Next Article in Journal
Previous Article in Journal

Int. J. Environ. Res. Public Health 2006, 3(4), 343-347;

Synergistic Effects of Copper and Butylic Ester of 2,4-Dichlorophenoxyacetic Acid (Esternon Ultra) on Amphibian Embryos
Cristina Silvia Pérez-Coll 1,2,3 and Jorge Herkovits 1,2,*
Programa de Seguridad Química, Instituto de Ciencias Ambientales y Salud (ICAS), Fundación PROSAMA, Paysandú 752, (1405) Buenos Aires, Argentina.
Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín
Correspondence to Dr. Jorge Herkovits. Email:
Received: 21 December 2005 / Accepted: 10 January 2006 / Published: 31 December 2006


: Cu2+ and butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) toxicity on Bufo arenarum embryos were evaluated by means of a short-term chronic toxicity test (AMPHITOX). The NOEC values for Cu and 2,4-D were 0.02 mg/L and 2 mg/L respectively. The toxicity profile curves for Cu and 2,4-D were reported. The interactions of the metal and the herbicide were evaluated by combined treatments with different concentrations of Cu and 2,4-D. Although in all cases, a synergistic effect between these chemicals was observed, the combination of concentrations exerting low level effects in isolated treatments resulted in more adverse embryonic survival. Considering that both products are extensively used in agroecosystems, this fact could be of concern for non target species like amphibians.
2,4-D; copper; amphibian embryos; synergistic effects


Copper and butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) are extensively employed as herbicides in the agroecosystems. Cu, an essential element, is involved both in the structure of several proteins and as enzymatic cofactor. At least 12 proteins require Cu in their structure [1]. However, in spite of its essential properties, concentrations of this heavy metal slightly higher than the homeostatic ones produce significant toxic effects mainly on reproductive processes, behaviour, skeleton and skin of different organisms. Concentrations as low as 1–2 μg Cu/L produce adverse effects on aquatic biota [2]. In this context, the Cu concentrations employed as herbicides could represent a risk for “no target” organisms [2, 3]. The main adverse effects of Cu at biochemical level occur on the structure and function of DNA and proteins, both in a direct way or mediated by means of oxidative stress mechanisms [4].

2,4-D is a phenoxyacetic herbicide that integrates one of the largest groups of herbicides sold in the world, widely used in agriculture and forestry to destroy broad leaved weeds [5]. Although its higher toxicity is on autotrophyc organisms there are numerous reports of adverse effects on a wide diversity of heterotrophyc species [2]. Gorzinski et al showed a significant diminution in the gain weight and functional and structural alterations of kidneys of rats subchronically treated with 2,4-D (15–150 mg/kg/day for 13 days) [6], and it was also reported that different 2,4-D formulations produce neurotoxic effects such as ataxia and failures in neuromuscular coordination [7, 8] probably related to changes in various neurotransmitter systems, such as serotonin and dopamine [9]. Its oxidative stress effects were reported as the diminution of glutathion and thiol-proteins, lipoperoxidation [10], increases in superoxide dismutase activity, changes in catalase, glutathione peroxidase and Glutathione S-transferase activities [11]. It has also a desacoplant effect on the oxidative phosphorilation in mitochondria [1213], and its genotoxicity was reported as chromatid and chromosome breaks, number of micronuclei and nuclear buds [5]. Most of these studies were conducted with 2,4-D in its technical grade form. In order to provide more relevant information for environmental conditions, in this study we employed “Esternon Ultra” a commercial formulate of 2,4-D because it is well known that the formulated herbicide exerts higher toxicity than its corresponding technical grade chemical.

Since the toxicity of a substance or complex mixture depends on the concentration and exposure time, threshold values taking into account these parameters are reported as Toxicity Profile curves (TOP) providing, within a systemic toxicity approach, a more appropriate set of data for hazard and risk assessment purposes [14–3]. Taking into account that the two chemicals could be frequently present in the same agroecosystems, and both exert oxidative stress, it could be relevant to know the toxicity of these chemicals in simultaneous exposure conditions. In this study we report the toxicity of copper and 2,4-D as TOP curves and evaluate the interaction between these chemicals on Bufo arenarum embryonic survival by means of the short-term chronic AMPHITOX toxicity test [1516]. The AMPHITOX test includes four different possibilities to evaluate adverse effects exerted by physico-chemical agents, including teratogenesis [1516]. The short-term chronic AMPHITOX toxicity test is not appropriate for teratogenic studies because the embryonic stage employed is post early morphogenetic processes; however it is useful to evaluate lethality as well as synergistic or antagonistic effect because the susceptibility of the embryo to noxious agents remains almost constant during the whole test period.

Material and Methods

Ovulation of Bufo arenarum females was induced by an i.p. injection of homologous hypophysis preserved according Pisanó [17]. Oocytes were fertilized “in vitro” with a sperm suspension in AMPHITOX Solution (AS); composition (in mg/L): NaCl: 36; KCl: 0.5; CaCl2: 1 and NaHCO3: 2. Embryos were maintained in AS at 20+/− 1°C until the complete operculum stage (S.25), [18]. At this developmental stage embryos were selected for the toxicity study conducting a 7 day-exposure test (short-term toxicity of AMPHITOX) [16]. Duplicate batches of 10 embryos were placed in covered 10cm glass Petri dishes containing 40 mL of AS with copper, butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) and copper plus 2,4-D in different concentrations at 20 1°C. Chemical concentrations were selected according to results obtained in preliminary studies (NOEC values for 2,4-D and Cu2+ for 7 days of exposure were 2 mg/L and 0,02 mg/L respectively). To evaluate Cu and 2,4-D toxicity, Bufo arenarum embryos were treated with Cu2+ (in mg/L): 0.03; 0.04; 0.06; 0.075 and with 2,4-D (in mg/L): 2.5; 3.5; 4.5. The toxicity of Cu and 2,4-D was reported as TOP curves from 24 and up to 168 hr. To evaluate the combined effects of the two chemicals, embryos were exposed to solutions containing Cu and 2,4-D in all the possible combinations of the concentrations stated above. Controls were duplicate groups of 10 embryos maintained in AS without additions. Survival was evaluated hour by hour during the first 12 hr of treatment and then each 24 hr up to 168 hr (7 days). Experimental solutions were prepared in AS from stock solutions of Cl2Cu (385 mg Cu2+/L), measured by means of atomic absorption spectrophotometry with flame and the commercial formulate herbicide “Esternon Ultra” (butylic ester of 2,4-dichlorophenoxyacetic acid: 797 g Eq. ac/L). Lethal concentrations were obtained by means of PROBIT analysis [19].

Results and Discussion

AMPHITOX is a standardized test employing amphibian embryos that can be used to evaluate toxicity for acute, short-term chronic, chronic, and early life stage exposure to hazardous substances and samples [15]. By means of AMPHITOX the toxicity of different substances and environmental samples such as surface, groundwater, soils, leaches and industrial effluents can be evaluated by adjusting the exposure period to the toxicity of the sample [1620].

Figure 1 shows the TOPs curves of Cu from 24 to 168 hr for Bufo arenarum embryos. For copper, the LC50 24 and 168 hr were 0.12 and 0.05 mg Cu2+/L respectively. The LCs are within the range obtained in previous studies [321]. The LC10 for 168 hr resulted in 0.022 mg/L. For freshwater invertebrates, 48-h L(E)C50 range from 5 μg Cu/L for a daphnid species to 5300 μg Cu/L for an ostracod [2]. For freshwater fish 96-h LC50s range from 3 μg Cu/L (artic grayling) to 7340 μg Cu/L (bluegill), [2]. Although the concentrations exerting adverse effects on Bufo arenarum embryos are within the range reported as maximal value found in unpolluted aquatic ecosystems, that is from 1 to 20 μg Cu/L [22], the bioavailability of copper as well as antagonistic effect of other compounds like Zn [3] could explain the presumably no adverse effect on amphibians in those pristine environments due to copper toxicity.

Figure 2 shows the TOPs curves of 2,4-D from 24 to 168 hr for Bufo arenarum embryos. The LC50 24 and 168 hr resulted in 4 and 3 mg 2,4-D/L respectively. Morgan et al., reported for another amphibian, Xenopus laevis, a LC50 of 254 mg/L 2,4-D [23]. This value, almost 70 times higher than the obtained for Bufo arenarum embryos in this study, could be related to different susceptibility inherent to each species. In a recent contribution, the difference in the susceptibility to physico-chemical stress was partially related to the record in living organism of evolutionary events during their phylogenetic process [24]. In addition, the fact that the Xenopus laevis study was conducted with FETAX [25], a different toxicity test, could also explain at least partially this difference by taking into account: i) the high salinity of the solution employed in FETAX test which usually reduce toxic effects [16]; ii) FETAX is a 96 hr test conducted at 24±2°C while the AMPHITOX test employed is for 168 hr at 20±1°C. In mammals, different formulations of 2,4-D administered in unique oral doses, result in a LD50 of 553–1090 mg/kg depending on its chemical form, while the NOEL value was 15 mg/kg/day [6]. As a whole, for Bufo arenarum, the results point out that copper is very significantly more toxic than 2,4-D (for example, 60 times at 168 hr of exposure).

Figure 3 shows examples of the interactions evaluated for both chemicals (2,4-D: 2.5 and 4.5 mg/L with Cu: 0.03 mg/l) on the survival of Bufo arenarum embryos allowing to easily appreciate the pattern of Cu and 2,4-D synergistic effect. For example, for the more lower concentrations of both chemicals, at 168 hr of exposure, the toxicity increased approximately twice respect to the corresponding additive effect while for the highest concentrations evaluated, an earlier toxic effect was registered with an average increment of 20% of lethality from 18hs of exposure onwards. The synergistic effect of CuCl2 and a substance with 2,4-D (the Dimethylammonium 2,4-dichlorophenoxyacetate (2,4-D.DMA), usually knows as U46 D Fluid was also demonstrated in vitro employing human fibroblasts. In that study, the pretreatment with copper in subtoxic (or very slight toxic) concentrations did not affect cellular survival and the capability to generate colonies. However the treatment with both chemicals exerted cellular growth inhibition, diminution in the DNA synthesis and failure in the DNA reparation process, alterations that indicate a synergistic effect [26]. In a subsequent study the same formulation showed a synergistic effect with copper on DNA structure. Whereas 2,4-D.DMA alone or CuCl2 alone did not show any or only a negligible effect, 2,4-D.DMA plus CuCl2 induced strand breaks in PM2, probably by means of redox reaction of Cu (II) and 2,4-D [27]. Ferri et al showed in pups exposed to 2,4-D through dam’s milk, that copper was the most altered ion (among others as Fe and Zn), increasing its level in serum, liver and some brain areas and decreasing in whole brain. A general decrease in the dam’s body brain and liver weight was also reported [28]. They also observed that undernourished pups were more vulnerable to the 2,4-D effects. The interaction of Cu and 2,4-D at enzyme level was reported in the mechanisms involved in the detoxification of the herbicide by a TfdA, a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of 2,4-D [29].

The main purpose of this study was to report lethality and the eventual interactions of Cu and a formulate of 2,4-D in a native amphibian specie from South America. Thus, the AMPHITOX test selected (short-term chronic), although appropriate for that purpose is not indicated for teratological studies. It is noteworthy that both chemicals exert teratogenic effects in different species including mammals and amphibians. For Xenopus laevis the EC50 was 0.16 mg Cu2+/L with eye, gut, facial, notochord, and cardiac anomalies [30], while in mammals (e.g., mice), there are studies reporting adverse effects at developmental stages such as reduced body weight, hydrocephaly, encephalocoeles, abnormalities of the ribs and vertebrae, from 1.3 up to 159 mg Cu/kg body weight per day [31, 32].

There is an increasing concern due to the decline of amphibian populations and the large number of malformations found in many geographic regions, a fact very probably related to the multiple developmental toxicants due to anthropogenic activities that decrease water quality [3336]. For instance, by means of early life stage toxicity test of copper to endangered and surrogate species, a safety factor of 0.5 was recommended to apply to the current chronic water quality criterion (WQC) values in order to protect the most sensitive fishes [37]. A similar criterion could be of high value for amphibian species. With respect to 2,4-D, although there is very limited information on its levels in surface waters in South America, in the case of the Traiguen river basin in Southern Chile in October, 2003 it was registered 2.9 μg/L of this herbicide which is three orders of magnitude below the lethal concentrations for Bufo arenarum embryos as obtained in this study [5].

Amphibian embryos can be more susceptible to chemical stress than fishes [38] and moreover some studies point out that even water quality features in unpolluted places could be also related to adverse effects on amphibian embryos [3940]. Taking into account that 2,4-D and copper could be employed in the same environmental scenarios, the information on their synergistic effect on no target species as in this case Bufo arenarum embryos, could be valuable for the protection of endangered species and for more customized environmental risk assessment.

Ijerph 03 00343f1 1024
Figure 1. Toxicity profile (TOP) curves of copper for Bufo arenarum embryos at complete operculum stage (S.25).

Click here to enlarge figure

Figure 1. Toxicity profile (TOP) curves of copper for Bufo arenarum embryos at complete operculum stage (S.25).
Ijerph 03 00343f1 1024
Ijerph 03 00343f2 1024
Figure 2. Toxicity profile (TOP) curves of 2,4-D for Bufo arenarum embryos at complete operculum stage (S.25).

Click here to enlarge figure

Figure 2. Toxicity profile (TOP) curves of 2,4-D for Bufo arenarum embryos at complete operculum stage (S.25).
Ijerph 03 00343f2 1024
Ijerph 03 00343f3 1024
Figure 3. Survival of Bufo arenarum embryos treated with 2,4-D and copper evaluated by means of the short-term toxicity of AMPHITOX test.

Click here to enlarge figure

Figure 3. Survival of Bufo arenarum embryos treated with 2,4-D and copper evaluated by means of the short-term toxicity of AMPHITOX test.
Ijerph 03 00343f3 1024


The authors wish to thank Olga Domínguez for skillful technical assistance and Dow Agro, Argentine for providing us the herbicide. This research was financially supported by the grant PIP 02316 of the National Council of Science and Technology (CONICET, Argentine).


  1. Linder, M. C.; Hazegh-Azam, M. Copper biochemistry and molecular biology. Am. J. Clin. Nutr 1996, 63(5), 797S–811S. [Google Scholar]
  2. IPCS International Programme on Chemical Safety. Copper; Environmental Health Criteria 200. World Health Organization: Genova, 1998.
  3. Herkovits, J.; Helguero, L. A. Copper toxicity and copper-zinc interactions in amphibian embryos. Sci. Total Environ 1998, 221, 1–10. [Google Scholar]
  4. Alt, E. R.; Sternlieb, I.; Goldfisher, S. The cytopathology of metal overload. Int. Rev. Exp. Pathol 1990, 31, 165–188. [Google Scholar]
  5. Palma, G.; Sanchez, A.; Olave, Y.; Encina, F.; Palma, R.; Barra, R. Pesticide levels in surface waters in an agricultural-forestry basin in Southern Chile. Chemosphere 2004, 57(8), 763–770. [Google Scholar]
  6. Gorzinsky, S. J.; Kociba, R. J.; Campbell, R. A.; Smith, F. A.; Nolan, R. J.; Eisenbrandt, D. L. Acute, pharmacokinetic and subchronic toxicological studies of 2,4-dichlorophenoxyacetic acid. Fundam. Appl. Toxicol 1987, 9(3), 423–435. [Google Scholar]
  7. Schulze, G. E. Neurobehavioral toxicity and tolerance to the herbicide 2,4-dichlorophenoxiacetic acid-n-butyl ester (2,4-D ester). Fundam. Appl. Toxicol 1988, 10(3), 413–424. [Google Scholar]
  8. Schulze, G. E. 2,4-D-n-butyl ester (2,4-D ester) induced ataxia in rats: role for n-butanol formation. Neurotoxicol. Teratol 1988, 10(1), 81–84. [Google Scholar]
  9. Bortolozzi, A. A.; Evangelista de Duffard, A.M.; Duffard, R. O.; Antonelli, M. C. Effects of 2,4-dichlorophenoxyacetic acid exposure on dopamine D2-like receptors in rat brain. Neurotoxicol Teratol 2004, 26(4), 599–605. [Google Scholar]
  10. Palmeira, C. M.; Moreno, A. J.; Madeira, V. M. Thiols metabolism is altered by the herbicides paraquat, dinoseb and 2,4-D: a study in isolated hepatocytes. Toxicol. Lett 1995, 81(2–3), 115–123. [Google Scholar]
  11. Oruc, E. O.; Sevgiler, Y.; Uner, N. Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl. Comp. Biochem. Physiol. C 2004, 137(1), 43–51. [Google Scholar]
  12. Palmeira, C. M.; Moreno, A. J.; Madeira, V. M. Metabolic alterations in hepatocytes prompted by the herbicides paraquat, dinoseb and 2,4-D. Arch. Toxicol 1994, 68(1), 24–31. [Google Scholar]
  13. Palmeira, C. M.; Moreno, A. J.; Madeira, V. M. Interactions of herbicides 2,4-D and dinoseb with liver mitochondrial bioenergetics. Toxicol. Appl. Pharmacol 1994, 127(1), 50–57. [Google Scholar]
  14. Herkovits, J.; Herkovits, F. D.; Pérez-Coll, C. S. Identification of aluminium toxicity and Al-Zn interaction in amphibian Bufo arenarum embryos. Environm. Sci 1997, 5(1), 57–64. [Google Scholar]
  15. Herkovits, J.; Pérez-Coll, C. S. Bioensayos para test de toxicidad con embriones de anfibio “ANFITOX” basado en Bufo arenarum. Test Agudo (ANFIAGU), Crónico corto (ANFICOR), Crónico (ANFICRO) y de Estadios Tempranos del Desarrollo (ANFIEMB). Ingeniería Sanitaria y Ambiental 1999, 42. [Google Scholar]
  16. Herkovits, J.; Pérez-Coll, C. S.; Herkovits, F. D. Ecotoxicological studies of environmental samples from Buenos Aires area using a standardized amphibian embryo toxicity test (AMPHITOX). Environ. Poll 2002, 116(1), 177–183. [Google Scholar]
  17. Pisanó, A. Efficienza funzionale e struttura dell’ipofisi di anfibio. Arch. Zool. Ital 1957, 42, 221–227. [Google Scholar]
  18. Del Conte, E.; Sirlin, L. The first stages of Bufo arenarum development. Acta Zool. Lilloana 1951, 12, 495–499. [Google Scholar]
  19. U.S. EPA. Users guide for a computer program for PROBIT analysis of data from acute and short-term chronic toxicity test with aquatic organisms. Biological Methods, Environmental monitoring and Support Lab 1988.
  20. Herkovits, J.; Pérez-Coll, C. S. AMPHITOX: A customized set of toxicity tests employing amphibian embryos. Symposium on multiple stressor effects in relation to declining amphibian populations”. In Multiple Stressor Effects in Relation to Decclining Amphibian Populations; Linder, G. L., Krest, S., Sparling, D., Little, E. E., Eds.; printed in USA. 2003; pp. 46–60. [Google Scholar]
  21. Pérez-Coll, C. S.; Herkovits, J.; Fridman, O.; D’Eramo, J. L.; Corró, L. Acclimation of Bufo arenarum embryos to copper: Effects on survival and the induction of metallothioneins. Abstract Book 21stAnnual Meeting Nashville Convention Center 2000, PWP140, 274. [Google Scholar]
  22. Nriagu, J. O. Global inventory of natural and anthropogenic emissions of trace metals to the atmosphere. Nature (Lond) 1979, 279, 409–411. [Google Scholar]
  23. Morgan, M. K.; Scheuerman, P. R.; Bishop, C. S.; Pyles, R. A. Teratogenic potential of atrazine and 2,4-D using FETAX. J. Toxicol. Environ. Health 1996, 48(2), 151–168. [Google Scholar]
  24. Herkovits, J. Evoecotoxicology: Oxygen. A major driver in the evolutionary process. VII SETAC LA Meeting. Abstract Book 2005, 121, 68. [Google Scholar]
  25. American Society for Testing and Materials. Standard guide for conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). E1439-91. Annual Book of ASTM Standards, Philadelphia, PA 1992, 11.04, 1199–1209.
  26. Jacobi, H.; Witte, I. Synergistic effects of U46 D fluid (dimethylammonium salt of 2,4-D) and CuCl2on cytotoxicity and DNA repair in human fibroblasts. Toxicol. Lett 1991, 58(2), 159–167. [Google Scholar]
  27. Jacobi, H.; Metzger, J.; Witte, I. Synergistic effects of Cu(II) and dimethylammonium 2,4-dichlorophenoxyacetate (U46 D fluid) on PM2 DNA and mechanism of DNA damage. Free Radic. Res. Commun 1992, 16(2), 123–130. [Google Scholar]
  28. Ferri, A.; Duffard, R.; Sturtz, N.; Evangelista de Duffard, A.M. Iron, zinc and copper levels in brain, serum and liver of neonates exposed to 2,4-dichlorophenoxyacetic acid. Neurotoxicol. Teratol 2003, 25(5), 607–613. [Google Scholar]
  29. Hegg, E. L.; Whiting, A. K.; Saari, R. E.; McCracken, J.; Hausinger, R. P.; Que, L., Jr. Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights. Biochemistry 1999, 38(50), 16714–16726. [Google Scholar]
  30. Luo, S. Q.; Plowman, M. C.; Hopfer, S. M.; Sunderman, F. W., Jr. Embryotoxicity and teratogenicity of Cu2+and Zn2+for Xenopus laevis, assayed by the FETAX procedure. Ann. Clin. Lab. Sci 1993, 23(2), 111–120. [Google Scholar]
  31. Kasama, T.; Tanaka, H. Effects of copper administration on fetal and neonatal mice. J. Nutr. Sci. Vitam 1988, 34, 595–605. [Google Scholar]
  32. Lecyk, M. Toxicity of copper sulphate in mice embryonic development. Zool. Pol 1980, 28, 101–105. [Google Scholar]
  33. Blaustein, A. R.; Wake, D. B.; Sousa, W. P. Amphibian declines: judging stability, persistence and susceptibility of populations to local and global extinctions. Conserv. Biol 1994, 8, 60–71. [Google Scholar]
  34. Boyer, R.; Grue, C. E. The need for water quality criteria for frogs. Environ. Health Perspect 1995, 103, 352–355. [Google Scholar]
  35. Reeder, A. L.; Ruiz, M. O.; Pessier, A.; Brown, L. E.; Levengood, J. M.; Phillips, C. A.; Wheeler, M. B.; Warner, R. E.; Beasley, V. R. Intersexuality and the cricket frog decline: historic and geographic trends. Environ. Health Persp 2005, 113(3), 261–265. [Google Scholar]
  36. Vismara, C.; Bacchetta, R.; Cacciatore, B.; Vailati, G.; Fascio, U. Paraquat embryotoxicity in the Xenopus laevis cleavage phase. Aquat. Toxicol 2001, 55(1–2), 85–93. [Google Scholar]
  37. Besser, J. M.; Dwyer, F. J.; Ingersoll, C. G.; Wang, N. Early Life-stage toxicity of copper to endangered and surrogate fish species; U.S. Environmental Protection Agency EPA/600/R-01/051. : Washington, DC, 2001. [Google Scholar]
  38. Herkovits, J.; Pérez-Coll, C S.; Herkovits, F. D. Ecotoxicity in Reconquista River (Province of Buenos Aires, Argentine): A preliminary study. Environm. Health Persp 1996, 104(2), 186–189. [Google Scholar]
  39. 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.D.; Lucier, G. Strategies for assessing the implications of malformed frogs for environmental health. Environ. Health Perspect 2000, 108(1), 83–90. [Google Scholar]
  40. Tietge, J. E.; Ankley, G. T.; DeFoe, D. L.; Holcombe, G. W.; Jensen, K. M. 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 2000, 19, 2114–2121. [Google Scholar]
Int. J. Environ. Res. Public Health EISSN 1660-4601 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert