Bull Environ Contam Toxicol (2014) 93:442–445 DOI 10.1007/s00128-014-1367-6
Sensitivity of Danio rerio (Teleostei, Cyprinidae) During Two Stages of Development Based on Acute Toxicity Tests R. Freiry • J. A. A. Stelzer • L. Maltchik A. Arenzon
•
Received: 4 December 2013 / Accepted: 21 August 2014 / Published online: 29 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The sensitivity of Danio rerio to three chemicals was compared at two growth stages [larval (10 ± 2 after hatching) and post-larval (60 ± 4 days after hatching)] based on acute toxicity tests. Thirty-nine 48 h acute toxicity tests were performed with the substances CuSO4, NaCl and KCl. The 48 h LC50 values at the two growth stages were compared by independent samples t-tests. The results showed a clear decrease in sensitivity when post-larval organisms were used. Since acute toxicity test methods for D. rerio that recommend using post-larval stage fish do not represent the most sensitive stage of the test organism, our study suggests a revision of the methods to use larval fish. Keywords Danio rerio Larval toxicity Acute toxicity tests Sensitivity Different methods are used to evaluate acute toxicity in fish beyond the early stages of development (embryonic and larval), such as OECD 203 (1992), ABNT NBR 15088 (2011) and ISO 7346-1 (1996). Some problems with using adult fish in toxicity tests include the reduced sensitivity of adult individuals to pollutants and the need for a large sample volume (Lammer et al. 2009). Usually fish are more sensitive during the post-hatching stage than the adult stage (Hutchinson 1998). Due to the different sensitivities, the
R. Freiry L. Maltchik Laboratory of Ecology and Conservation of Aquatic Ecosystems, University of Vale do Rio dos Sinos (UNISINOS), Unisinos Av., 950, Sa˜o Leopoldo, RS 93022-000, Brazil J. A. A. Stelzer A. Arenzon (&) Laboratory of Ecotoxicology, Ecology Center, Federal University of Rio Grande do Sul, Bento Gonc¸alves Av., 9500, Porto Alegre, RS 91501-970, Brazil e-mail: [email protected]
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Environmental Protection Agency (US EPA) recommends that toxicity tests should be performed with organisms that are in the early stages of development, which ensures that the most sensitive stages are tested (USEPA 2002). The chorion of the embryonic stage of Danio rerio protects the embryo from toxic substances, which makes this stage less sensitive than the larval stage (Henn and Braunbeck 2011). However, recent toxicity tests methods have proposed substituting juvenile and adult fish with fish in the early stages of development, such as the OECD 236 Guideline that uses recently fertilized embryos in acute toxicity tests (OECD 2013a), ISO 15088 (2007) and DIN 38415-T-6 (2001) (Henn and Braunbeck 2011). Additionally, OECD 210 (2013b) developed a protocol that includes the larval stage of D. rerio in a 96 h acute toxicity test that begins soon after fertilization. D. rerio is one of the species most commonly used in the acute toxicity test worldwide. However, such assays in many cases are still using organisms that are almost adults, as suggested by protocols such as OECD 203 (1992), ABNT NBR 15088 (2011) and ISO 7346-1 (1996). The main argument for this is the commercial availability of these organisms in the later stages of development. There are few publications that present in a direct way the differences in sensitivity between the stages of development for this species. Therefore, the objective of this study was to compare the differences in sensitivity between two phases of development of D. rerio, larval (10 ± 2 days after hatching) and post-larval (60 ± 4 days after hatching) stages, in acute toxicity tests.
Materials and Methods Acute toxicity tests of D. rerio were performed during two development stages. The study organisms were
Bull Environ Contam Toxicol (2014) 93:442–445
raised and maintained in the laboratory. Recently hatched fish were separated in groups based on the date of hatching. Each group was evaluated at two different moments: larval [(10 ± 2 days after hatching (dah)] and post-larval (60 ± 4 dah). The latter age is the necessary age to reach a length of 2 ± 1 cm, which is the length required in OECD 203 and ABNT NBR 15088/2011. All procedures performed were in compliance with institutional guidelines for research on animals. A total of 39 acute toxicity tests of 48 h duration were run in a static exposure system (without renewing the test solution) to compare the sensitivity at the two growth stages. Stock solutions were prepared by dissolving copper sulfate pentahydrate (CuSO45H2O - Sigma-Aldrich, St. Louis, MO, USA), sodium chloride (NaCl—Sigma–Aldrich) and potassium chloride (KCl; Merck, Darmstadt, DE) to obtain a concentration of 20 mg L-1 of Cu, 100 g L-1 of NaCl, and 100 g L-1 of KCl. The stock solutions were further diluted in reconstituted water (total hardness of 100 mg CaC03 L-1, and pH 7.0 to 7.4) in volumetric flasks to obtain working solutions at designated nominal concentrations. Conductivity measurements (WTW LF 197, Wellhein, DE) were used to indicate the quality of dilutions in the tests with NaCl and KCl. The maximum variability observed in the conductivities of each dilution were below the natural variability found in results of biological assays, namely 1.6 % and 5.1 % for NaCl and KCl, respectively. Copper concentrations were measured by ICP-MS (Hewlett-Packard -HP 4500, USA), and were within 80 % of nominal. Each toxicity test consisted of at least five concentrations in addition to the control group, with two replicates of each concentration. For the 10 ± 2 dah toxicity tests, 5 fish larvae were placed in each container of 250 mL test solution. For the 60 ± 4 dah toxicity tests, five fish were placed in each container with 1,500 mL test solution. All of the toxicity tests were performed at a temperature of 25 ± 2°C. The toxicity tests with 60 ± 4 fish were carried out with fish with an approximate length of 19 ± 6 mm and average weight of 0.07 g. The procedures for all of the toxicity tests followed specifications of OECD 203 (1992) (which are reproduced in ABNT NBR 15088/2011 (2011), except for the size of organisms in the early developmental stages. The 48 h LC50 value for each toxicity test was calculated by the computerized trimmed Spearman-Karber method, ver. 1.5 (Hamilton et al. 1978). The average sensitivities to the three reference substances for each developmental stage were compared with independent sample t-tests. In all of the toxicity tests that were executed, mortality in the control group never exceeded 10 %.
443
Fig. 1 LC50 values for D. rerio exposed to NaCl for 48 h at ages 10 ± 2 (n = 7; CV = 2.3 %) and 60 ± 4 (n = 7; CV = 4.7 %) days after hatching
Fig. 2 LC50 values for D. rerio exposed to KCl for 48 h at ages 10 ± 2 (n = 7; CV = 14.0 %) and 60 ± 4 (n = 7; CV = 13.7 %) days after hatching
Results and Discussion The results of the LC50 s and their 95 % confidence intervals suggested that the larval stage (10 ± 2 dah) of D. rerio was more sensitive than the post-larval stage (60 ± 4 dah) (p \ 0.001) to the three chemicals evaluated (Figs. 1, 2, 3). Our results were similar to those found by Dave and Liu (1991), Hutchinson (1998) and U.S. Environmental Protection Agency (USEPA) (2002). These researchers stated that the test organisms have different sensitivities throughout their development.
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444
Fig. 3 LC50 values for D. rerio exposed to CuSO4 for 48 h at ages 10 ± 2 (n = 5; CV = 19.9 %) and 60 ± 4 (n = 6; CV = 22.1 %) days after hatching
There are several distinct advantages in adopting the use of younger larval fish in acute toxicity test protocols. In order to respect the density limit (maximum of 1 g fish per liter water/sample), the tests with post-larval fish are carried out in larger containers, which require more space for test exposure, a larger sample volume and more discarded chemical residues. Lammer et al. (2009) emphasize the practicality of using fish in early developmental stages in toxicity tests to reduce the period in which the test organisms must be kept in the laboratory until reaching the size required for tests with post-larval fish. Furthermore, transferring post-larval specimens is more difficult due to the size of the organisms, while transferring larvae is a more simplified procedure. Test fish source may have an effect upon toxicity test results. Freiry (2012) performed toxicity tests with fish taken from commercial establishments (the usual practice for toxicity tests with D. rerio in Brazil), and found average 48 h LC50 values of 9.90, 1.28 and 0.24 mg L-1 for NaCl, KCl and CuSO4, respectively. These results suggest a significant decrease in sensitivity in test organisms that are not raised in laboratories. This result indicates that the acquisition of test organisms should be given careful consideration in ecotoxicological evaluations. The Brazilian test protocol in ABNT NBR 15088 (2011) addresses neither the external acquisition of test organisms nor the compulsory cultivation of these organisms in the laboratory. Domingues and Bertoletti (2006) states that raising commercially available species, such as D. rerio, is not a good alternative for laboratories that do not frequently perform toxicity tests. However, this author may not be taking into consideration the reduced sensitivity of these
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Bull Environ Contam Toxicol (2014) 93:442–445
organisms and the other problems related to the use of organisms from unreliable sources (lack of uniformity in cultivation conditions and not knowing the age of the organisms). The organisms used in these tests should be of approximately the same age, and from the same group. Therefore, using the total length of a test subject as a requirement for toxicity tests is inappropriate (U.S. Environmental Protection Agency (USEPA) 2002). The length of a fish does not indicate its age, since growth can be affected by culture conditions (Sipa´uba et al. 1995). Total length of the postlarval fish (60 ± 4 dah) in this study varied from 13 to 25 mm, which means that fish of the same age may have different lengths, even when they are raised under similar conditions. The present study demonstrated that the post-larval developmental stage of D. rerio used today by ABNT NBR 15088 (2011), by the international standards OECD 203 (1992) and ISO 7346-3 (1996) is not this test organism’s most sensitive stage. Thus, considering that the goal of toxicity tests is to protect the biota of the natural aquatic ecosystem, the use of post-larval D. rerio in 48 h acute toxicity tests may be questioned. Our study highlights the need to review methods that use post-larval D. rerio as test organisms in toxicity tests.
References Associac¸a˜o Brasileira de Normas Te´cnicas (2011). ABNT NBR 15088: Aquatic ecotoxicology - Acute toxicity - Test with fish Dave G, Liu R (1991) Toxicity of mercury, copper, nickel, lead, and cobalt to embryos and larvae of zebrafish, Brachydanio rerio. Arch Environ Contam Toxicol 21:126–134. doi:10.1007/ BF01055567 Freiry R (2012) Diferenc¸a na sensibilidade entre duas fases de desenvolvimento de Danio rerio (Teleostei, Cyprinidae) e comparac¸a˜o com Pimephales promelas (Teleostei, Cyprinidae), utilizando ensaios de toxicidade aguda. Dissertation, Universidade do Vale do Rio dos Sinos Hamilton MA et al (1978) Trimmed Spearman-Karber method for estimating median lethal concentration in toxicity bioassays. Environ Sci Technol 11:714–719 Correction 12, 417 Henn K, Braunbeck T (2011) Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp Biochem Physiol C: Toxicol Pharmacol 153(1):91–98. doi:10.1016/j.cbpc.2010.09.003 Hutchinson T (1998) Analysis of the ecetoc aquatic toxicity (eat) database iii - comparative toxicity of chemical substances to different life stages of aquatic organisms. Chemosphere 36(1):129–142. doi:10.1016/S0045-6535(97)10025-X ISO (1996): ISO 7346-1. Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] Lammer E et al (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C: Toxicol Pharmacol 149(2):196–209. doi:10.1016/j.cbpc.2008.11.006
Bull Environ Contam Toxicol (2014) 93:442–445 Organisation for Economic Co-operation and Development (1992) OECD guideline for testing of chemicals: fish acute toxicity test. OECD 203, Paris, France Organisation for Economic Co-operation and Development (2013) OECD guideline for testing of chemicals: fish, early-life stage toxicity test. OECD 210, Paris, France Organisation for Economic Co-operation and Development (2013) OECD guideline for testing of chemicals: Fish embryo toxicity (FET) Test. OECD 236, Paris, France Sipa´uba L et al (1995) Variac¸a˜o de alguns paraˆmetros limnolo´gicos em um viveiro de piscicultura em func¸a˜o da luz. Acta Limnol. Brasil. 7:138–150
445 U.S. Environmental Protection Agency (USEPA) (2002) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms, fourth ed. EPA-821-R02-013. US Environmental Protection Agency, Washington, DC Domingues DF, Bertoletti E (2006) Selec¸a˜o, Manutenc¸a˜o e Cultivo de organismos aqua´ticos. In: Zagatto PA, Bertoletti E (eds) Ecotoxicologia aqua´tica: princı´pios e aplicac¸o˜es. RIMA, Sa˜o Carlos, pp 153–184
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Sensitivity of Danio rerio (Teleostei, Cyprinidae) During Two Stages of Development Based on Acute Toxicity Tests R. Freiry • J. A. A. Stelzer • L. Maltchik A. Arenzon
•
Received: 4 December 2013 / Accepted: 21 August 2014 / Published online: 29 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The sensitivity of Danio rerio to three chemicals was compared at two growth stages [larval (10 ± 2 after hatching) and post-larval (60 ± 4 days after hatching)] based on acute toxicity tests. Thirty-nine 48 h acute toxicity tests were performed with the substances CuSO4, NaCl and KCl. The 48 h LC50 values at the two growth stages were compared by independent samples t-tests. The results showed a clear decrease in sensitivity when post-larval organisms were used. Since acute toxicity test methods for D. rerio that recommend using post-larval stage fish do not represent the most sensitive stage of the test organism, our study suggests a revision of the methods to use larval fish. Keywords Danio rerio Larval toxicity Acute toxicity tests Sensitivity Different methods are used to evaluate acute toxicity in fish beyond the early stages of development (embryonic and larval), such as OECD 203 (1992), ABNT NBR 15088 (2011) and ISO 7346-1 (1996). Some problems with using adult fish in toxicity tests include the reduced sensitivity of adult individuals to pollutants and the need for a large sample volume (Lammer et al. 2009). Usually fish are more sensitive during the post-hatching stage than the adult stage (Hutchinson 1998). Due to the different sensitivities, the
R. Freiry L. Maltchik Laboratory of Ecology and Conservation of Aquatic Ecosystems, University of Vale do Rio dos Sinos (UNISINOS), Unisinos Av., 950, Sa˜o Leopoldo, RS 93022-000, Brazil J. A. A. Stelzer A. Arenzon (&) Laboratory of Ecotoxicology, Ecology Center, Federal University of Rio Grande do Sul, Bento Gonc¸alves Av., 9500, Porto Alegre, RS 91501-970, Brazil e-mail: [email protected]
123
Environmental Protection Agency (US EPA) recommends that toxicity tests should be performed with organisms that are in the early stages of development, which ensures that the most sensitive stages are tested (USEPA 2002). The chorion of the embryonic stage of Danio rerio protects the embryo from toxic substances, which makes this stage less sensitive than the larval stage (Henn and Braunbeck 2011). However, recent toxicity tests methods have proposed substituting juvenile and adult fish with fish in the early stages of development, such as the OECD 236 Guideline that uses recently fertilized embryos in acute toxicity tests (OECD 2013a), ISO 15088 (2007) and DIN 38415-T-6 (2001) (Henn and Braunbeck 2011). Additionally, OECD 210 (2013b) developed a protocol that includes the larval stage of D. rerio in a 96 h acute toxicity test that begins soon after fertilization. D. rerio is one of the species most commonly used in the acute toxicity test worldwide. However, such assays in many cases are still using organisms that are almost adults, as suggested by protocols such as OECD 203 (1992), ABNT NBR 15088 (2011) and ISO 7346-1 (1996). The main argument for this is the commercial availability of these organisms in the later stages of development. There are few publications that present in a direct way the differences in sensitivity between the stages of development for this species. Therefore, the objective of this study was to compare the differences in sensitivity between two phases of development of D. rerio, larval (10 ± 2 days after hatching) and post-larval (60 ± 4 days after hatching) stages, in acute toxicity tests.
Materials and Methods Acute toxicity tests of D. rerio were performed during two development stages. The study organisms were
Bull Environ Contam Toxicol (2014) 93:442–445
raised and maintained in the laboratory. Recently hatched fish were separated in groups based on the date of hatching. Each group was evaluated at two different moments: larval [(10 ± 2 days after hatching (dah)] and post-larval (60 ± 4 dah). The latter age is the necessary age to reach a length of 2 ± 1 cm, which is the length required in OECD 203 and ABNT NBR 15088/2011. All procedures performed were in compliance with institutional guidelines for research on animals. A total of 39 acute toxicity tests of 48 h duration were run in a static exposure system (without renewing the test solution) to compare the sensitivity at the two growth stages. Stock solutions were prepared by dissolving copper sulfate pentahydrate (CuSO45H2O - Sigma-Aldrich, St. Louis, MO, USA), sodium chloride (NaCl—Sigma–Aldrich) and potassium chloride (KCl; Merck, Darmstadt, DE) to obtain a concentration of 20 mg L-1 of Cu, 100 g L-1 of NaCl, and 100 g L-1 of KCl. The stock solutions were further diluted in reconstituted water (total hardness of 100 mg CaC03 L-1, and pH 7.0 to 7.4) in volumetric flasks to obtain working solutions at designated nominal concentrations. Conductivity measurements (WTW LF 197, Wellhein, DE) were used to indicate the quality of dilutions in the tests with NaCl and KCl. The maximum variability observed in the conductivities of each dilution were below the natural variability found in results of biological assays, namely 1.6 % and 5.1 % for NaCl and KCl, respectively. Copper concentrations were measured by ICP-MS (Hewlett-Packard -HP 4500, USA), and were within 80 % of nominal. Each toxicity test consisted of at least five concentrations in addition to the control group, with two replicates of each concentration. For the 10 ± 2 dah toxicity tests, 5 fish larvae were placed in each container of 250 mL test solution. For the 60 ± 4 dah toxicity tests, five fish were placed in each container with 1,500 mL test solution. All of the toxicity tests were performed at a temperature of 25 ± 2°C. The toxicity tests with 60 ± 4 fish were carried out with fish with an approximate length of 19 ± 6 mm and average weight of 0.07 g. The procedures for all of the toxicity tests followed specifications of OECD 203 (1992) (which are reproduced in ABNT NBR 15088/2011 (2011), except for the size of organisms in the early developmental stages. The 48 h LC50 value for each toxicity test was calculated by the computerized trimmed Spearman-Karber method, ver. 1.5 (Hamilton et al. 1978). The average sensitivities to the three reference substances for each developmental stage were compared with independent sample t-tests. In all of the toxicity tests that were executed, mortality in the control group never exceeded 10 %.
443
Fig. 1 LC50 values for D. rerio exposed to NaCl for 48 h at ages 10 ± 2 (n = 7; CV = 2.3 %) and 60 ± 4 (n = 7; CV = 4.7 %) days after hatching
Fig. 2 LC50 values for D. rerio exposed to KCl for 48 h at ages 10 ± 2 (n = 7; CV = 14.0 %) and 60 ± 4 (n = 7; CV = 13.7 %) days after hatching
Results and Discussion The results of the LC50 s and their 95 % confidence intervals suggested that the larval stage (10 ± 2 dah) of D. rerio was more sensitive than the post-larval stage (60 ± 4 dah) (p \ 0.001) to the three chemicals evaluated (Figs. 1, 2, 3). Our results were similar to those found by Dave and Liu (1991), Hutchinson (1998) and U.S. Environmental Protection Agency (USEPA) (2002). These researchers stated that the test organisms have different sensitivities throughout their development.
123
444
Fig. 3 LC50 values for D. rerio exposed to CuSO4 for 48 h at ages 10 ± 2 (n = 5; CV = 19.9 %) and 60 ± 4 (n = 6; CV = 22.1 %) days after hatching
There are several distinct advantages in adopting the use of younger larval fish in acute toxicity test protocols. In order to respect the density limit (maximum of 1 g fish per liter water/sample), the tests with post-larval fish are carried out in larger containers, which require more space for test exposure, a larger sample volume and more discarded chemical residues. Lammer et al. (2009) emphasize the practicality of using fish in early developmental stages in toxicity tests to reduce the period in which the test organisms must be kept in the laboratory until reaching the size required for tests with post-larval fish. Furthermore, transferring post-larval specimens is more difficult due to the size of the organisms, while transferring larvae is a more simplified procedure. Test fish source may have an effect upon toxicity test results. Freiry (2012) performed toxicity tests with fish taken from commercial establishments (the usual practice for toxicity tests with D. rerio in Brazil), and found average 48 h LC50 values of 9.90, 1.28 and 0.24 mg L-1 for NaCl, KCl and CuSO4, respectively. These results suggest a significant decrease in sensitivity in test organisms that are not raised in laboratories. This result indicates that the acquisition of test organisms should be given careful consideration in ecotoxicological evaluations. The Brazilian test protocol in ABNT NBR 15088 (2011) addresses neither the external acquisition of test organisms nor the compulsory cultivation of these organisms in the laboratory. Domingues and Bertoletti (2006) states that raising commercially available species, such as D. rerio, is not a good alternative for laboratories that do not frequently perform toxicity tests. However, this author may not be taking into consideration the reduced sensitivity of these
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Bull Environ Contam Toxicol (2014) 93:442–445
organisms and the other problems related to the use of organisms from unreliable sources (lack of uniformity in cultivation conditions and not knowing the age of the organisms). The organisms used in these tests should be of approximately the same age, and from the same group. Therefore, using the total length of a test subject as a requirement for toxicity tests is inappropriate (U.S. Environmental Protection Agency (USEPA) 2002). The length of a fish does not indicate its age, since growth can be affected by culture conditions (Sipa´uba et al. 1995). Total length of the postlarval fish (60 ± 4 dah) in this study varied from 13 to 25 mm, which means that fish of the same age may have different lengths, even when they are raised under similar conditions. The present study demonstrated that the post-larval developmental stage of D. rerio used today by ABNT NBR 15088 (2011), by the international standards OECD 203 (1992) and ISO 7346-3 (1996) is not this test organism’s most sensitive stage. Thus, considering that the goal of toxicity tests is to protect the biota of the natural aquatic ecosystem, the use of post-larval D. rerio in 48 h acute toxicity tests may be questioned. Our study highlights the need to review methods that use post-larval D. rerio as test organisms in toxicity tests.
References Associac¸a˜o Brasileira de Normas Te´cnicas (2011). ABNT NBR 15088: Aquatic ecotoxicology - Acute toxicity - Test with fish Dave G, Liu R (1991) Toxicity of mercury, copper, nickel, lead, and cobalt to embryos and larvae of zebrafish, Brachydanio rerio. Arch Environ Contam Toxicol 21:126–134. doi:10.1007/ BF01055567 Freiry R (2012) Diferenc¸a na sensibilidade entre duas fases de desenvolvimento de Danio rerio (Teleostei, Cyprinidae) e comparac¸a˜o com Pimephales promelas (Teleostei, Cyprinidae), utilizando ensaios de toxicidade aguda. Dissertation, Universidade do Vale do Rio dos Sinos Hamilton MA et al (1978) Trimmed Spearman-Karber method for estimating median lethal concentration in toxicity bioassays. Environ Sci Technol 11:714–719 Correction 12, 417 Henn K, Braunbeck T (2011) Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp Biochem Physiol C: Toxicol Pharmacol 153(1):91–98. doi:10.1016/j.cbpc.2010.09.003 Hutchinson T (1998) Analysis of the ecetoc aquatic toxicity (eat) database iii - comparative toxicity of chemical substances to different life stages of aquatic organisms. Chemosphere 36(1):129–142. doi:10.1016/S0045-6535(97)10025-X ISO (1996): ISO 7346-1. Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] Lammer E et al (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C: Toxicol Pharmacol 149(2):196–209. doi:10.1016/j.cbpc.2008.11.006
Bull Environ Contam Toxicol (2014) 93:442–445 Organisation for Economic Co-operation and Development (1992) OECD guideline for testing of chemicals: fish acute toxicity test. OECD 203, Paris, France Organisation for Economic Co-operation and Development (2013) OECD guideline for testing of chemicals: fish, early-life stage toxicity test. OECD 210, Paris, France Organisation for Economic Co-operation and Development (2013) OECD guideline for testing of chemicals: Fish embryo toxicity (FET) Test. OECD 236, Paris, France Sipa´uba L et al (1995) Variac¸a˜o de alguns paraˆmetros limnolo´gicos em um viveiro de piscicultura em func¸a˜o da luz. Acta Limnol. Brasil. 7:138–150
445 U.S. Environmental Protection Agency (USEPA) (2002) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms, fourth ed. EPA-821-R02-013. US Environmental Protection Agency, Washington, DC Domingues DF, Bertoletti E (2006) Selec¸a˜o, Manutenc¸a˜o e Cultivo de organismos aqua´ticos. In: Zagatto PA, Bertoletti E (eds) Ecotoxicologia aqua´tica: princı´pios e aplicac¸o˜es. RIMA, Sa˜o Carlos, pp 153–184
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