- Email: [email protected]
Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax
Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax A. Tornambè n, L. Manfra, S. Canepa, F. Oteri, G. Martuccio, A.M. Cicero, E. Magaletti ISPRA, Italian Institute for Environmental Protection and Research, via Vitaliano Brancati 48, 00144 Rome, Italy
ar t ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2015 Received in revised form 4 December 2015 Accepted 25 December 2015
The OECD TG 215 method (2000) (C.14 method of EC Regulation 440/2008) was developed on the rainbow trout (Oncorynchus mykiss) to assess chronic toxicity (28 d) of chemicals on fish juveniles. It contemplates to use other well documented species identifying suitable conditions to evaluate their growth. OECD proposes the European sea bass (Dicentrarchus labrax, L. 1758) as Mediterranean species among vertebrates recommended in the OECD guidelines for the toxicity testing of chemicals. In this context, our study is aimed to proposing the adaptation of the growth test (OECD TG 215, 2000) to D. labrax. For this purpose toxicity tests were performed with sodium dodecyl sulfate, a reference toxicant commonly used in fish toxicity assays. The main aspects of the testing procedure were reviewed: fish size (weight), environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding (food type and ration), test validity criteria. The experience gained from growth tests with the sea bass allows to promote its inclusion among the species to be used for the C.14 method. & 2016 Published by Elsevier Inc.
Keywords: OECD 215 Dicentrarchus labrax Fish growth toxicity test Sodium dodecyl sulfate
1. Introduction The EC Regulation 440/2008 (Council Regulation (EC), 2008) lays down test methods pursuant to European REACH Regulation for registration, evaluation, authorization and restriction of chemicals. It reports the fish growth toxicity test (classified as C.14 method) as “a replicate of the OECD TG 215 (2000)”. This method is designed to assess the effects of prolonged exposure to chemicals on fish juveniles and it has been developed on the freshwater species rainbow trout (Oncorynchus mykiss) but other well documented species such as Danio rerio (zebrafish) and Oryzias latipes (medaka) may be used adapting the procedure. However, the use of a target marine species may be considered more appropriate to assess the impact of chemical anthropogenic substances on the marine environment, especially if the ultimate goal is to establish a regulatory Predicted No-Effect Concentration (PNEC) that should be protective of potentially more sensitive species of this compartment (ECHA, 2008). The marine species most globally used for short- and long-term toxicity assessments by standardized methods are of North American origin, such as Cyprinodon variegatus, Menidia beryllina and Atherinops affinis (US EPA, 2002; ASTM, 2013), that cannot be considered representative n Correspondence to: ISPRA, Italian Institute for Environmental Protection and Research, via di Castel Romano 100, 00128 Rome, Italy. E-mail address: [email protected] (A. Tornambè).
species of the Mediterranean Sea. On the other hand, in the last decade the European species Dicentrarchus labrax Linnaeus 1758 (Osteichthyes, Perciformes, Moronidae) has been proposed as biological indicator in acute toxicity tests (Cicero et al., 2001; Gelli et al., 2004; Magaletti et al., 2006; Mariani et al., 2004, 2006; El-Sayed et al., 2009; Tornambè et al., 2012; Manfra et al., 2015), biomarker analyses (Caruso et al., 2005; Faucher et al., 2008; Giari et al., 2007, 2008; Jebali et al., 2008; Gorbi et al., 2009; Greco et al., 2010; Almeida et al., 2010; Ferreira et al., 2010; Kerambrun et al., 2012; Della Torre et al., 2012; De Domenico et al., 2013) and bioaccumulation studies (Serrano et al., 2004; Carubelli et al., 2007; Antunes et al., 2007, 2008; Trocino et al., 2009; Danion et al., 2011). D. labrax is widespread in Mediterranean waters and broadly reared in fish farms for its commercial importance. It is an euryaline (4–40 psu) and eurythermal (2–30 °C) species, with an optimal range of salinity [20–30 psu] and temperature [14–28 °C], easy to maintain under laboratory conditions and widely available for much of the year. It is one of the recommended fish species in acute toxicity tests for the Italian approval of oil dispersants and absorbents to be used at sea in case of oil spills (Magaletti et al., 2006; Decreto Ministeriale 116 del 25 Febbraio, 2011), and OECD has recently proposed its inclusion among species recommended in the OECD guidelines for the toxicity testing of chemicals (Conti et al., 2015). The general principle of the C.14 test method consists of exposing for 28 d fish juveniles to a range of sublethal concentrations of the test substance preferably under flow-through or
http://dx.doi.org/10.1016/j.ecoenv.2015.12.032 0147-6513/& 2016 Published by Elsevier Inc.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
appropriate semi-static (static-renewal) conditions. Fish are selected in exponential growth phase and weighed before their use. During the test fish are fed daily, the food ration is based on their initial weight and it may be recalculated after 14 d to induce acceptable growth rate. At the end of the test the fish are weighed again and growth rate effects are analyzed. The aim of the present work is to revise and define the main aspects of this growth method (i.e. initial fish weight, environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding, test validity criteria) by exposing D. labrax to sodium dodecyl sulfate (SDS). It is an anionic surfactant widely employed in industry, agriculture and domestic use and therefore it may be found in abundance in the environment, particularly in the sea. It is commonly used as reference organic toxicant in ecotoxicity tests (〈http://echa.europa.eu/〉). The proposed method has been applied for assessing the chronic toxicity of diethylene glycol (DEG), an organic compound used as anticorrosive agent during offshore oil activities (Manfra et al., 2015).
2. Materials and methods 2.1. Materials and chemicals Two polypropylene tanks (300 Liter) were used for fish acclimatization prior to the test. Inert plastic vessels (11 Liter) were used as test chambers with 10 Liter effective volume of test solution. Reconstituted sea water was used both for acclimatization as for test medium. It was prepared by dissolving a commercial sea salt mixture (Instant Ocean™) into distilled water, prepared and aerated for at least 24 h before use. Temperature (20 72 °C), salinity (20 7 1 psu), pH (87 0.5) and dissolved oxygen ( Z60%) were daily checked. The concentrations of ammonia (maximum acceptable value MAV ¼5 mg/l), nitrite (MAV¼0.5 mg/l) and nitrate (MAV¼50 mg/l) were measured twice a week both in stabulation tanks and in test vessels. SDS (Nzytech, Portugal; CAS registry number 151-21-3 with ultra purity 499%) was used as reference toxicant during the experiments. 2.2. Fish holding About 500 juveniles of D. labrax (60–80 d old, length: 4.23 70.37 cm, weight: 0.78 70.25 g) were obtained from the fish farm “Agroittica Caldoli” of Lesina (Italy). They were transferred to the laboratory in polyethylene containers (30 Liter) filled with breeding water and equipped with air diffusers to maintain the level of dissolved oxygen in the water above 60% of saturation. The transfer operations were performed trying to minimize manipulation of the organisms. Fish were acclimatized into two tanks for ten days prior to the test, in order to detect any pathological manifestations and/or mortality due to stress of transport and acclimatization to the new conditions of breeding. The fish tanks operated in a closed cycle with a system of mechanical and biological filtration, and with a water chiller device for the maintenance of constant temperature. Tanks were illuminated with fluorescent lamps that provided 500–800 lx on the surface water, with a photoperiod of 14 h of light and 10 h of darkness. The continuous aeration in the water was maintained using electric air pumping compressors. Fish were fed daily with commercial marine fish food (Aller Futura™ Gr.1) with a high protein content (64%), with daily ration of 2% body weight. During the acclimatization period fry mortality was 4%. A subsample of 30 organisms was weighed and measured to estimate the mean
length and the mean weight of juveniles before the test. Fish were not fed for 24 h prior to the start of the tests. 2.3. Toxicity test method Three tests were performed on juveniles exposed to SDS: 1) an acute test (96 h) by applying the C.1 method (OECD TG 203, 1992), aimed at checking the sensitivity of the fish stock; 2) one preliminary 28-d test (test 1) according to the C.14 method (OECD TG 215, 2000), to explore sublethal concentrations and find the appropriate feeding; 3) one final 28-d test (test 2) at the conditions defined in the preliminary 28-d test, to validate the proposed method. Test solutions were prepared daily by dissolving aliquots of a fresh stock solution (1 g/l SDS in deionized water) into dilution water. The acute test (96 h) was carried out using only three concentrations (14, 7, and 3.5 mg/l) selected in a known range of toxicity (Mariani et al., 2006), with two replicates for each concentration and one control (dilution water without SDS). Test 1 was performed using five concentrations (7, 3.5, 1.75, 0.87, and 0.44 mg/l) with two replicates for each concentration and the control, in order to reduce the number of utilized organisms according to European guidelines (Halder et al., 2014). Test 2 was carried out using the above-mentioned five concentrations, since they showed to be sublethal concentrations with effect on fish growth. In this case, three replicates for each concentration and for the control were used. The SDS concentration was weekly measured, both at the time of preparation and prior to renewal (about 24 h later) of the test solutions by colorimetric MBAS assay using Hach Lange LCK 332 kit analysis for anionic surfactants through DR2800 Hach Lange spectrophotometer (λ ¼ 650 nm)(George and White, 1999). In all the measurements, SDS degradation after 24 h was less than 20% (6.072.2%). Before the test fish were individually weighed as wet weights (blotted dry, without using anesthetics) to the precision of 10 mg and randomly distributed between the different test vessels. The initial range of individual weights was within 7 25% of the arithmetic mean body weight of all used organisms in test 1, and within 7 10% in test 2. The loading rate was less than 1 g/l and the stocking density was of 7 individuals in 10 Liter (for each vessel). The sum of wet weights of each vessel was recorded and used to calculate the initial daily food ration. All the procedure was done trying to avoid stressing test animals as much as possible. The tests were conducted in a temperature-controlled room at 2071 °C, with illumination of 500–800 lx on the water surface, provided by ‘cool-white’ fluorescent lamps, with a photoperiod of 14 h light: 10 h dark. Survival and possible onset of abnormal behaviors of the organisms were daily checked. The renewal of the test solutions was performed by transferring the fish with small nets in other test vessels containing new fresh solutions. The feeding was provided in a single daily ration one hour after the renewal of the test solutions, consisting of the same dry food used in the acclimatization period. The rations, based on the initial total fish body weight, were increased from a minimum of 1% in the first two weeks up to a maximum of 2% in the last two weeks in test 1, while from 2% in the first two weeks up to 3% in the third week and 4% in the last week in test 2. At 28 d, surviving fish were individually weighed again after being anesthetized with clove oil (40 ppt) and dried on absorbent paper. During the acute test fish were not weighed and fed, surviving fish were counted after 96 h according to the C.1 method. 2.4. Data analysis For the 96 h test, the concentration that would cause 50%
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3
mortality (LC50) was calculated by the Trimmed Spearman Karber method (USEPA TSK software, version 1.5). The test was considered valid with a control mortality percentage of r10%. For the 28 d tests, average specific growth rate was calculated for each vessel using the following equation:
r2 =
logew2 − logew1 t2 − t1
× 100
(1)
where: w1, w2 ¼ weights of a particular fish at times t1 and t2, respectively log w1 ¼ average of the logarithms of the values w1 for the fish in the vessel at the beginning of the test log w2 ¼ average of the logarithms of the values w2 for the fish in the tank at the end of the test t1, t2 ¼time (days) at the beginning and end of test, respectively The estimation of the concentration that would cause a x% variation of growth rate (ECx) was calculated by non-linear regression analysis (Logit-Hill model) using the Benchmark Dose Software 2.4.0 (US EPA, 2012). Alternatively, the percentages of growth inhibition were compared with control values in order to determine the no observed effect concentration (NOEC) using Dunnett test. The 28-d tests were considered valid when the following conditions were met (OECD TG 215, 2000): – the mortality in the controls did not exceed 10% at the end of the test, – the mean weight of fish in the controls increased enough to allow the detection of the minimum variation of growth rate considered as significant, – the dissolved oxygen concentration was at least 60% of the air saturation value (ASV) throughout the test, – the water temperature did not differ by more than 71 °C between test chambers at any one time during the test and was maintained within a range of 2 °C within the temperature ranges specified for the test species.
3. Results and discussion The acute toxicity test was considered valid since the control mortality percentage was r10%, as required by the C.1 method, and 96hLC50 value of 9.90 mg/l was comparable to acute results available in literature for SDS (Mariani et al., 2006; Conti et al., 2015). This toxicity value is of the same order of magnitude of the estimated effect concentrations found for other fish species (Ribelles et al., 1995; Rosety et al., 2001) and for other organisms (see Rosety et al. (2001) and references therein), thus confirming the sensitivity of the fish batch used in this study. The test validity criteria were met in both 28-d tests, except for the mean weight increase in the test 1. No fish mortality was observed at the used SDS concentrations. The mean weight increase in the controls was 28.5% (cv ¼42.5%) and 81.4% (cv ¼4.9%) in the test 1 and test 2, respectively. The dissolved oxygen concentration ranged between 76% and 85% of saturation during both tests and the temperature was 207 1 °C in all test chambers. The control weight increase was not enough to estimate a statistically significant effect of SDS on fish growth in test 1, however the weight increase was clearly inhibited at the highest concentration tested (7 mg/l) with respect to control (13.0% vs. 28.5%) (Fig. 1a). On the basis of these results, the test 2 was conducted using the same five SDS concentrations as in test 1, but the range of initial fish weights was narrower and the weekly food ration (from 2%
Fig. 1. 28-d SDS toxicity tests results with D. labrax: (a) comparison between test 1 and test 2 average weight increase at different SDS concentrations and control (CTR); (b) comparison between test 1 and test 2 average specific fish growth rates.
minimum to 4% maximum) was increased, based on the observation of actual consumption by fish (rate of consumption, presence or absence of food residues on the water surface and on the bottom of test chambers). Due to these changes, a double control growth rate (2.15 70.09% d 1) was obtained in the test 2 respect to that in test 1 (1.03 70.30% d 1) (Fig. 1b); similar values of control growth rates were obtained in the two other 28-d tests conducted to assess the chronic toxicity of DEG (1.86 7 0.12% d 1 and 1.727 0.31% d 1, Manfra et al., 2015) following this method. At the highest SDS concentration, the average specific growth rate (1.29 70.12% d 1) showed a statistically significant difference (p o0.05) compared to the control growth rate. The significant increase of control mean weight, together with the validity of the other parameters (mortality, oxygen concentration and temperature value), allowed to consider acceptable the test 2. Calculated 28dEC20 (6.61 mg/l, c.l. 3.95–9.27) and 28dNOEC (3.5 mg/l) values for SDS are consistent with those reported for mortality tests with embryo and sac-fry stages of the freshwater fish Pimephales promelas (28dLC20¼4.5 mg/l and 28dNOECo3.8 mg/l) and for growth tests with P. promelas juveniles (42dNOEC41.357 mg/l) (〈http://apps.echa.europa.eu/registered/ data/dossiers/DISS-9d80a3c6-17a5-3287-e044 00144f67d249/DISS9d80a3c6-17a5-3287-e044-00144f67d249_DISS-9d80a3c6-17a53287-e044-00144f67d249.html〉). The acute and chronic SDS toxicity recorded in our study for D. labrax has already been observed for other marine fish such as Sparus aurata and Scophthalmus maximus (see review of Venkatesh and Ashok (2010)). In those cases changes in morphology, metabolism and swimming capacity were found in association with increased SDS concentrations and length of exposure. Sub-lethal chronic (15–30 d) effects of SDS on the survival, metabolism and growth of juveniles of Centropomus parallelus have also been reported (Rocha et. al., 2007).
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
Table 1 Comparison of main test conditions between three OECD-recommended freshwater species and the marine species D. labrax. Recommended species
Oncorhynchus mykiss Rainbow trout
Danio rerio Zebrafish
Oryzias latipes Ricefish (Medaka)
Dicentrarchus labrax European sea bass
Type of test Recommended weight (g) Required measurement precision Temperature pH Light intensity Photoperiod (hrs) O2 saturation Dilution water
Semi-static/Flow through 1.0–5.0 to nearest 100 mg
Semi-static/Flow through 0.050–0.100 to nearest 1 mg
Semi-static/Flow through 0.050–0.100 to nearest 1 mg
Semi-static renewal 0.5–1.0 to nearest 10 mg
12.5–16 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
21–25 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
21–25 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
Volume test solution (Liter) Loading rate (g/Liter) Stocking density Food type
40 1.2–2.0 4/ten litres Dry proprietary salmonid fry food
Food ration Test duration Test validity criteria
4% mean body weight Z 28 d - Control mortality o 10% Control mean weight 450%
1 0.2–1.0 5–10/one litre Live food (Brachionus, Artemia) Not specified Z 28 d Control mortality o 10%
1 0.2–1.0 5–20 /one litre Live food (Brachionus, Artemia) Not specified Z 28 d Control mortality o 10%
18–22 °C 7.5–8.5 500–800 lx 14 light 460% Synthetic sea water 20 psu (Istant Ocean™) 10 0.35–1.0 7–10/ten litres Dry proprietary fry food (high protein content) 2–4% mean body weight Z28 d Control mortality o 10% Control mean weight450%
The OECD guideline allows that well documented species may be used other than rainbow trout, leaving uncertain some aspects of the procedure. However whereas the growth response in fish is a critical endpoint of toxicity (Woltering, 1984), we considered necessary to explore and fix some experimental variables in order to obtain a reliable method for D. labrax. The main experimental conditions for conducting 28-d growth tests with fish juvenile of D. labrax are reported in Table 1, compared with the recommended conditions for freshwater species (OECD TG 215, 2000), highlighting the differences, and then discussed. 3.1. Fish size The initial average weight was about 0.6–0.7 g, that D. labrax reaches about 80–100 days after the hatching under intensive rearing conditions (fish farmer personal communication). This size was chosen on the basis of laboratory practical experience: smaller organisms are too delicate and susceptible to manipulation especially for long term tests, while bigger organisms require too large water volumes during static-renewal tests. The required measurement precision was recalculated with respect to the chosen fish size. As recommended by the protocol, it is very important to maximize the initial size homogeneity in each vessel because larger fish tend to eat more food by subtracting it from the smaller and then grow faster thus increasing in-tank final weight differences. 3.2. Environmental conditions The suggested rearing and test temperature (20 72 °C) is usually used for acute toxicity test with this species (Mariani et al., 2004, 2006; Tornambè et al. 2012) and has proved optimal for growth of D. labrax during nursery periods (Mylonas et al., 2003). The indicated pH value is typically marine and should remain constant with small oscillations around 8.0. As for other parameters, the light intensity during the test should be similar as during acclimatization period in order to minimize unwanted sources of stress for the organisms. 3.3. Dilution water Suitability of Instant Ocean™ reconstituted sea water as rearing
and test medium was amply demonstrated in previous cited studies. Furthermore, due to its euryhalinity, toxicity test with this species can be conducted in a wide range of salinity after a short conditioning period of the animals. 3.4. Experimental design Five concentrations of toxicant for three replicates with 7 fish in each vessel (10 Liter test volume), were planned trying to reach a good balance between practical issue, statistical requirements and minimum use of animals. In fact this test design can be considered a minimum to apply a dose-response model based on regression analysis to estimate the ECx and to calculate NOEC/ LOEC values using analysis of variance with a good statistical significance (see ISO (2006) for NOEC/ECx implications for test design). 3.5. Loading rate and stocking density The OECD 203 (1992) recommendations were followed using the maximum loading of 1.0 g fish/Liter for static and semi-static (acute) tests and the minimum of seven fish at each test concentration (replicate in this case). 3.6. Food ration The food rations should be recalculate once a week on the basis of observation of feed consumption and the increased size of organisms. They start from a minimum of 2% up to a maximum of 4% of total body weight, to reach a significant increase of the mean weight of fish in the control, at least above 50% for this species. The first test week can be considered of acclimatization to the new test conditions. In this phase the maintaining of a good fish welfare is very important for a successful growth test (without mortality). The environmental conditions in the test chambers should be very similar to those during stabulation to avoid additional stress to organisms (in particular with regard the water quality, temperature and light intensity). In this sense it is better to start with the usual food ration and then increase it on the basis of effective consumption by organisms, since uneaten food, if not promptly removed, may deteriorate the water quality over the time before the next renewal of test solution.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3.7. Test validity criteria and treatment of results The OECD 215 indicates three alternative ways to calculate the growth rate of fish (individual specific growth rate r1, average specific growth rate r2, and pseudo specific growth rate r3) and gives an indication of acceptable increase of control growth rate only for O. mykiss. We have chosen to calculate the average specific growth rate r2 (Eq. 1) because r1 is not applicable to little size fish (impossible fish marking), while r3 can be considered less reliable, especially in the case of NOEC/LOEC assessment, as fish weight variability within-tank is generally higher than between-tank at the end of the test. This trend was indeed seen in our study where the coefficient of variation (cv) of r2 ranged from 1.9 to 4.5% while cv of r3 ranged from 17.6 to 126.9% between test concentrations. A good weight gain of the control fish is critical to the success of the test. Our results confirm for D. labrax the need to overcome the 50% of increase at the end of test. This result can be obtained only through a careful weekly recalculation of the portions of food based primarily on actual consumption.
4. Conclusions D. labrax can be considered a species suitable for C.14 method and has the advantage of being a Mediterranean species very well known and studied, ready available most of the year, easy to handle, resistant to physical stress but sensitive to chemicals. Moreover, being a marine species of great commercial and ecological importance can be considered more appropriate for risk assessments studies in sea environments than the recommended freshwater species.
Statement The fish used in this study were kept in authorized breeding room pursuant to Italian Legislative Decree 116/92 (Decreto Legislativo, 1992), implementation of Directive n. 86/609/EEC (Council Directive, 1986) on the protection of animals used for experimental and other scientific purposes. This study was performed according to the protocol approved under Decree 116/92.
Acknowledgments The authors are grateful to Dr. Stefania Balzamo for providing working facilities, Dr. Andrea Paina for his technical assistance, and to Dr. Federica Savorelli for many useful suggestions.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.12. 032.
References Almeida, J.R., Oliveira, C., Gravato, C., Guilhermino, L., 2010. Linking behavioural alterations with biomarkers responses in the European seabass Dicentrarchus labrax L. exposed to the organophosphate pesticide fenitrothion. Ecotoxicology 19 (8), 1369–1381. http://dx.doi.org/10.1007/s10646-010-0523-y, Epub 2010 Aug 5. PubMedPMID: 2068692. Antunes, P., Gil, O., Reis-Henriques, M.A., 2007. Evidence for higher biomagnification factors of lower chlorinated PCBs in cultivated seabass. Sci. Total Environ. 377, 36–44.
5
Antunes, P., Hendriks, A.J., Huijbregts, M.A.J., Gil, O., Reis-Henriques, M.A., 2008. Organ-specific accumulation and elimination patterns of PCBs in adult seabass (Dicentrarchus labrax). Sci. Total Environ. 407, 204–210. ASTME1022-94, 2013. Standard guide for conducting bioconcentration tests with fishes and saltwater bivalve mollusks, ASTM International, West Conshohocken, PA, 2013. www.astm.org DOI: 10.1520/E1022-94R13. Carubelli, G., Fanelli, R., Mariani, G., Nichetti, S., Crosa, G., Calamari, D., Fattore, E., 2007. PCB contamination in farmed and wild sea bass (Dicentrarchus labrax L .) from a coastal wetland area in central Italy. Chemosphere 68, 1630–1635. Caruso, G., Genovese, L., Maricchiolo, G., Modica, A., 2005. Haematological, biochemical and immunological parameters as stress indicators in Dicentrarchus labrax and Sparus aurata farmed in off-shore cages. Aquac. Int. 13, 67–73. Cicero, A.M., Savorelli, F., Gelli, F., Palazzi, D., Pregnolato, L., Roncarati, A., Casazza, G., 2001. Use of sea bass (Dicentrarchus labrax) larvae, obtained from controlled reproduction, as biological indicator in acute toxicity tests. In: Proceedings of the 6th International Conference of the Aquatic Ecosystem Health and Management Society. 4–7 November, 2001. Amsterdam, The Netherlands. Conti, D., Balzamo, S., Paina, A., Martone, C., Raso, E., Cadoni, F., Savorelli, F., Croppo, M., Bellaria, V., Pati, A., 2015. European sea bass (Dicentrarchus labrax L . 1758) as a sentinel species in europe to study the effects of contaminants. Annu. Res. Rev. Biol. 8 (4), 1–13. Council Directive 86/609/EEC, 1986. Council Directive 86/609/EEC of 24 November, 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Council Regulation (EC) No. 440/2008, 2008. Council Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (2008) OJ L 142/1. Danion, M., Le Floch, S., Kanan, R., Lamour, F., Quentel, C., 2011. Effects of in vivo chronic hydrocarbons pollution on sanitary status and immune system in sea bass (Dicentrarchus labrax L.). Aquat. Toxicol. 105 (3–4), 300–311. http://dx.doi. org/10.1016/j.aquatox.2011.06.022, Epub 2011 Jun 28. PubMed PMID: 21781655. Decreto Legislativo 27 gennaio 1992, n. 116. Protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici. G.U. n. 40, 18 febbraio 1992. Attuazione direttiva CEE n. 609/86. Decreto Ministeriale 116 del 25 Febbraio 2011. Definizione delle procedure per il riconoscimento di idoneità dei prodotti assorbenti e disperdenti da impiegare in mare per la bonifica dalla contaminazione da idrocarburi petroliferi. Ministero dell’Ambiente e della Tutela del Territorio e del Mare - DG Protezione della Natura e del Mare. De Domenico, E., Mauceri, A., Giordano, D., Maisano, M., Giannetto, A., Parrino, V., Natalotto, A., D’Agata, A., Cappello, T., Fasulo, S., 2013. Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol. Environ. Saf. 97, 114–123. http://dx.doi.org/10.1016/j. ecoenv.2013.07.015, Epub 2013 Aug 14. PubMed PMID: 23953925. Della Torre, C., Tornambè, A., Cappello, S., Mariottini, M., Perra, G., Giuliani, S., Amato, E., Falugi, C., Crisari, A., Yakimov, M.M., Magaletti, E., 2012. Modulation of CYP1A and genotoxic effects in European seabass (Dicentrarchus labrax) exposed to weathered oil: a mesocosm study. Mar. Environ. Res. 76, 48–55. http: //dx.doi.org/10.1016/j.marenvres.2011.08.006, Epub 2011 Sep 8. PubMed PMID: 21963215. ECHA, 2008. Guidance on information requirements and chemical safety assessment. Chapter R.10: Characterisation of dose [concentration]-response for environment. Guidance for the Implementation of REACH. 2008, p. 65. El-Sayed, Y.S., Khalil, H.R., Saad, T.T., 2009. Acute toxicity of ochratoxin-A in marine water-reared sea bass (Dicentrarchus labrax L .). Chemosphere 75, 878–882. Faucher, K., Fichet, D., Miramand, P., Lagardère, J.P., 2008. Impact of chronic cadmium exposure at environmental dose on escape behaviour in sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae). Environ. Pollut. 151 (1), 148–157, Epub 2007 May 10. PubMed PMID: 17493718. Ferreira, M., Caetano, M., Antunes, P., Costa, J., Gil, O., Bandarra, N., Pousào-Ferreira, P., Vale, C., Reis-Henriques, M.A., 2010. Assessment of contaminants and biomarkers of exposure in wild and farmed seabass. Ecotoxicol. Environ. Saf. 73, 579–588. Gelli, F., Cicero, A.M., Melotti, P., Roncarati, A., Pregnolato, L., Savorelli, F., Palazzi, D., Casazza, G., 2004. A proposal of method to evaluate the quality of marine waters: optimization of 7 days bioassays using Dicentrarchus labrax (L.) juveniles. Chem. Ecol. 20 (Suppl 1), S225–S229. George, A.L., White, G.F., 1999. Optimization of the methylene blue assay for anionic surfactants added to estuarine and marine water. Environ. Toxicol. Chem. 18 (10), 2232–2236. Giari, L., Manera, M., Simoni, E., Dezfuli, B.S., 2007. Cellular alterations in different organs of European sea bass Dicentrarchus labrax L. exposed to cadmium. Chemosphere 67, 1171–1181. Giari, L., Simoni, E., Manera, M., Dezfuli, B.S., 2008. Histo-cytological responses of (Dicentrarchus labrax L.) following mercury exposure. Ecotoxicol. Environ. Saf. 70, 400–410. Gorbi, S., Benedetti, M., Virno Lamberti, C., Pisanelli, B., Moltedo, G., Regoli, F., 2009. Biological effects of diethylene glycol ((Almeida et al., 2010)DEG) and produced waters (PWs) released from offshore activieties: a multi-biomarker approach with the sea bass Dicentrarchus labrax. Environ. Pollut. 157, 3166–3173. Greco, L., Serrano, R., Blanes, M.A., Serrano, E., Capri, E., 2010. Bioaccumulation markers and biochemical responses in European sea bass (Dicentrarchus labrax) raised under different environmental conditions. Ecotoxicol. Environ. Saf. 73,
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
6
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
38–45. Halder, M., Kienzler, A., Whelan, M., Worth, A., 2014. EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing. European Commission, Joint Research Centre, Institute for Health and Consumer Protectionhttp://dx.doi.org/10.2788/084219, Luxembourg, European Union, EUR 26973 EN. ISO/TS, 2006. Water quality-Guidance document on the statistical analysis of ecotoxicity data. Ref. n. ISO/TS 20281 2006 252. Jebali, J., Banni, M., Gerbej, H., Boussetta, H., Lòpez-Barea, J., Alhama, J., 2008. Metallothionein induction by Cu, Cd and Hg in Dicentrarchus labrax L. liver: assessment by RP-HPLC with fluorescence detection and spectrophotometry. Mar. Environ. Res. 65, 358–363. Kerambrun, E., Le Floch, S., Sanchez, W., Thomas Guyon, H., Meziane, T., Henry, F., Amara, R., 2012. Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers. Chemosphere 87 (7), 692–702. http://dx.doi.org/10.1016/j.chemosphere.2011.12.059, Epub 2012 Jan 10. PubMed PMID: 22236592. E., Magaletti, Onorati, F., Faraponova, O., Mariani, L., Tornambè, A., Cicero, A.M., 2006. Ecotoxicological tests for the approval of oil dispersants and absorbents to be used at sea. In: Proceedings of the SETAC Europe 16th Annual Meeting. The Hague, The Netherlands 7–11 May 2006. Abstract Book: 96, MO1/KL/P168. Manfra, L., Tornambè, A., Savorelli, F., Rotini, A., Canepa, S., Mannozzi, M., Cicero, A. M., 2015. Ecotoxicity of diethylene glycol and risk assessment for marine environment. J. Hazard. Mater. 284, 130–135. Mariani, L., Manfra, L., Maggi, C., Savorelli, F., Di Mento, R., Cicero, A.M., 2004. Produced formation waters: a preliminary study on chemical characterization and acute toxicity by using fish larvae (Dicentrarchus labrax L . 1758). Fresenius Environ. Bull. 13, 1427–1432. Mariani, L., De Pascale, D., Faraponova, O., Tornambè, A., Sarni, A., Giuliani, S., Ruggiero, G., Onorati, F., Magaletti, E., 2006. The use of a test battery in marine ecotoxicology: the acute toxicity of sodium dodecylsulfate. Environ. Toxicol. 21, 373–379. Mylonas, C.C., Anezaki, L., Divanach, P., Zanuy, S., Piferrer, F., Ron, B., Peduel, A., Ben Atia, I., Gorshkov, S., Tandler, A., 2003. Influence of rearing temperature at two periods during early life on growth and sex differentiation of two strains of European sea bass. Fish. Physiol. Biochem. 28 (1–4), 167–168. OECD, 1992. Test No. 203: Fish, Acute Toxicity Test, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Parishttp://dx.doi.org/10.1787/ 9789264069961-en. OECD, 2000. Test No. 215: Fish, Juvenile Growth Test, OECD Guidelines for the
Testing of Chemicals, Section 2. OECD Publishing, Parishttp://dx.doi.org/ 10.1787/9789264070202-en. Ribelles, A., Carrasco, C., Rosety, M., Aldana, M., 1995. A histochemical study of the biological effects of sodium dodecyl sulfate on the intestine of gilthead seabream, Sparus aurata. Ecotoxicol. Environ. Saf. 32, 131–138. Rocha, A.J., Gomes, V., Ngan, P.V., Passos, M.J., Furia, R.R., 2007. Effects of anionic surfactant and salinity on the bioenergetics of juveniles of Centropomus parallelus (Poey). Ecotoxicol. Environ. Saf. 3, 397–404. Rosety, R.M., Ordonez, F.J., Rosety, M., Rosety, J.M., Rosety, I., Ribelles, A., Carrasco, C., 2001. Morphohistochemical changes in the gills of turbot, Scophthalmus maximus L ., induced by sodium dodecyl sulfate. Ecotoxicol. Environ. Saf. 3, 223–228. Serrano, R., Simal-Juliàn, A., Pitarch, E., Hernàndez, F., 2004. Response to comment on “Biomagnification study on organochlorine compounds in marine aquaculture: the Sea Bass (Dicentrarchus labrax) as a Model”. Environ. Sci. Technol. 38, 1263. Trocino, A., Majolini, D., Xiccato, G., 2009. PCBs contamination in farmed European seabass from different Italian rearing systems. Chemosphere 76 (2), 2504. http: //dx.doi.org/10.1016/j.chemosphere.2009.03.017, Epub 2009 Apr 5. PubMed PMID: 19349063. Tornambè, A., Manfra, L., Mariani, L., Faraponova, O., Onorati, F., Savorelli, F., Cicero, A.M., Virno Lamberti, C., Magaletti, E., 2012. Toxicity evaluation of diethylene glycol and its combined effects with produced waters of off-shore gas platforms in the Adriatic Sea (Italy): bioassays with marine/estuarine species. Mar. Environ. Res. 77, 141–149. US EPA, 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, fifth ed.Washington, DC 20460, 821-R-02-012. EPA. US EPA, 2012. Benchmark Dose Technical Guidance. Risk Assessment Forum, U.S. Environmental Protection Agency, Washington, DC 20460EPA/100/R-12/001 June 2012. Venkatesh, C., Ashok, K., 2010. Toxicity of sodium dodecyl sulfate in fishes and animals. a review. Int. J. Appl. Biol. Pharm. Technol. 1 (2), 630–633. Woltering, D.M., 1984. The growth response in fish chronic and early life stage toxicity tests: A critical review. Aquat. Toxicol. 5 (1), 1–21. http://dx.doi.org/ 10.1016/0166-445X(84)90028-6. 〈http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d80a3c6-17a5-3287e044 00144f67d249/DISS-9d80a3c6-17a5-3287-e044-00144f67d249_DISS9d80a3c6-17a5-3287-e044-00144f67d249.html〉. Last access 16 March 2015.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax A. Tornambè n, L. Manfra, S. Canepa, F. Oteri, G. Martuccio, A.M. Cicero, E. Magaletti ISPRA, Italian Institute for Environmental Protection and Research, via Vitaliano Brancati 48, 00144 Rome, Italy
ar t ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2015 Received in revised form 4 December 2015 Accepted 25 December 2015
The OECD TG 215 method (2000) (C.14 method of EC Regulation 440/2008) was developed on the rainbow trout (Oncorynchus mykiss) to assess chronic toxicity (28 d) of chemicals on fish juveniles. It contemplates to use other well documented species identifying suitable conditions to evaluate their growth. OECD proposes the European sea bass (Dicentrarchus labrax, L. 1758) as Mediterranean species among vertebrates recommended in the OECD guidelines for the toxicity testing of chemicals. In this context, our study is aimed to proposing the adaptation of the growth test (OECD TG 215, 2000) to D. labrax. For this purpose toxicity tests were performed with sodium dodecyl sulfate, a reference toxicant commonly used in fish toxicity assays. The main aspects of the testing procedure were reviewed: fish size (weight), environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding (food type and ration), test validity criteria. The experience gained from growth tests with the sea bass allows to promote its inclusion among the species to be used for the C.14 method. & 2016 Published by Elsevier Inc.
Keywords: OECD 215 Dicentrarchus labrax Fish growth toxicity test Sodium dodecyl sulfate
1. Introduction The EC Regulation 440/2008 (Council Regulation (EC), 2008) lays down test methods pursuant to European REACH Regulation for registration, evaluation, authorization and restriction of chemicals. It reports the fish growth toxicity test (classified as C.14 method) as “a replicate of the OECD TG 215 (2000)”. This method is designed to assess the effects of prolonged exposure to chemicals on fish juveniles and it has been developed on the freshwater species rainbow trout (Oncorynchus mykiss) but other well documented species such as Danio rerio (zebrafish) and Oryzias latipes (medaka) may be used adapting the procedure. However, the use of a target marine species may be considered more appropriate to assess the impact of chemical anthropogenic substances on the marine environment, especially if the ultimate goal is to establish a regulatory Predicted No-Effect Concentration (PNEC) that should be protective of potentially more sensitive species of this compartment (ECHA, 2008). The marine species most globally used for short- and long-term toxicity assessments by standardized methods are of North American origin, such as Cyprinodon variegatus, Menidia beryllina and Atherinops affinis (US EPA, 2002; ASTM, 2013), that cannot be considered representative n Correspondence to: ISPRA, Italian Institute for Environmental Protection and Research, via di Castel Romano 100, 00128 Rome, Italy. E-mail address: [email protected] (A. Tornambè).
species of the Mediterranean Sea. On the other hand, in the last decade the European species Dicentrarchus labrax Linnaeus 1758 (Osteichthyes, Perciformes, Moronidae) has been proposed as biological indicator in acute toxicity tests (Cicero et al., 2001; Gelli et al., 2004; Magaletti et al., 2006; Mariani et al., 2004, 2006; El-Sayed et al., 2009; Tornambè et al., 2012; Manfra et al., 2015), biomarker analyses (Caruso et al., 2005; Faucher et al., 2008; Giari et al., 2007, 2008; Jebali et al., 2008; Gorbi et al., 2009; Greco et al., 2010; Almeida et al., 2010; Ferreira et al., 2010; Kerambrun et al., 2012; Della Torre et al., 2012; De Domenico et al., 2013) and bioaccumulation studies (Serrano et al., 2004; Carubelli et al., 2007; Antunes et al., 2007, 2008; Trocino et al., 2009; Danion et al., 2011). D. labrax is widespread in Mediterranean waters and broadly reared in fish farms for its commercial importance. It is an euryaline (4–40 psu) and eurythermal (2–30 °C) species, with an optimal range of salinity [20–30 psu] and temperature [14–28 °C], easy to maintain under laboratory conditions and widely available for much of the year. It is one of the recommended fish species in acute toxicity tests for the Italian approval of oil dispersants and absorbents to be used at sea in case of oil spills (Magaletti et al., 2006; Decreto Ministeriale 116 del 25 Febbraio, 2011), and OECD has recently proposed its inclusion among species recommended in the OECD guidelines for the toxicity testing of chemicals (Conti et al., 2015). The general principle of the C.14 test method consists of exposing for 28 d fish juveniles to a range of sublethal concentrations of the test substance preferably under flow-through or
http://dx.doi.org/10.1016/j.ecoenv.2015.12.032 0147-6513/& 2016 Published by Elsevier Inc.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
appropriate semi-static (static-renewal) conditions. Fish are selected in exponential growth phase and weighed before their use. During the test fish are fed daily, the food ration is based on their initial weight and it may be recalculated after 14 d to induce acceptable growth rate. At the end of the test the fish are weighed again and growth rate effects are analyzed. The aim of the present work is to revise and define the main aspects of this growth method (i.e. initial fish weight, environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding, test validity criteria) by exposing D. labrax to sodium dodecyl sulfate (SDS). It is an anionic surfactant widely employed in industry, agriculture and domestic use and therefore it may be found in abundance in the environment, particularly in the sea. It is commonly used as reference organic toxicant in ecotoxicity tests (〈http://echa.europa.eu/〉). The proposed method has been applied for assessing the chronic toxicity of diethylene glycol (DEG), an organic compound used as anticorrosive agent during offshore oil activities (Manfra et al., 2015).
2. Materials and methods 2.1. Materials and chemicals Two polypropylene tanks (300 Liter) were used for fish acclimatization prior to the test. Inert plastic vessels (11 Liter) were used as test chambers with 10 Liter effective volume of test solution. Reconstituted sea water was used both for acclimatization as for test medium. It was prepared by dissolving a commercial sea salt mixture (Instant Ocean™) into distilled water, prepared and aerated for at least 24 h before use. Temperature (20 72 °C), salinity (20 7 1 psu), pH (87 0.5) and dissolved oxygen ( Z60%) were daily checked. The concentrations of ammonia (maximum acceptable value MAV ¼5 mg/l), nitrite (MAV¼0.5 mg/l) and nitrate (MAV¼50 mg/l) were measured twice a week both in stabulation tanks and in test vessels. SDS (Nzytech, Portugal; CAS registry number 151-21-3 with ultra purity 499%) was used as reference toxicant during the experiments. 2.2. Fish holding About 500 juveniles of D. labrax (60–80 d old, length: 4.23 70.37 cm, weight: 0.78 70.25 g) were obtained from the fish farm “Agroittica Caldoli” of Lesina (Italy). They were transferred to the laboratory in polyethylene containers (30 Liter) filled with breeding water and equipped with air diffusers to maintain the level of dissolved oxygen in the water above 60% of saturation. The transfer operations were performed trying to minimize manipulation of the organisms. Fish were acclimatized into two tanks for ten days prior to the test, in order to detect any pathological manifestations and/or mortality due to stress of transport and acclimatization to the new conditions of breeding. The fish tanks operated in a closed cycle with a system of mechanical and biological filtration, and with a water chiller device for the maintenance of constant temperature. Tanks were illuminated with fluorescent lamps that provided 500–800 lx on the surface water, with a photoperiod of 14 h of light and 10 h of darkness. The continuous aeration in the water was maintained using electric air pumping compressors. Fish were fed daily with commercial marine fish food (Aller Futura™ Gr.1) with a high protein content (64%), with daily ration of 2% body weight. During the acclimatization period fry mortality was 4%. A subsample of 30 organisms was weighed and measured to estimate the mean
length and the mean weight of juveniles before the test. Fish were not fed for 24 h prior to the start of the tests. 2.3. Toxicity test method Three tests were performed on juveniles exposed to SDS: 1) an acute test (96 h) by applying the C.1 method (OECD TG 203, 1992), aimed at checking the sensitivity of the fish stock; 2) one preliminary 28-d test (test 1) according to the C.14 method (OECD TG 215, 2000), to explore sublethal concentrations and find the appropriate feeding; 3) one final 28-d test (test 2) at the conditions defined in the preliminary 28-d test, to validate the proposed method. Test solutions were prepared daily by dissolving aliquots of a fresh stock solution (1 g/l SDS in deionized water) into dilution water. The acute test (96 h) was carried out using only three concentrations (14, 7, and 3.5 mg/l) selected in a known range of toxicity (Mariani et al., 2006), with two replicates for each concentration and one control (dilution water without SDS). Test 1 was performed using five concentrations (7, 3.5, 1.75, 0.87, and 0.44 mg/l) with two replicates for each concentration and the control, in order to reduce the number of utilized organisms according to European guidelines (Halder et al., 2014). Test 2 was carried out using the above-mentioned five concentrations, since they showed to be sublethal concentrations with effect on fish growth. In this case, three replicates for each concentration and for the control were used. The SDS concentration was weekly measured, both at the time of preparation and prior to renewal (about 24 h later) of the test solutions by colorimetric MBAS assay using Hach Lange LCK 332 kit analysis for anionic surfactants through DR2800 Hach Lange spectrophotometer (λ ¼ 650 nm)(George and White, 1999). In all the measurements, SDS degradation after 24 h was less than 20% (6.072.2%). Before the test fish were individually weighed as wet weights (blotted dry, without using anesthetics) to the precision of 10 mg and randomly distributed between the different test vessels. The initial range of individual weights was within 7 25% of the arithmetic mean body weight of all used organisms in test 1, and within 7 10% in test 2. The loading rate was less than 1 g/l and the stocking density was of 7 individuals in 10 Liter (for each vessel). The sum of wet weights of each vessel was recorded and used to calculate the initial daily food ration. All the procedure was done trying to avoid stressing test animals as much as possible. The tests were conducted in a temperature-controlled room at 2071 °C, with illumination of 500–800 lx on the water surface, provided by ‘cool-white’ fluorescent lamps, with a photoperiod of 14 h light: 10 h dark. Survival and possible onset of abnormal behaviors of the organisms were daily checked. The renewal of the test solutions was performed by transferring the fish with small nets in other test vessels containing new fresh solutions. The feeding was provided in a single daily ration one hour after the renewal of the test solutions, consisting of the same dry food used in the acclimatization period. The rations, based on the initial total fish body weight, were increased from a minimum of 1% in the first two weeks up to a maximum of 2% in the last two weeks in test 1, while from 2% in the first two weeks up to 3% in the third week and 4% in the last week in test 2. At 28 d, surviving fish were individually weighed again after being anesthetized with clove oil (40 ppt) and dried on absorbent paper. During the acute test fish were not weighed and fed, surviving fish were counted after 96 h according to the C.1 method. 2.4. Data analysis For the 96 h test, the concentration that would cause 50%
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3
mortality (LC50) was calculated by the Trimmed Spearman Karber method (USEPA TSK software, version 1.5). The test was considered valid with a control mortality percentage of r10%. For the 28 d tests, average specific growth rate was calculated for each vessel using the following equation:
r2 =
logew2 − logew1 t2 − t1
× 100
(1)
where: w1, w2 ¼ weights of a particular fish at times t1 and t2, respectively log w1 ¼ average of the logarithms of the values w1 for the fish in the vessel at the beginning of the test log w2 ¼ average of the logarithms of the values w2 for the fish in the tank at the end of the test t1, t2 ¼time (days) at the beginning and end of test, respectively The estimation of the concentration that would cause a x% variation of growth rate (ECx) was calculated by non-linear regression analysis (Logit-Hill model) using the Benchmark Dose Software 2.4.0 (US EPA, 2012). Alternatively, the percentages of growth inhibition were compared with control values in order to determine the no observed effect concentration (NOEC) using Dunnett test. The 28-d tests were considered valid when the following conditions were met (OECD TG 215, 2000): – the mortality in the controls did not exceed 10% at the end of the test, – the mean weight of fish in the controls increased enough to allow the detection of the minimum variation of growth rate considered as significant, – the dissolved oxygen concentration was at least 60% of the air saturation value (ASV) throughout the test, – the water temperature did not differ by more than 71 °C between test chambers at any one time during the test and was maintained within a range of 2 °C within the temperature ranges specified for the test species.
3. Results and discussion The acute toxicity test was considered valid since the control mortality percentage was r10%, as required by the C.1 method, and 96hLC50 value of 9.90 mg/l was comparable to acute results available in literature for SDS (Mariani et al., 2006; Conti et al., 2015). This toxicity value is of the same order of magnitude of the estimated effect concentrations found for other fish species (Ribelles et al., 1995; Rosety et al., 2001) and for other organisms (see Rosety et al. (2001) and references therein), thus confirming the sensitivity of the fish batch used in this study. The test validity criteria were met in both 28-d tests, except for the mean weight increase in the test 1. No fish mortality was observed at the used SDS concentrations. The mean weight increase in the controls was 28.5% (cv ¼42.5%) and 81.4% (cv ¼4.9%) in the test 1 and test 2, respectively. The dissolved oxygen concentration ranged between 76% and 85% of saturation during both tests and the temperature was 207 1 °C in all test chambers. The control weight increase was not enough to estimate a statistically significant effect of SDS on fish growth in test 1, however the weight increase was clearly inhibited at the highest concentration tested (7 mg/l) with respect to control (13.0% vs. 28.5%) (Fig. 1a). On the basis of these results, the test 2 was conducted using the same five SDS concentrations as in test 1, but the range of initial fish weights was narrower and the weekly food ration (from 2%
Fig. 1. 28-d SDS toxicity tests results with D. labrax: (a) comparison between test 1 and test 2 average weight increase at different SDS concentrations and control (CTR); (b) comparison between test 1 and test 2 average specific fish growth rates.
minimum to 4% maximum) was increased, based on the observation of actual consumption by fish (rate of consumption, presence or absence of food residues on the water surface and on the bottom of test chambers). Due to these changes, a double control growth rate (2.15 70.09% d 1) was obtained in the test 2 respect to that in test 1 (1.03 70.30% d 1) (Fig. 1b); similar values of control growth rates were obtained in the two other 28-d tests conducted to assess the chronic toxicity of DEG (1.86 7 0.12% d 1 and 1.727 0.31% d 1, Manfra et al., 2015) following this method. At the highest SDS concentration, the average specific growth rate (1.29 70.12% d 1) showed a statistically significant difference (p o0.05) compared to the control growth rate. The significant increase of control mean weight, together with the validity of the other parameters (mortality, oxygen concentration and temperature value), allowed to consider acceptable the test 2. Calculated 28dEC20 (6.61 mg/l, c.l. 3.95–9.27) and 28dNOEC (3.5 mg/l) values for SDS are consistent with those reported for mortality tests with embryo and sac-fry stages of the freshwater fish Pimephales promelas (28dLC20¼4.5 mg/l and 28dNOECo3.8 mg/l) and for growth tests with P. promelas juveniles (42dNOEC41.357 mg/l) (〈http://apps.echa.europa.eu/registered/ data/dossiers/DISS-9d80a3c6-17a5-3287-e044 00144f67d249/DISS9d80a3c6-17a5-3287-e044-00144f67d249_DISS-9d80a3c6-17a53287-e044-00144f67d249.html〉). The acute and chronic SDS toxicity recorded in our study for D. labrax has already been observed for other marine fish such as Sparus aurata and Scophthalmus maximus (see review of Venkatesh and Ashok (2010)). In those cases changes in morphology, metabolism and swimming capacity were found in association with increased SDS concentrations and length of exposure. Sub-lethal chronic (15–30 d) effects of SDS on the survival, metabolism and growth of juveniles of Centropomus parallelus have also been reported (Rocha et. al., 2007).
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
Table 1 Comparison of main test conditions between three OECD-recommended freshwater species and the marine species D. labrax. Recommended species
Oncorhynchus mykiss Rainbow trout
Danio rerio Zebrafish
Oryzias latipes Ricefish (Medaka)
Dicentrarchus labrax European sea bass
Type of test Recommended weight (g) Required measurement precision Temperature pH Light intensity Photoperiod (hrs) O2 saturation Dilution water
Semi-static/Flow through 1.0–5.0 to nearest 100 mg
Semi-static/Flow through 0.050–0.100 to nearest 1 mg
Semi-static/Flow through 0.050–0.100 to nearest 1 mg
Semi-static renewal 0.5–1.0 to nearest 10 mg
12.5–16 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
21–25 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
21–25 °C 6.5–8.5 Not specified 12–16 light 460% Fresh water
Volume test solution (Liter) Loading rate (g/Liter) Stocking density Food type
40 1.2–2.0 4/ten litres Dry proprietary salmonid fry food
Food ration Test duration Test validity criteria
4% mean body weight Z 28 d - Control mortality o 10% Control mean weight 450%
1 0.2–1.0 5–10/one litre Live food (Brachionus, Artemia) Not specified Z 28 d Control mortality o 10%
1 0.2–1.0 5–20 /one litre Live food (Brachionus, Artemia) Not specified Z 28 d Control mortality o 10%
18–22 °C 7.5–8.5 500–800 lx 14 light 460% Synthetic sea water 20 psu (Istant Ocean™) 10 0.35–1.0 7–10/ten litres Dry proprietary fry food (high protein content) 2–4% mean body weight Z28 d Control mortality o 10% Control mean weight450%
The OECD guideline allows that well documented species may be used other than rainbow trout, leaving uncertain some aspects of the procedure. However whereas the growth response in fish is a critical endpoint of toxicity (Woltering, 1984), we considered necessary to explore and fix some experimental variables in order to obtain a reliable method for D. labrax. The main experimental conditions for conducting 28-d growth tests with fish juvenile of D. labrax are reported in Table 1, compared with the recommended conditions for freshwater species (OECD TG 215, 2000), highlighting the differences, and then discussed. 3.1. Fish size The initial average weight was about 0.6–0.7 g, that D. labrax reaches about 80–100 days after the hatching under intensive rearing conditions (fish farmer personal communication). This size was chosen on the basis of laboratory practical experience: smaller organisms are too delicate and susceptible to manipulation especially for long term tests, while bigger organisms require too large water volumes during static-renewal tests. The required measurement precision was recalculated with respect to the chosen fish size. As recommended by the protocol, it is very important to maximize the initial size homogeneity in each vessel because larger fish tend to eat more food by subtracting it from the smaller and then grow faster thus increasing in-tank final weight differences. 3.2. Environmental conditions The suggested rearing and test temperature (20 72 °C) is usually used for acute toxicity test with this species (Mariani et al., 2004, 2006; Tornambè et al. 2012) and has proved optimal for growth of D. labrax during nursery periods (Mylonas et al., 2003). The indicated pH value is typically marine and should remain constant with small oscillations around 8.0. As for other parameters, the light intensity during the test should be similar as during acclimatization period in order to minimize unwanted sources of stress for the organisms. 3.3. Dilution water Suitability of Instant Ocean™ reconstituted sea water as rearing
and test medium was amply demonstrated in previous cited studies. Furthermore, due to its euryhalinity, toxicity test with this species can be conducted in a wide range of salinity after a short conditioning period of the animals. 3.4. Experimental design Five concentrations of toxicant for three replicates with 7 fish in each vessel (10 Liter test volume), were planned trying to reach a good balance between practical issue, statistical requirements and minimum use of animals. In fact this test design can be considered a minimum to apply a dose-response model based on regression analysis to estimate the ECx and to calculate NOEC/ LOEC values using analysis of variance with a good statistical significance (see ISO (2006) for NOEC/ECx implications for test design). 3.5. Loading rate and stocking density The OECD 203 (1992) recommendations were followed using the maximum loading of 1.0 g fish/Liter for static and semi-static (acute) tests and the minimum of seven fish at each test concentration (replicate in this case). 3.6. Food ration The food rations should be recalculate once a week on the basis of observation of feed consumption and the increased size of organisms. They start from a minimum of 2% up to a maximum of 4% of total body weight, to reach a significant increase of the mean weight of fish in the control, at least above 50% for this species. The first test week can be considered of acclimatization to the new test conditions. In this phase the maintaining of a good fish welfare is very important for a successful growth test (without mortality). The environmental conditions in the test chambers should be very similar to those during stabulation to avoid additional stress to organisms (in particular with regard the water quality, temperature and light intensity). In this sense it is better to start with the usual food ration and then increase it on the basis of effective consumption by organisms, since uneaten food, if not promptly removed, may deteriorate the water quality over the time before the next renewal of test solution.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3.7. Test validity criteria and treatment of results The OECD 215 indicates three alternative ways to calculate the growth rate of fish (individual specific growth rate r1, average specific growth rate r2, and pseudo specific growth rate r3) and gives an indication of acceptable increase of control growth rate only for O. mykiss. We have chosen to calculate the average specific growth rate r2 (Eq. 1) because r1 is not applicable to little size fish (impossible fish marking), while r3 can be considered less reliable, especially in the case of NOEC/LOEC assessment, as fish weight variability within-tank is generally higher than between-tank at the end of the test. This trend was indeed seen in our study where the coefficient of variation (cv) of r2 ranged from 1.9 to 4.5% while cv of r3 ranged from 17.6 to 126.9% between test concentrations. A good weight gain of the control fish is critical to the success of the test. Our results confirm for D. labrax the need to overcome the 50% of increase at the end of test. This result can be obtained only through a careful weekly recalculation of the portions of food based primarily on actual consumption.
4. Conclusions D. labrax can be considered a species suitable for C.14 method and has the advantage of being a Mediterranean species very well known and studied, ready available most of the year, easy to handle, resistant to physical stress but sensitive to chemicals. Moreover, being a marine species of great commercial and ecological importance can be considered more appropriate for risk assessments studies in sea environments than the recommended freshwater species.
Statement The fish used in this study were kept in authorized breeding room pursuant to Italian Legislative Decree 116/92 (Decreto Legislativo, 1992), implementation of Directive n. 86/609/EEC (Council Directive, 1986) on the protection of animals used for experimental and other scientific purposes. This study was performed according to the protocol approved under Decree 116/92.
Acknowledgments The authors are grateful to Dr. Stefania Balzamo for providing working facilities, Dr. Andrea Paina for his technical assistance, and to Dr. Federica Savorelli for many useful suggestions.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.12. 032.
References Almeida, J.R., Oliveira, C., Gravato, C., Guilhermino, L., 2010. Linking behavioural alterations with biomarkers responses in the European seabass Dicentrarchus labrax L. exposed to the organophosphate pesticide fenitrothion. Ecotoxicology 19 (8), 1369–1381. http://dx.doi.org/10.1007/s10646-010-0523-y, Epub 2010 Aug 5. PubMedPMID: 2068692. Antunes, P., Gil, O., Reis-Henriques, M.A., 2007. Evidence for higher biomagnification factors of lower chlorinated PCBs in cultivated seabass. Sci. Total Environ. 377, 36–44.
5
Antunes, P., Hendriks, A.J., Huijbregts, M.A.J., Gil, O., Reis-Henriques, M.A., 2008. Organ-specific accumulation and elimination patterns of PCBs in adult seabass (Dicentrarchus labrax). Sci. Total Environ. 407, 204–210. ASTME1022-94, 2013. Standard guide for conducting bioconcentration tests with fishes and saltwater bivalve mollusks, ASTM International, West Conshohocken, PA, 2013. www.astm.org DOI: 10.1520/E1022-94R13. Carubelli, G., Fanelli, R., Mariani, G., Nichetti, S., Crosa, G., Calamari, D., Fattore, E., 2007. PCB contamination in farmed and wild sea bass (Dicentrarchus labrax L .) from a coastal wetland area in central Italy. Chemosphere 68, 1630–1635. Caruso, G., Genovese, L., Maricchiolo, G., Modica, A., 2005. Haematological, biochemical and immunological parameters as stress indicators in Dicentrarchus labrax and Sparus aurata farmed in off-shore cages. Aquac. Int. 13, 67–73. Cicero, A.M., Savorelli, F., Gelli, F., Palazzi, D., Pregnolato, L., Roncarati, A., Casazza, G., 2001. Use of sea bass (Dicentrarchus labrax) larvae, obtained from controlled reproduction, as biological indicator in acute toxicity tests. In: Proceedings of the 6th International Conference of the Aquatic Ecosystem Health and Management Society. 4–7 November, 2001. Amsterdam, The Netherlands. Conti, D., Balzamo, S., Paina, A., Martone, C., Raso, E., Cadoni, F., Savorelli, F., Croppo, M., Bellaria, V., Pati, A., 2015. European sea bass (Dicentrarchus labrax L . 1758) as a sentinel species in europe to study the effects of contaminants. Annu. Res. Rev. Biol. 8 (4), 1–13. Council Directive 86/609/EEC, 1986. Council Directive 86/609/EEC of 24 November, 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Council Regulation (EC) No. 440/2008, 2008. Council Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (2008) OJ L 142/1. Danion, M., Le Floch, S., Kanan, R., Lamour, F., Quentel, C., 2011. Effects of in vivo chronic hydrocarbons pollution on sanitary status and immune system in sea bass (Dicentrarchus labrax L.). Aquat. Toxicol. 105 (3–4), 300–311. http://dx.doi. org/10.1016/j.aquatox.2011.06.022, Epub 2011 Jun 28. PubMed PMID: 21781655. Decreto Legislativo 27 gennaio 1992, n. 116. Protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici. G.U. n. 40, 18 febbraio 1992. Attuazione direttiva CEE n. 609/86. Decreto Ministeriale 116 del 25 Febbraio 2011. Definizione delle procedure per il riconoscimento di idoneità dei prodotti assorbenti e disperdenti da impiegare in mare per la bonifica dalla contaminazione da idrocarburi petroliferi. Ministero dell’Ambiente e della Tutela del Territorio e del Mare - DG Protezione della Natura e del Mare. De Domenico, E., Mauceri, A., Giordano, D., Maisano, M., Giannetto, A., Parrino, V., Natalotto, A., D’Agata, A., Cappello, T., Fasulo, S., 2013. Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol. Environ. Saf. 97, 114–123. http://dx.doi.org/10.1016/j. ecoenv.2013.07.015, Epub 2013 Aug 14. PubMed PMID: 23953925. Della Torre, C., Tornambè, A., Cappello, S., Mariottini, M., Perra, G., Giuliani, S., Amato, E., Falugi, C., Crisari, A., Yakimov, M.M., Magaletti, E., 2012. Modulation of CYP1A and genotoxic effects in European seabass (Dicentrarchus labrax) exposed to weathered oil: a mesocosm study. Mar. Environ. Res. 76, 48–55. http: //dx.doi.org/10.1016/j.marenvres.2011.08.006, Epub 2011 Sep 8. PubMed PMID: 21963215. ECHA, 2008. Guidance on information requirements and chemical safety assessment. Chapter R.10: Characterisation of dose [concentration]-response for environment. Guidance for the Implementation of REACH. 2008, p. 65. El-Sayed, Y.S., Khalil, H.R., Saad, T.T., 2009. Acute toxicity of ochratoxin-A in marine water-reared sea bass (Dicentrarchus labrax L .). Chemosphere 75, 878–882. Faucher, K., Fichet, D., Miramand, P., Lagardère, J.P., 2008. Impact of chronic cadmium exposure at environmental dose on escape behaviour in sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae). Environ. Pollut. 151 (1), 148–157, Epub 2007 May 10. PubMed PMID: 17493718. Ferreira, M., Caetano, M., Antunes, P., Costa, J., Gil, O., Bandarra, N., Pousào-Ferreira, P., Vale, C., Reis-Henriques, M.A., 2010. Assessment of contaminants and biomarkers of exposure in wild and farmed seabass. Ecotoxicol. Environ. Saf. 73, 579–588. Gelli, F., Cicero, A.M., Melotti, P., Roncarati, A., Pregnolato, L., Savorelli, F., Palazzi, D., Casazza, G., 2004. A proposal of method to evaluate the quality of marine waters: optimization of 7 days bioassays using Dicentrarchus labrax (L.) juveniles. Chem. Ecol. 20 (Suppl 1), S225–S229. George, A.L., White, G.F., 1999. Optimization of the methylene blue assay for anionic surfactants added to estuarine and marine water. Environ. Toxicol. Chem. 18 (10), 2232–2236. Giari, L., Manera, M., Simoni, E., Dezfuli, B.S., 2007. Cellular alterations in different organs of European sea bass Dicentrarchus labrax L. exposed to cadmium. Chemosphere 67, 1171–1181. Giari, L., Simoni, E., Manera, M., Dezfuli, B.S., 2008. Histo-cytological responses of (Dicentrarchus labrax L.) following mercury exposure. Ecotoxicol. Environ. Saf. 70, 400–410. Gorbi, S., Benedetti, M., Virno Lamberti, C., Pisanelli, B., Moltedo, G., Regoli, F., 2009. Biological effects of diethylene glycol ((Almeida et al., 2010)DEG) and produced waters (PWs) released from offshore activieties: a multi-biomarker approach with the sea bass Dicentrarchus labrax. Environ. Pollut. 157, 3166–3173. Greco, L., Serrano, R., Blanes, M.A., Serrano, E., Capri, E., 2010. Bioaccumulation markers and biochemical responses in European sea bass (Dicentrarchus labrax) raised under different environmental conditions. Ecotoxicol. Environ. Saf. 73,
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i
6
A. Tornambè et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
38–45. Halder, M., Kienzler, A., Whelan, M., Worth, A., 2014. EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing. European Commission, Joint Research Centre, Institute for Health and Consumer Protectionhttp://dx.doi.org/10.2788/084219, Luxembourg, European Union, EUR 26973 EN. ISO/TS, 2006. Water quality-Guidance document on the statistical analysis of ecotoxicity data. Ref. n. ISO/TS 20281 2006 252. Jebali, J., Banni, M., Gerbej, H., Boussetta, H., Lòpez-Barea, J., Alhama, J., 2008. Metallothionein induction by Cu, Cd and Hg in Dicentrarchus labrax L. liver: assessment by RP-HPLC with fluorescence detection and spectrophotometry. Mar. Environ. Res. 65, 358–363. Kerambrun, E., Le Floch, S., Sanchez, W., Thomas Guyon, H., Meziane, T., Henry, F., Amara, R., 2012. Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers. Chemosphere 87 (7), 692–702. http://dx.doi.org/10.1016/j.chemosphere.2011.12.059, Epub 2012 Jan 10. PubMed PMID: 22236592. E., Magaletti, Onorati, F., Faraponova, O., Mariani, L., Tornambè, A., Cicero, A.M., 2006. Ecotoxicological tests for the approval of oil dispersants and absorbents to be used at sea. In: Proceedings of the SETAC Europe 16th Annual Meeting. The Hague, The Netherlands 7–11 May 2006. Abstract Book: 96, MO1/KL/P168. Manfra, L., Tornambè, A., Savorelli, F., Rotini, A., Canepa, S., Mannozzi, M., Cicero, A. M., 2015. Ecotoxicity of diethylene glycol and risk assessment for marine environment. J. Hazard. Mater. 284, 130–135. Mariani, L., Manfra, L., Maggi, C., Savorelli, F., Di Mento, R., Cicero, A.M., 2004. Produced formation waters: a preliminary study on chemical characterization and acute toxicity by using fish larvae (Dicentrarchus labrax L . 1758). Fresenius Environ. Bull. 13, 1427–1432. Mariani, L., De Pascale, D., Faraponova, O., Tornambè, A., Sarni, A., Giuliani, S., Ruggiero, G., Onorati, F., Magaletti, E., 2006. The use of a test battery in marine ecotoxicology: the acute toxicity of sodium dodecylsulfate. Environ. Toxicol. 21, 373–379. Mylonas, C.C., Anezaki, L., Divanach, P., Zanuy, S., Piferrer, F., Ron, B., Peduel, A., Ben Atia, I., Gorshkov, S., Tandler, A., 2003. Influence of rearing temperature at two periods during early life on growth and sex differentiation of two strains of European sea bass. Fish. Physiol. Biochem. 28 (1–4), 167–168. OECD, 1992. Test No. 203: Fish, Acute Toxicity Test, OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Parishttp://dx.doi.org/10.1787/ 9789264069961-en. OECD, 2000. Test No. 215: Fish, Juvenile Growth Test, OECD Guidelines for the
Testing of Chemicals, Section 2. OECD Publishing, Parishttp://dx.doi.org/ 10.1787/9789264070202-en. Ribelles, A., Carrasco, C., Rosety, M., Aldana, M., 1995. A histochemical study of the biological effects of sodium dodecyl sulfate on the intestine of gilthead seabream, Sparus aurata. Ecotoxicol. Environ. Saf. 32, 131–138. Rocha, A.J., Gomes, V., Ngan, P.V., Passos, M.J., Furia, R.R., 2007. Effects of anionic surfactant and salinity on the bioenergetics of juveniles of Centropomus parallelus (Poey). Ecotoxicol. Environ. Saf. 3, 397–404. Rosety, R.M., Ordonez, F.J., Rosety, M., Rosety, J.M., Rosety, I., Ribelles, A., Carrasco, C., 2001. Morphohistochemical changes in the gills of turbot, Scophthalmus maximus L ., induced by sodium dodecyl sulfate. Ecotoxicol. Environ. Saf. 3, 223–228. Serrano, R., Simal-Juliàn, A., Pitarch, E., Hernàndez, F., 2004. Response to comment on “Biomagnification study on organochlorine compounds in marine aquaculture: the Sea Bass (Dicentrarchus labrax) as a Model”. Environ. Sci. Technol. 38, 1263. Trocino, A., Majolini, D., Xiccato, G., 2009. PCBs contamination in farmed European seabass from different Italian rearing systems. Chemosphere 76 (2), 2504. http: //dx.doi.org/10.1016/j.chemosphere.2009.03.017, Epub 2009 Apr 5. PubMed PMID: 19349063. Tornambè, A., Manfra, L., Mariani, L., Faraponova, O., Onorati, F., Savorelli, F., Cicero, A.M., Virno Lamberti, C., Magaletti, E., 2012. Toxicity evaluation of diethylene glycol and its combined effects with produced waters of off-shore gas platforms in the Adriatic Sea (Italy): bioassays with marine/estuarine species. Mar. Environ. Res. 77, 141–149. US EPA, 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, fifth ed.Washington, DC 20460, 821-R-02-012. EPA. US EPA, 2012. Benchmark Dose Technical Guidance. Risk Assessment Forum, U.S. Environmental Protection Agency, Washington, DC 20460EPA/100/R-12/001 June 2012. Venkatesh, C., Ashok, K., 2010. Toxicity of sodium dodecyl sulfate in fishes and animals. a review. Int. J. Appl. Biol. Pharm. Technol. 1 (2), 630–633. Woltering, D.M., 1984. The growth response in fish chronic and early life stage toxicity tests: A critical review. Aquat. Toxicol. 5 (1), 1–21. http://dx.doi.org/ 10.1016/0166-445X(84)90028-6. 〈http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d80a3c6-17a5-3287e044 00144f67d249/DISS-9d80a3c6-17a5-3287-e044-00144f67d249_DISS9d80a3c6-17a5-3287-e044-00144f67d249.html〉. Last access 16 March 2015.
Please cite this article as: Tornambè, A., et al., Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax. Ecotoxicol. Environ. Saf. (2016), http://dx.doi.org/10.1016/j.ecoenv.2015.12.032i