.E., Ankley, G.T., DeFoe, D.L, Holcombe, G.W., Jensen, K.M, 2000. Effects of water quality on development of Xenopus laevis: a frog embryo teratogenesis assay-Xenopus assessment of surface water associated with malformations in native anurans. Environ. Toxicol. Chem. 19, 2114–2121. US EPA, 1988. Users guide for a computer program for Probit analisis of data from acute and short-term chronic toxicity test with aquatic organisms. Biological Methods, Environmental Monitoring and Support Lab. US EPA, 1991. Technical support document for water quality-based toxic control. EPA 505-2-90-001. Environmental Protection Agency, Washington, DC. Vismara, C., Bernardini, G., Bonfanti, P., Colombo, A., Camatini, M., 1993. The use of in vitro fertilization in the frog embryo teratogenesis assay in Xenopus (FETAX) and its applications to ecotoxicology. Sci. Total Environ.1993, 787–790.
Ecotoxicological studies of environmental samples from Buenos Aires area using a standardized amphibian embryo toxicity test (AMPHITOX) Jorge Herkovitsa,*,1, Cristina Perez-Colla,1, Francisco D. Herkovitsa a
Programa de Seguridad Quı´mica, Instituto de Ciencias Ambientales y Salud (ICAS). Fundacio´n PROSAMA, Paysandu´ 752, (1405) Buenos Aires, Argentina. Received 28 December 2000; accepted 1 May 2001
‘‘Capsule’’: AMPHITOX was found to be useful in evaluating toxicity of a wide variety of samples. Abstract The toxicity of 34 environmental samples from potentially polluted and reference stations were evaluated by means of the AMPHITOX test from acute to chronic exposure according to the toxicity found in each sample. The samples were obtained from surface and ground water, leaches, industrial effluents and soils. The data, expressed in acute, short-term chronic and chronic Toxicity Units (TUa, TUstc and TUc) resulted in a maximal value of 1000 TUc, found in a leach, while the lower toxicity value was 1.4 TUa corresponding to two surface water samples. In five samples (four providing from reference places) no toxicity was detected. The results point out the possibility of evaluating the toxicity of a wide diversity of samples by means of AMPHITOX as a customized toxicity test. The fact that almost all samples with suspected toxicity in rivers and streams from the Metropolitan area of Buenos Aires city resulted toxic, indicates the need of enhanced stewardship of chemical substances for environmental and human health protection purposes. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: AMPHITOX; Toxicity test; Amphibian embryos; Environmental pollution; Buenos Aires Metropolitan area
1. Introduction Environmental contamination in general has increased in concordance with the growing pressures on the Earth resources and life support systems resulting from escalating human numbers and activities combined with inappropriate environmental management (Cairns, 1994; Strong, 1994; Herkovits et al., 1996). These facts are of major concern because ecosystem services, essential to the quality of human life, could be severely affected while and, on the other hand, there is an ethical responsibility to also preserve attributes important for species diversity conservation purposes (Black, 1994; Cairns and Niederlehner, 1994, Costanza et al., 1997; Herkovits, 1997).
* Corresponding author. Tel.: +54-11-4431-2445; fax: +54-114432-1111. E-mail address: [email protected] (J. Herkovits). 1 Members of the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina.
The aquatic environment is particularly important as a recipient, transporting medium and sink for the majority of xenobiotic chemicals. The studies on the fate and transport of pollutants in the environment point out the close-in problems for air, soil, sediments, surface and groundwater as interrelated compartments. In order to cope with such a wide range of environmental situations and to have the possibility to compare results from different matrixes, there is a need for more sensitive, cost-effective and short-term biological test methods. For water quality-based toxicity control purposes, the US EPA found a good predictive correlation between embryo-larval survival and independent ecological parameters, such as species richness and diversity (US EPA, 1991). Moreover, by focusing on the most sensitive life-cycle stages, the US EPA suggested that chronic toxicity tests could be shortened to 7-day exposure protocols for the evaluation of industrial effluents. A worldwide decline of amphibian populations linked to the increasing environmental degradation is widely documented (Simms, 1969; Cook, 1981; Baringa, 1990; Blaustein et al., 1994; Boyer and Grue, 1995; Burkhart
0269-7491/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00167-1
178
J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183
et al., 1998) and adverse effects, such as malformations and disruption of metamorphosis, could be related to either to the presence of developmental toxicants or the absence of essential micronutrients (Dawson et al., 1985; Herkovits et al., 1989, Vismara et al., 1993; Fort et al., 1999, Burkhart et al., 2000; Tietge et al., 2000). Although aquatic toxicology has focused mainly on concerns for fishes, invertebrates and algae (e.g. US EPA, 1991), studies exploring the response of amphibians to environmental pollutants found that they are usually highly sensitive and appropriate for testing single chemicals
and complex mixtures (Pe´rez-Coll et al., 1988, Pe´rezColl and Herkovits, 1990; Herkovits and Pe´rez-Coll, 1990, 1991, 1993; Herkovits and Helguero, 1998; Herkovits et al., 1996, 1997, 2000; Burkhart et al., 2000; Tietge et al., 2000). AMPHITOX is a set of four standardized tests employing amphibian embryos that can be used to evaluate toxicity for acute (AMPHIACUT), shortterm chronic (AMPHISHORT), chronic (AMPHICHRO) and early life stage (AMPHIEMB) exposure to hazardous substances and samples (Herkovits and Pe´rez-Coll, 1999). In the present study, we report the
Fig. 1. Location of the samples obtained from rivers and streams in Buenos Aires neighborhood.
J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183
evaluation of toxicity of different environmental samples such as surface, groundwater, leaches, soils and industrial effluents by adjusting the AMPHITOX exposure period according to the toxicity of the sample.
2. Material and methods 2.1. Sample collection Environmental samples were obtained from the neighborhood of Buenos Aires City and were grouped in (1) surface water, (2) groundwater, (3) industrial effluents, (4) leaches (obtained from landfills and solid industrial wastes), and (5) soils. Samples of surface water were collected at the following stations far away from industrial or municipal discharge points (Fig 1): Table 1 Comparison of FETAX, artificial water and AMPHITOX solution water Ion
FETAX solution (mg/l)
Artificial watera (mg/l)
AMPHITOX solution (mg/l)
Na+ K+ Ca2+ Mg2+ Cl SO42 NO3 HCO3
272.0 15.7 19.4 15.2 403.0 93.3 0 69.7
0.72 0.72 8.48 1.57 0.66 26.5 0.04 1.88
14.75 0.26 0.36 0 22.71 0 0 1.45
Embryos/volume
25/10ml
a
From Tietge et al., 2000.
10/50ml
179
five samples from the Reconquista River (R1–R5) being the first of them (R1) a reference sample, five from Moron Stream (MS), a tributary of Reconquista River and nine from the Matanza-Riachuelo River and tributaries (MR1–MR9). Surface water samples were collected near the shore by means of a manual pump at a depth of 0.5 m. Groundwater samples were collected in two reference (G4 and G5) and three presumably polluted stations (G1, G2 and G3). Industrial effluents (E1–E4), leaches (L1–L4) and soil samples (S1, S2) were provided to our facility by a consultant firm in clean glass bottles maintained at 4 C. S1 was a reference sample and the industrial effluents were provided as blind samples from different activities. In the cases of soil and solid industrial wastes, a 24-h extraction procedure was applied (EPA, 1980) and toxicity test were performed with the processed material. The samples were obtained during 1994–1997 period and with the exception of surface water they were provided to the laboratory as coded samples. For each, 4 l or 1 kg (in the case of solids) were transported to the laboratory and maintained at 4 C; the toxicity tests were accomplished within the next 96 h. 2.2. Obtainment of Bufo arenarum (Hensel) embryos Bufo arenarum adult females weighing around 200– 250 g were collected in Lobos (Buenos Aires Province). Ovulation was induced by i.p injection of a suspension of one female homologous hypophysis in 2 ml of AMPHITOX’s solution, (AS), see Table 1. Oocytes were ‘‘in vitro’’ fertilized with a sperm suspension in AS. After fertilization, embryos were maintained in AS at
Fig. 2. Examples of toxicity values obtained from leaches and industrial effluents by means of AMPHITOX.
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20 1 C until the last stages of embryonic development (S. 23–25, Del Conte and Sirlin, 1951). 2.3. Toxicity tests The toxicity tests conducted to evaluate the environmental samples include three of the standardized conditions of the AMPHITOX tests: AMPHIACUT (96-h exposure), AMPHISHORT (7-day exposure) and AMPHICHRO (14 day-exposure) to evaluate acute, short-term chronic and chronic toxicity respectively (Herkovits and Perez-Coll, 1999). Batches of 10 embryos, in triplicate, were placed in 10-cm diameter glass petridishes containing 40 ml of medium and maintained at 20 1 C. Renewal of the solutions were done every 24 h. Embryonic survival was registered daily and dead embryos were removed. The exposure period was determined by the initial toxicity found in each sample. In the case of samples exerting a high toxicity, acute and short-term chronic data were reported. For samples with low toxicity, the exposure period was extended up to 14 days. Based on the preliminary toxicity results, tests employing dilutions of the samples were performed to determine the LC50 (acute) and NOEC values (short-term chronic and chronic tests). Solutions were prepared by diluting the samples with AS. Conductivity and pH were measured with a Luftman C400 conductimeter and a Luftman P300 pH meter, respectively. These measurements provided information about the water quality needed for control conditions of the toxicity bioassays. As an additional quality control for the toxicity test method, a reference
toxicant (cadmium) and the minimum criteria of acceptability including the percent coefficient of variation were evaluated. Data were statistically analyzed by means of PROBIT (US EPA, 1988) and transformed into acute Toxicity Units (TUa= 100/LC50) and short term-chronic or chronic Toxicity Units (TUstc or TUc=100/NOEC). For TUa and TUstc or TUc the maximal values recommended by USEPA for water quality control of industrial effluents are 0.3 and 1 respectively (US EPA, 1991).
3. Results and discussion Fig. 1 provides a view of the Metropolitan area of Buenos Aires city and the location of the 19 surface water samples obtained from rivers and streams in the region. The toxicity in all the samples evaluated ranged in a wide spectrum as illustrated in Figs. 2 and 3 which represent examples of the toxicity results obtained in this study. The highest toxicity was found in a sample of a leach (L2) that produced 100% lethality at a concentration of 0.9% within 24 h of exposure. The lowest toxicity (excluding no toxic samples) was registered in soil (S2), surface (R5) and groundwater (G2) samples, which exerted slight lethality only after 12 days of exposure. The coefficient of variation as a within laboratory precision data for acute, short-term chronic and chronic exposure conditions ranged between 16 and 48% that is within the accepted values for toxicity test purposes (US EPA, 1991).
Fig. 3. Examples of toxicity values obtained from surface and ground water samples by means of AMPHITOX.
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Because toxicity involves an inverse relationship to effective concentration (EC), it is helpful to view the magnitude of the hazard due to chemical stress by translating concentration-based toxicity measurements into toxicity units. In Table 2 the toxicity data for acute, short-term chronic and chronic exposure of embryos to the environmental samples evaluated are summarized. Taking into account the US EPA criteria for water quality-based toxicity control (US EPA, 1991), from a total of 34 samples studied, 19 exerted toxicity within the initial 96 h of exposure, while after 7 days of exposure the number of samples exerting lethal effects rose to 26. By expanding the exposure period to 14 days, only five samples did not exert toxicity. Among these samples with no toxicity detected, four belong to reference places (R1, G4 and G5 and S1) and therefore, 97% of the non-reference environmental samples had some measurable toxicity. The acute toxicity of the samples eval-
uated in most cases was around 2 TUa and represent 7 times over the maximal value recommended by US EPA for industrial effluents. For short-term chronic exposures in most cases the TUstc was between 1.2 and 20 times higher than that reference value for US EPA. For samples which exerted toxic effects only after chronic exposure, the toxicity ranged between 1.11 and 1.43 times over the maximal value recommended by US EPA for water quality-based toxic control in the case of chronic exposure. This result supports the suggestion from EPA that chronic toxicity test could be shortened to 7-day exposure protocols by focusing on the most sensitive life-stages (US EPA, 1991). As expected, the TUc/TUa ratios were 1 or higher, and the increase in this value could reflect a lower uptake rate of the pollutants, their delayed toxic effects on the embryos or both. The MR6 sample, a tributary of Matanza-Riachuelo River (Del Rey Stream), was not toxic although the
Table 2 Acute, short-term chronic and chronic toxicity data of different environmental samples obtained by means of AMPHITOX testa Sample (Group)
R1(a)b R2 (a) R3(a) R4(a) R5(a) MS1(a) MS2(a) MS3(a) MS4(a) MS5(a) MR1(a) MR2(a) MR3(a) MR4(a) MR5(a) MR6(a) MR7(a) MR8(a) MR9(a) G1(b) G2(b G3(b) G4(b)b G5(b)b E1 (c) E2 (c) E3(c) E4(c) L1(d) L2 (d) L3(d) L4(d) S1 (e)b S2 (e)
TUa (96 h) (Acute test)
TUstc (7 days) (Short-term chronic test)
TUc (14 days) (Chronic test)
No toxic No toxic No toxic No toxic No toxic 2.50 3.30 2.20 1.42 2.85 2.85 1.25 2 2.50 3.30 No toxic 1.42 1.33 1.67 No toxic No toxic No toxic No toxic No toxic 0.77 1.67 No toxic 5.88 10 200 No toxic 5.88 No toxic No toxic
No toxic 3.33 2 1.25 No toxic 14.29 20 14.29 8.33 16.67 5 5 5 2.5 10 No toxic 1.67 1.67 1.67 2 No toxic 1.33 No toxic No toxic 20 2.22 1.43 20 100 1000 1.11 10 No toxic No toxic
No toxic 10 5 2.5 1.25 Ne Ne Ne Ne Ne Ne Ne Ne Ne Ne No toxic Ne Ne Ne 5 1.43 2.5 No toxic No toxic Ne Ne 2 Ne Ne Ne 1.18 Ne No toxic 1.11
TUstc/TUa
– – – – – 5.72 6.06 6.50 5.87 5.85 1.75 4 2.5 1 3 – 1.18 1.26 1 – – – – – 25.97 1.33 4.77 3.40 10 5 – 1.72 – –
a Results are expressed as TUa, TUstc, TUc and TUstc/TUa ratios. (a) surface water; (b) groundwater; (c) industrial effluents; (d) leaches; (e) soil. Ne: No evaluated due to the toxicity found in acute and short-term chronic tests. b Reference samples.
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water of this place appeared with a non-pristine brown color. The results obtained conducting AMPHITOX as a customized test according to the toxicity of the sample points out the possibility to evaluate toxicity of samples with a wide range of different features. The toxicity of some of the samples (R1–R5) were also evaluated by means of a fish toxicity test employing Cnesterodon decemmaculatus, a sympatric specie of Bufo arenarum and in all cases the amphibian embryos were more sensitive at least by one order of magnitude (data not shown). Similar levels of higher susceptibility for amphibian embryos compared to fishes was found in studies conducted with single chemicals (Herkovits and Pe´rez-Coll, 1998). Based on stage-dependent susceptibility criteria, several studies have been performed to find out the most susceptible developmental stages to xenobiotics. Although, the last developmental stages of the embryos are not the most susceptible to xenobiotics (Pe´rez-Coll et al., 1988, Herkovits and Perez-Coll, 1993; Pe´rez-Coll and Herkovits, 1996), in our experience, they provide several advantages for routine toxicity screening purposes: (1) the relatively constant sensitivity to xenobiotics of embryos from stage 23 onwards which allows to conduct the preliminary and definitive test with embryos providing from the same couple (2) the very low mortality baseline (less than 10%) registered during all this period of about 20 days which facilitates to extend the toxicity assessment at least to 14 days of exposure, (3) the convenience of the biological material which allows register mortality with a limited expertise compared to teratological studies. In a recent study, Tietge et al. (2000) found out that inherent variability in water quality of field-collected samples could result in artifactual developmental effects when using FETAX. These artifacts were related to the low volume and high concentration of certain salts in the maintaining media of FETAX test, conditions which do not apply in the case of AMPHITOX (Table 1). The toxicity found in the samples from rivers and streams in the Metropolitan area of Buenos Aires city points out the need for enhanced stewardship of chemical substances for environment and human health protection in this urban ecosystem.
Acknowledgements The AMPHITOX test standardization was performed with a grant from the Pan American Health Organization and the Secretary of Science and Technology, Argentina. This study was financially supported by the Grupo de Investigaciones en Seguridad Quı´mica (CONICET) and Fundacio´n PROSAMA. The authors wish to thank Peter Daniel, Monica Stampacchio, Marcelo Merino and Miguel Angel Gomez Peral for their cooperation during the surface water sampling and
to Oscar Jimenez and Jorge Aragon from GEOCONSULTA S.A. for their contribution in the digitalization of the Buenos Aires metropolitan map. We also wish to thank the comments and editorial help provided by the reviewers. References Baringa, M., 1990. Where have all the froggies gone? Science 247, 1033–1034. Black, H., 1994. The price of progress: environmental health in Latin America. Environ. Health Perspect. 102, 1024–1028. Blaustein, A.R., Wake, D.B., Sousa, W.P., 1994. Amphibian declines: judging stability, persistence and susceptibility of populations to local and global extinctions. Conserv. Biol. 8, 60–71. Boyer, R., Grue, C.E., 1995. The need for water quality criteria for frogs. Environ. Health Perspect. 103, 352–355. Burkhart, J.G., Helgen, J.C., Fort, D., Gallagher, K., Bowers, D., Propst, T.L., Gemes, M., Magner, J., Shelby, M.D., Lucier, G., 1998. Induction of Xenopus laevis embryo mortality and malformation by water sources associated with field frog deformities. Environ. Health Perspect. 106, 841–848. Burkhart, J.G., Ankley, G., Bell, H., Carpenter, H., Fort, D., Gardiner, D., Gardner, H., Hale, R., Helgen, J.C., Jepson, P., Johnson, D., Lannoo, M., Lee, D., Lary, J., Levey, R., Magner, J., Meteyer, C., Shelby, M., Lucier, G., 2000. Strategies for assessing the implications of malformed frogs for environmental health. Environ. Health Perspect. 108 (1), 83–90. Cairns Jr., J., 1994. Third wave ecotoxicology. Ecotoxicology 3, 1–3. Cairns, J., Niederlehner, B.R., 1994. Predictive ecotoxicology. Handbook of Ecotoxicology. CRC Press, Lewis Publishers, Boca Raton, pp. 667–677. Cook, A.S., 1981. Tadpoles as indicators of harmful levels of pollution in the field. Environ. Poll. 25, 123–133. Costanza, R., d’Arge, R., de Groot, R., Farber, S., Grasso, M., Hannon, B., Limburg, K., Naeem, S., O’Neill, R., Paruelo, J., Raskion, R.G., Sutton, P., van der Bel, M., 1997. The value of the world ecosystem services and natural capital. Nature 387, 253–260. Dawson, D.A., McCornick, C.A., Bantle, J.A., 1985. Detection of teratogenic substances in acidic mine water samples using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). J. Appl. Toxicol. 5, 234–244. Del Conte, E., Sirlin, L., 1951. The first stages of Bufo arenarum development. Acta Zool. Lilloana 12, 495–499. EPA. SW 846 (1980) Test methods for evaluating solid wastes. Fort, D.J., Rogers, R.L., Copley, H.F., Bruning, L.A., Stover, E.L., Helgen, J.C., Burkhart, J.G., 1999. Progress toward identifying causes of maldevelopment induced in Xenopus by pond water and sediment extracts from Minnesota, USA. Env. Toxicol. Chem. 18 (10), 2316–2324. Herkovits, J., Pe´rez-Coll, C., Zeni, S., 1989. Protective effect of zinc against spontaneous malformations and lethality in Bufo arenarum embryos. Biol. Trace Elem. Res. 25, 247–251. Herkovits, J., Pe´rez-Coll, C.S., 1990. Zinc protection against delayed development produced by cadmium. Biol. Trace Elem. Res. 24, 217– 221. Herkovits, J., Pe´rez-Coll, C.S., 1991. Antagonism and synergism between lead and zinc in amphibian larvae. Environ. Poll. 69, 217– 221. Herkovits, J., Pe´rez-Coll, C.S., 1993. Stage-dependent susceptibility to cadmium on Bufo arenarum embryos. Bull. Environ. Contam. Toxicol. 50, 608–611. Herkovits, J., Pe´rez-Coll, C.S., Herkovits, F.D., 1996. Ecotoxicity in Reconquista River (Province of Buenos Aires): a preliminary study. Environ. Health Perspect 104 (2), 186–189.
J. Herkovits et al. / Environmental Pollution 116 (2002) 177–183 Herkovits, J., 1997. Sustentabilidad: preservacio´n de los servicios del ecosistema. Gerencia Ambiental. 34, 274–276. and 343. Herkovits, J., Herkovits, F.D., Perez-Coll, C.S., 1997. Identification of aluminum hazard and aluminum-zinc antagonism in amphibian (Bufo arenarum) embryos. Environ. Sciences 5 (1), 57–64. Herkovits, J., Helguero, A.L., 1998. Copper toxicity and copperzinc interactions in amphibian embryos. Sci. Total Environ. 221, 1–10. Herkovits, J., Pe´rez-Coll, C.S., 1998. Toxicidad comparada de metales para Cnesterodon decemmaculatus., Abstract Book TR1O, 1st SETAC Meeting, Latinamerican Branch, Argentina Section, pp. 42. Herkovits, J., Pe´rez-Coll, C.S., 1999. Bioensayos para test de toxicidad con embriones de anfibio (‘‘ANFITOX’’). Ing. Sanitaria y Ambiental 42 (24–30) and 43 (50–55). Herkovits, J., Pe´rez-Coll, C.S., Herkovits, F.D., 2000. Evaluation of nickel-zinc interactions by means of bioassays with amphibian embryos. Ecotoxicol. Environ. Saf. 45, 266–273. Pe´rez-Coll, C.S., Herkovits, J., Salibia´n, A., 1988. Embryotoxicity of lead on Bufo arenarum. Bull. Environm. Contam. Toxicol. 41, 247– 252. Pe´rez-Coll, C.S., Herkovits, J., 1990. Stage-dependent susceptibility to lead in Bufo arenarum. embryos. Environ. Poll. 63, 239–245.
183
Pe´rez-Coll, C.S., Herkovits, J., 1996. Stage-dependent uptake of cadmium in Bufo arenarum embryos. Bull. Environ. Contam. Toxicol. 56 (4), 663–669. Simms, C., 1969. Indications of the decline of breeding amphibians at an isolated pond in marginal land, 1954–1967. Br. J. Herpetol. 4, 93–96. Strong M.F.(1994). Science and a sustainable earth. Professor C.G. Bernhard Lecture, Royal Swedish Academy of Science. Tietge, J.E., Ankley, G.T., DeFoe, D.L, Holcombe, G.W., Jensen, K.M, 2000. Effects of water quality on development of Xenopus laevis: a frog embryo teratogenesis assay-Xenopus assessment of surface water associated with malformations in native anurans. Environ. Toxicol. Chem. 19, 2114–2121. US EPA, 1988. Users guide for a computer program for Probit analisis of data from acute and short-term chronic toxicity test with aquatic organisms. Biological Methods, Environmental Monitoring and Support Lab. US EPA, 1991. Technical support document for water quality-based toxic control. EPA 505-2-90-001. Environmental Protection Agency, Washington, DC. Vismara, C., Bernardini, G., Bonfanti, P., Colombo, A., Camatini, M., 1993. The use of in vitro fertilization in the frog embryo teratogenesis assay in Xenopus (FETAX) and its applications to ecotoxicology. Sci. Total Environ.1993, 787–790.