Effect of copper contaminated food on the life cycle and secondary production of Daphnia laevis

Ecotoxicology and Environmental Safety 133 (2016) 235–242

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Effect of copper contaminated food on the life cycle and secondary production of Daphnia laevis Giseli S. Rocha a,n, Alessandra E. Tonietto b, Ana T. Lombardi b, Maria da G.G. Melão a a Departamento de Hidrobiologia, Centro de Ciências Biológicas e da Saúde (CCBS), Universidade Federal de São Carlos (UFSCar), Rodovia Washington Luís, Km 235, CEP 13565-905 São Carlos, SP, Brazil b Departamento de Botânica, CCBS, UFSCar, Rodovia Washington Luís, Km 235, CEP 13565-905 São Carlos, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 3 May 2016 Received in revised form 7 July 2016 Accepted 11 July 2016

In aquatic environments, copper (Cu) plays important physiological roles in planktonic food chain, such as electron transfer in photosynthesis and constituting proteins that transport oxygen in some arthropods, while at higher concentrations it is toxic on these organisms and higher trophic levels. The combined effects of natural (e.g. volcanic activity) and anthropogenic sources (e.g. mining waste) contribute to the increase in copper pollution in different ecosystems and regions around the world. In the present study, we evaluated the bioaccumulation and effect of Cu on Raphidocelis subcapitata (freshwater algae), and the influence of Cu-contaminated food (algae) on Daphnia laevis (tropical cladoceran). The amount of copper accumulated in microalgae and cladoceran was quantified, and life-history parameters of D. laevis such as growth, reproduction and longevity were measured. The cell density of Cu exposed R. subcapitata declined, and cladoceran fed with contaminated food had lower longevity, production of eggs and neonates, and reduced secondary production. A concentration dependent increase in Cu accumulation was observed in the microalgae, while the opposite occurred in the animal, indicating a cellular metal regulatory mechanism in the latter. However, this regulation seems not to be sufficient to avoid metal induced damages in the cladoceran such as decreased longevity and reproduction. We conclude that diet is an important metal exposure route to this cladoceran, and the assessment of chronic contamination during the complete life cycle of cladoceran provides results that are similar to those observed in natural environments, especially when native organisms are investigated. & 2016 Elsevier Inc. All rights reserved.

Keywords: Freshwater zooplankton Metal contamination Chronic toxicity Raphidocelis subcapitata Secondary production

1. Introduction In aquatic ecosystems, metals precipitate and accumulate in sediments, compete with nutrients, are chelated by organic substances, taken up and transferred via the food chain, and have toxic effects on several organisms (Sigg and Behra, 2005). The interactions between capture, transport and excretion of metals by organisms affect their bioaccumulation pattern (Rainbow, 2002; Qiu et al., 2015). Some metals, such as copper (Cu), have important functions in organisms and are thus considered as essential metals. Copper supports electron transfer in photosynthesis and redox processes in microalgae (Bossuyt and Janssen, 2005) and is present in haemocyanin, a copper-protein that transports oxygen in molluscs and arthropods (Markl, 2013). However, when present at high concentrations, Cu can cause toxic effects such as decreased respiration, chlorophyll-a production, somatic growth and reproduction (Mason and Jenkins, 1995; Prosnier et al., 2015). Free n

Corresponding author. E-mail address: [email protected] (G.S. Rocha).

http://dx.doi.org/10.1016/j.ecoenv.2016.07.011 0147-6513/& 2016 Elsevier Inc. All rights reserved.

metal ions are the bioavailable fraction that can be taken up by the organisms, presenting a higher toxic potential than organic and inorganic complexed metal forms (Santos et al., 2008; Mendes et al., 2013). Zooplankton transfer energy from primary producers to higher trophic levels, and the quality and quantity of food can affect their fitness (Rose et al., 2002). Metal exposure routes to zooplankton in aquatic ecosystems are water and food, with waterborne exposure being the most evaluated. Kainz et al. (unpublished data) observed that aqueous form of methylmercury was more effective for predicting toxicity in freshwater zooplankton (cladocerans and copepods) than particulate diet sources, thereby, suggesting a trophic level jump. Similarly, aqueous forms of cadmium (Cd) were more toxic to Ceriodaphnia dubia and Daphnia magna than particulate forms (Barata et al., 2002; Rodgher and Espíndola, 2008). Metals taken up via dietary sources may cause deleterious effects to zooplankton including lowered filtration rates, survival, growth and reproductive production, and altered biochemical composition (Rodgher et al., 2009; Souza et al., 2014). To date, results of dietborne metal exposure are not comprehensive (DeForest and Meyer, 2015). The link between metal contaminated

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and toxicity to zooplankton is not fully understood, and some animals are able to recover from metal contamination by removing the contaminants, or when supplied with sufficient and highquality food (Mangas-Ramírez et al., 2004). In addition, the speciation of metals affect their toxicity to organisms, with free ions being bioavailable and more toxic, and the influence of speciation on metal uptake or toxicity is currently not understood (Wang, 2013a). Chronic exposures to dietborne metal contaminants during the complete life cycle of organisms are less investigated in standardized protocols (De Schamphelaere and Janssen, 2004; Kolts et al., 2009), and studies evaluating secondary production under metal exposure are scarce. However, in natural environments, animals can be exposed to chronic contamination, and some metal induced effects can only be detected after 20 days in the laboratory (GussoChoueri et al., 2012). Thus, the aim of present study was to evaluate the effects of Cu-contaminated freshwater algae (Raphidocelis subcapitata) on the full life cycle of Daphnia laevis, in order to understand the effects of copper at sublethal concentrations in two trophic levels – producers (algae – acute exposure) and consumers (Cladocera – chronic exposure) – of a planktonic food chain.

2. Materials and methods 2.1. Algal culture and toxicity tests The freshwater microalga Raphidocelis subcapitata (Chlorophyceae) was obtained from the Algae Culture Collection of the Department of Botany, Federal University of São Carlos (São Carlos, SP, Brazil). Stock cultures were kept in LC Oligo medium (AFNOR, 1980), which has the following composition: Ca(NO3)2
4H2O (1.7  10  4 M), KNO3 (1.0  10  3 M), MgSO4
7H2O (1.2  10  4 M), K2HPO4 (2.3  10  4 M), CuCl2
H2O (6.0  10  8 M), (NH4)6Mo7O24
4H2O (2.4  10  8 M), ZnSO4
7H2O (1.0  10  7 M), CoCl2
6H2O (1.3  10  7 M), Mn(NO3)2
H2O (1.5  10  7 M), C6H8O7
H2O (1.4  10  7 M), H3BO3 (4.9  10  7 M), C6H5FeO7
H2O (3.1  10  6 M), FeCl3
6H2O (1.9  10  6 M), FeSO4
7H2O 6 (1.1  10 M) and NaHCO3 (1.8  10  4 M). The algae were cultured at pH 7.0, under controlled conditions of light intensity (150 mmol m  2 s  1), photoperiod (16:8 h light:dark cycle) and temperature (22 72 °C). Every 12 h, the cultures were gently shaken. The culture medium was kept sterile through autoclaving for 20 min at 121 °C. Sterile conditions were maintained throughout the experiment to avoid culture contamination. Laboratory materials were washed with neutral detergent and kept for 7 days in 10% HCl for metal cleaning. Exponential phase growing R. subcapitata cells were exposed for 96 h to different Cu concentrations: 0.011 (Control), 0.034 (T1), 0.067 (T2) and 0.134 mg L  1 (T3), which corresponded to 0.6  10  7 (C), 2.5  10  7 (T1), 5.0  10  7 (T2) and 7 1 10  10 mol L (T3), respectively. These copper concentrations were defined after preliminary tests where we observed that algae did not grow in concentrations higher than used in this study (10  10  7 mol Cu L  1). Copper solutions were made by serial dilutions of CuCl2 Titrisol 1000 mg L  1 (Merck) with ultrapure water (Barnstead Easy Pure II, Thermo Scientific, Dubuque, IA, USA). Toxicity tests were performed with three experimental replicates per treatment in 500 mL polycarbonate Erlenmeyer flasks containing 200 mL of culture medium. Experiments started with an initial cell density of approximately 5  104 cell mL  1. Cell densities were monitored every 24 h. Samples were fixed with acetic acid lugol, and cells counted under an optical microscope (Leica, DMLS) using an Improved Neubauer-Bright Line haemocytometer.

Fig. 1. Calibration curve for the copper ion selective electrode system reported as log free copper concentration (mol L  1) plotted as function of potential reading (mV).

Free Cu ions in the culture medium were measured from 0 h using a pH meter (Analion AN 2000, Brazil) equipped with Cu ion selective electrode (ISE; Analion Cu-641, Brazil) in conjunction with a glass double-junction reference electrode (Ag/AgCl; Analion R-684, Brazil), under controlled conditions (22 7 1 °C; 0.1 mol L  1 ionic strength adjusted with NaNO3 – Fluka, Switzerland, pH  7.0). A calibration curve was obtained by adding different amounts of Cu, ranging from 1  10  6 to 5  10  5 mol Cu L  1, corresponding to a log Cu range from  6 to  4.3. The metal ion buffer solution comprised 4.5  10  4 mol Cu L  1, 9.1  10  4 mol NTA L  1 (nitrile triacetic acid) and 3  10  3 mol borax L  1, which extended the detection limit for free copper ions to 2.2  10  13 mol L  1 (log Cu 11.65) (See Fig. 1). 2.2. Zooplankton The water flea Daphnia laevis (Crustacea, Cladocera) was collected from culture tanks in the Experimental Reservoir of the Ecology and Evolutionary Biology Department, Federal University of São Carlos, São Carlos-SP, Brazil. The organisms were collected using a plankton net (68 mm mesh size) and transferred to the laboratory. They were gradually acclimated in reconstituted water (40 mg CaCO3 L  1 hardness, pH 7.2), with partial renewals twice per week. After acclimation, the cladocerans were maintained under controlled conditions (227 2 °C, photoperiod 16:8 h light/ dark) in reconstituted water having electrical conductivity of 160 mS cm  1 (ABNT, 2005). After 96 h of exposure, culture aliquots were centrifuged at 1000 rpm (Eppendorf 5702 R, Germany), the supernatant was discarded and the pellet re-suspended in reconstituted water and provided as food for the cladoceran at an initial cell density of  1  105 cells animal  1. The algal cell suspension was stored in the dark at 4 °C throughout the chronic toxicity testing period. The cladocerans (o24 h old) were kept individually in 80 mL polypropylene beakers containing 50 mL of reconstituted water and 1  105 cells of R. subcapitata in a Nova Ética culture chamber (Nova Ética, 411-D, Brazil) set to 22 °C and 16:8 h dark:light cycle. The size of the organisms was measured, and the number of molts, eggs and/or neonates was counted under a stereomicroscope (Leica MZ6; Germany). Culture medium was replaced daily because preliminary tests revealed that the cladocerans did not survive when renewal was done on alternate and every three days. There were ten replicates per treatment, and the control had cladocerans that were fed with uncontaminated algae (1  105 cell ind  1).

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counter electrode. Deoxygenation of DPASV samples was performed with high purity N2 for 10 min before analysis. Furthermore, prior to each analysis, the solution was deoxygenated for 2 min. Three replicate analyses were performed for all determinations. From the digestion of the samples, the ionic strength was maintained at 0.01 mol L  1. After purging the sample with supra pure N2, copper were deposited at the Hg electrode using a  1.2 V potential for 1–5 min, depending on metal concentration, while stirring with a 10 s rest time. Anodic scanning was performed at a scan rate of 8 mV s  1 and a pulse height of 50 mV (Tonietto et al., 2014). A standard addition technique was used for the determination of total metal concentration. The concentration of copper measured was considered as the total amount of metal accumulated (adsorbed onto algal surface and absorbed by the organisms) and expressed as g Cu cell  1 (algae) or g Cu ind  1 (cladoceran). Three previously acid washed filters were digested and analyzed as blanks. 2.4. Statistical analysis Fig. 2. Correlation of dry weight (mg) and length (mm) for different phases (neonates, juveniles, adults and ovate adults) of the cladoceran Daphnia laevis used for estimation of secondary production.

The biomass of each animal was estimated based on linear regression obtained using length-weight relationship of D. laevis placed on previously baked glass fiber filter papers (400 °C 24 h). For linear regression analysis, the length of the organisms were measured, dried in an oven at 60 °C for 48 h, cooled down to room temperature in a desiccator and weighed (Sartorius MC21S, 71 μg). The linear regression between weight and length was calculated using data obtained from 150 neonates (5 replicates of 30 organisms each), 40 juveniles (2 replicates of 20), 50 adults without eggs (5 replicates of 10) and adults with a total of 89 eggs (Fig. 2). The secondary production of D. laevis was calculated according to Edmondson and Winberg (1971). Total production (P) per cladoceran was calculated by summing growth (Pg) and reproductive (Pr) production during the lifetime of each animal. Growth production (Pg) was estimated by converting daily length to dry weight using the equation derived from the linear regression shown in Fig. 2, and daily increment was calculated using the differences in biomass per day [biomass day x biomass day (x 1)]. Reproductive production (Pr) was estimated by multiplying the total number of eggs produced per female by the mean weight of eggs (0.04 mg in present study; Choueri et al., 2007). 2.3. Metal determination Total Cu accumulated in R. subcapitata was obtained after 96 h of exposure to the metal. Algal culture aliquots were filtered through previously acid washed (24 h; HNO3 1 mol L  1) 0.22 mm cellulose membrane filters, and D. laevis (n¼ 10 per treatment) were placed on the cellulose acetate filters. The filters containing algae and Daphnia were dried at 65 °C for 24 h. The dried filters were placed in screw-capped Teflon vials containing 2 mL of 3 mol L  1 HNO3/1 mol L  1 HCl (Ultrapure acids, J. T. Baker), and allowed to digest for 48 h at 90 °C (Gusso-Choueri et al., 2012; Souza et al., 2014). The pH of digested filters was adjusted to 2 with 0.1 mol L  1 NaOH, and the volume adjusted to 10 mL with ultrapure water. Total particulate Cu concentration in the digested filters was measured using differential pulse anodic stripping voltammetry (DPASV). A DPASV static mercury drop electrode (EG&G PARC Model 303 A) system connected to a potentiostat EG&G Model 394 electrochemical trace analyzer was used throughout. Saturated Ag/ AgCl/KCl was used as a reference electrode, and Pt was used as a

Data obtained for R. subcapitata (free copper ions in culture medium, cell density and metal accumulation) and D. laevis (accumulated copper per animal, lengths during the life cycle, production of eggs and neonates, longevity and secondary production) were tested for normality (Kolmogorov-Smirnov test) and homogeneity (Bartlett test). The data were analyzed using oneway ANOVA, and Tukey post-hoc test (po 0.05) was used to detect significant differences in response of metal exposed organisms.

3. Results Free Cu ions (Cu2 þ ) concentration in the algal culture medium was measured at time 0 h. In the control (0.6  10  7 mol Cu L  1), we observed 0.7  10  8 mol Cu2 þ L  1; in T1 treatment (2.5  10  7 mol Cu L  1), 4.1-fold of the amount of Cu present in control medium, we obtained 3  10  8 mol Cu2 þ L  1. In T2 treatment (5  10  7 mol Cu L  1), we obtained 7.3  10  8 mol Cu2 þ L, while in T3 (10  10  7 mol Cu L  1), the value was 1.3  10  7 mol Cu2 þ L  1. The cell density results of R. subcapitata exposed to Cu for 96 h are shown in Fig. 3. The cell density of the microalga decreased with increasing Cu concentration. The control had the highest cell density (  6.4  105 cell mL  1), while T2 and T3 had the lowest ( 1.7  105 cell mL  1) and T1 intermediate (  3.5  105 cell mL  1) values. Fig. 4 shows that the amount of Cu accumulated per algal cell was significantly different between the treatments. Specifically, a concentration dependent accumulation of Cu was observed, where 0.6  10  15 g Cu cell  1 was found in control, 1.3  10  15 g Cu cell  1 in T1 treatment and 2.7  10  15 g Cu cell  1 in T2 treatment, while in the T3 treatment, there was 4.7-fold increase in the amount of the metal accumulated (11.6  10  15 g Cu cell  1) compared to T2 treatment. Table 1 shows the accumulated Cu and life history parameters of D. laevis exposed to Cu-contaminated food. There were no statistical differences in accumulated Cu in adult animals (1.8 2.1  10  9 g Cu ind  1). Although the length at primipara was higher in the T1 treatment (1.24 7 0.01 mm) compared to other treatments ( 1.08 mm), it was not significantly different between the treatments. Maximum length was recorded in the T1 treatment (2.01 70.03 mm), which was significantly higher than those of the control and T2 ( 1.8 mm) treatments, while T3 had the lowest length (1.55 70.05 mm). With regard to longevity, the highest values were observed in T1 and T2 treatments (  31 days), which were significantly higher than those of the control (  28

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Table 1 Mean values 7 SD of life history parameters of Daphnia laevis exposed to copper during the lifetime, for control (0.6  10  7 M added; 0.7  10  8 M free copper) and treatments T1 (2.5  10  7 M added; 3.0  10  8 M free copper), T2 (5.0  10  7 M added; 7.3  10  8 M free copper) and T3 (10  10  7 M added; 1.3  10  7 M free copper). Lines with same superscript letters are not significantly different (p 40.05).

Accumulated copper (10  9 g Cu mg ind  1) Neonate mean length (mm) Age at primipara (days) Length at primipara (mm) Mean clutch size Maximum length (mm) Longevity (days) Fig. 3. Cell density (  105 cell mL  1) of Raphidocelis subcapitata exposed up to 96 h to copper. Control refers to the copper present in LC Oligo medium (0.6  10  7 M added; 0.7  10  8 M free copper); T1: 2.5  10  7 M added; 3.0  10  8 M free copper; T2: 5.0  10  7 M added; 7.3  10  8 M free copper and T3: 10  10  7 M added; 1.3  10  7 M free copper. Values are means 7 SD for n ¼3. Bars with different superscript letters are significantly different (p o 0.05).

Control

T1

T2

T3

1.82 7 0.25

1.91 70.19

2.11 70.11

1.92 7 0.24

0.62 7 0.01

0.647 0.03

0.63 7 0.02

0.60 7 0.02

10.20 73.00

8.90 7 3.00

9.20 7 3.70

9.20 7 1.30

1.08 7 0.01

a

4.45 7 1.06 1.86 7 0.04a

1.247 0.01

b

4.3571.20 2.017 0.03b

a

1.077 0.01a

3.157 0.92 1.85 70.04a

2.317 0.40 1.55 7 0.05c

1.08 70.01

28.337 0.06a 31.677 0.07b 31.337 0.04b 24.007 0.01c

number eggs as those of the control but with a lower hatch rate. In the T2 treatment, the number of eggs produced was lower than in the control; and the females in the T3 treatment produced significantly lower number of eggs and had a lower hatch rate. Growth production did not significantly differ between the control, T1 and T2 (  0.13 mg DW ind day  1) treatments, while it was lowest in the T3 (  0.09 mg DW ind day  1) treatment (Fig. 6A, Table 1). Based on the number of eggs and neonates produced per individual (Fig. 5), the control organisms had the highest investments in reproductive production (  2.9 mg DW ind  1), which was significantly higher (p o0.05) than those of other treatments T1 (  2.7 mg DW ind  1), T2 (  1.9 mg DW ind  1) and T3 (  1 mg DW ind  1) (Fig. 6B). As shown in Fig. 6A and B, the organisms invested more energy in reproduction than growth, and total production followed the same pattern observed for reproductive production (Control 4T1 4T2 4T3) (Fig. 6C).

4. Discussion

Fig. 4. Accumulated copper (10  15 g cell  1) by algae Raphidocelis subcapitata exposed up to 96 h to copper. Control refers to the copper present in LC Oligo medium (0.6  10  7 M added; 0.7  10  8 M free copper); T1: 2.5  10  7 M added; 3.0  10  8 M free copper; T2: 5.0  10  7 M added; 7.3  10  8 M free copper and T3: 10  10  7 M added; 1.3  10  7 M free copper. Values are means7 SD for n¼3. Bars with different superscript letters are significantly different (po 0.05).

days) and T3 (  24 days) treatments. No significant differences were found between different treatments for the broods. Fig. 5 shows the mean number of eggs (5A) and neonates (5B) produced by female cladocerans during their lifetime. Production of eggs in the control and T1 treatments was similar ( 65 eggs female  1), while it significantly (p o0.05) decreased in T2 (  32 eggs female  1) and T3 ( 21 eggs female  1) treatments (Fig. 5A). Furthermore, there was a Cu concentration dependent decrease in neonates produced per female (Fig. 5B). Based on the number of eggs and neonates produced per female, we calculated percentage hatching, which revealed that the control and T2 treatments had similar hatchings ( 91%). However, significantly lowered hatchings were recorded in T1 (  84%) and T3 ( 65%) treatments. Furthermore, the animals in T1 treatment produced similar

High concentration of contaminants and nutrient limitation can result in poor food quality and lower ingestion rate by the animals (DeMott, 1986). This affects growth rates (Kilham et al., 1997); increase metabolic costs of food assimilation (De Schamphelaere et al., 2007); and change the fatty acids of cladocerans (Brett et al., 2006). The contamination of algae can inhibit the ability of cladoceran to feed (Rodgher and Espíndola, 2008). In addition, contaminants can change lipid levels, affect growth and reproduction (Wacker and Martin-Creuzburg, 2007; Jordão et al., 2016). These physiological alterations are sometimes reversible, when food quality is improved by removing the contaminant or adding more nutrients (e.g. phosphorus and nitrogen). Copper is an essential element required for the correct metabolism, and if the diet does not supplement the appropriate amount, the fitness of the organism can be affected (Kainz and Fisk, 2009). However, when present in higher concentrations it can be toxic, affecting aquatic food chain directly – when producers and consumers are affected by the contaminant present in water – or indirectly, when consumers ingest contaminated food. When metals are internalized and located in tissues, they are available to trophic transfer (Vijver et al., 2004) and this is, apparently, the predominating copper uptake route (Croteau and Luoma, 2009). In concentrations above those required, copper can affect growth, photosynthesis and biomolecules production such as protein, carbohydrates and lipids in algae (Perales-Vela et al., 2007, Kainz

G.S. Rocha et al. / Ecotoxicology and Environmental Safety 133 (2016) 235–242

Fig. 5. Production of eggs (A) and neonates (B) per female of Daphnia laevis exposed to copper during the lifetime. Control (full circles) refers to the animals fed algae cultured in LC Oligo medium (0.6  10  7 M added; 0.7  10  8 M free copper); T1 (open circles): 2.5  10  7 M added; 3.0  10  8 M free copper; T2 (full triangles): 5.0  10  7 M added; 7.3  10  8 M free copper and T3 (open triangles): 10  10  7 M added; 1.3  10  7 M free copper. Values are means for n¼ 10. Different letters in the end of lines means that the treatments are significantly different (ANOVA, Tukey’s post-test, p o 0.05).

and Fisk, 2009, Kumar et al., 2010) and affect zooplankton through modification of its filtration rates, survival (Rodgher et al., 2008), reproduction (Koch et al., 2009) and egg lipid content (Wacker and Martin-Creuzburg, 2007). The life cycles and predator-prey interactions may also be affected by excess copper (Timmermans, 1993). Chronic copper exposures are important and can elucidate if any acclimation/adaptation processes occur and how it affects the fitness of organisms exposed to contaminants. Due to the higher free ionic copper recorded in the treatments when compared to control, the cell density of R. subcapitata decreased by  50% in the T1 treatment ( 3.5  105 cell mL  1) compared to the control (  6.4  105 cell mL  1), and  75% in T2 and T3 (1.7  105 cell mL  1) treatments. These results corroborate previous literature results. Specifically, Tripathi and Gaur (2006) and Perales-Vela et al. (2007) observed a decrease in cell density of Scenedesmus spp. with increasing copper concentration in the media. On the other hand, Bossuyt and Janssen (2004) showed

239

Fig. 6. Secondary production of Daphnia laevis. Production invested in growth (Pg; A); in reproduction (Pr; B) and total (P; C) per individual of Daphnia laevis exposed to copper during the lifetime. Control (full circles) refers to the animals fed algae cultured in LC Oligo medium (0.6  10  7 M added; 0.7  10  8 M free copper); T1 (open circles): 2.5  10  7 M added; 3.0  10  8 M free copper; T2 (full triangles): 5.0  10  7 M added; 7.3  10  8 M free copper and T3 (open triangles): 10  10  7 M added; 1.3  10  7 M free copper. Values are means for n ¼ 10. Different letters in the end of lines means that the treatments are significantly different (ANOVA, Tukey’s post-test, p o 0.05).

that the acclimation of Pseudokirchneriella subcapitata to Cu increased its resistance to the metal. The growth of the microalga was affected in the presence of copper, probably due to the internalization of the metal (De Schamphelaere et al., 2005). The accumulated copper in the algae in the T1 treatment (1.3  10  15 g cell  1) was significantly higher than in the control (0.6  10  15 g cell  1; po0.05), which is in agreement with previous results that higher levels of the metal are accumulated in P. subcapitata (De Schamphelaere and Janssen, 2004; Tripathi and Gaur, 2006; Rodgher et al., 2008). These changes indicate that the algae adsorb and absorb Cu. According to Bossuyt and Janssen (2005), more than 80% of total cellular copper can be located intracellularly and transferred to higher trophic levels.

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The accumulation of metals, essentials or not, can be different between invertebrate species. When exposed to several metals such as copper and zinc, the organism can regulate the amount in its body (regulator), i.e. keep the metals in the body at a constant concentration by altering the rate of excretion, or accumulate and detoxified the metals with or without excretion (Rainbow and Dallinger, 1993; Rainbow, 2002). Other parameters, such as assimilation efficiency, filtration rate and excretion rate can affect the bioaccumulation pattern of metals in organisms (Pan and Wang, 2009). Based on our results (Table 1), there was no significant difference in the concentration of copper accumulated in the cladoceran, under the different treatments conditions in the different broods. We suggest that Daphnia laevis is a regulator species, which agrees with published results on other Daphnid species. For example, Bossuyt and Janssen (2005) concluded that Daphnia magna regulates the internal concentration of copper and is able to acclimate to the concentrations of the metal in the environment (Bossuyt and Janssen, 2004), while Hook and Fisher (2002) observed a similar regulation of zinc by copepods. We did not observe changes in neonate length, age at primipara or in the mean clutch size between treatments (p 40.05), which could be a consequence of the constant concentration of Cu in Daphnia laevis. We observed that first brood started in 6 days old animals in the control, 7 days old in treatment T1 and 8 days old in T2 and T3 treatments. However, these changes in brooding time was not statistical different between the treatments. These results differ from previous studies that demonstrated that copper affected the mean clutch size and age at primipara of Ceriodaphnia cornuta (Gusso-Choueri et al., 2012), nickel caused changes in brood sizes and neonate lengths after first brood of Daphnia magna (Evens et al., 2009), and cadmium affected the age at primipara of Simocephalus serrulatus (Souza et al., 2014). De Schamphelaere et al. (2007) did not observe changes in age at primipara of Daphnia magna exposed to copper, however there was a delay in the second and third reproduction of copper treated animals compared to the control. We observed differences in longevity, maximum length and length at primipara of cladocerans exposed to copper contaminated food. The highest length at primipara and maximum length were obtained in organisms in the T1 treatment. The highest longevities were found at intermediate Cu concentrations (T1 and T2; 31 days), compared to the control (  28 days) and T3 (  24 days) treatments. The higher longevity recorded in copper treatments agrees with the results of Gusso-Choueri et al. (2012). The authors showed that the presence of copper stimulated the longevity of C. cornuta. On the contrary, De Schamphelaere et al. (2007) did not observe changes in survival of D. magna exposed to copper for 21 days, while Souza et al. (2014) noted that the cadmium exposure decreased the longevity of S. serrulatus. In chronic tests, reproductive success is an important parameter for evaluating ecological effects of a toxicant in populations and communities (De Schamphelaere and Janssen, 2004). The production of eggs and neonates was significantly lower in T2 and T3 treatments than the control and T1 treatment, which corroborates with the results of previous studies. Sobral et al. (2001) reported that Cu contaminated water had toxic effects on Daphnia magna, and De Schamphelaere et al. (2007) recorded a 50% decrease in the reproduction of the same species when fed with copper contaminated algae for 21-days. Sofyan et al. (2007) observed deleterious effects in Ceriodaphnia dubia exposed to copper and cadmium via water, food and the combination of both exposure sources during three broods, and suggested that the metal could affect eggs produced and reproductive organs such as oocytes. However, with regard to the effects of copper on cladocerans and differing from the results of the present study, Koivisto and Ketola (1995) did not observe changes in Daphnia pulex exposed to

the metal during 4 broods (13–17 days); Kolts et al. (2009) did not record changes in reproduction of Ceriodaphnia dubia fed with algae contaminated with Ag and Cu; and Gusso-Choueri et al. (2012) reported that changes in the reproduction of Ceriodaphnia cornuta started after day 12 (4th brood), a period longer than recommended time frames (3 broods) in some standardized protocols for chronic toxicity assays. On the other hand, De Schamphelaere and Janssen (2004) observed increased growth and reproduction of Daphnia magna exposed to food particles contaminated with copper for 21 days. Toxicant effects can be extended over generations of exposed organisms, and evaluations of different generations are recommended (Coutellec and Barata, 2013; Campos et al., 2015). As an ecotoxicological metabolic endpoint, secondary production can be altered by anthropogenic activities (Carlisle and Clements, 2003). The effect of this alteration can be extended from individuals to population and community levels (Bayona et al., 2014; Benke, 2010; Faupel et al., 2012). Metals in food and water can affect energy flow in the food web (Wang et al., 2010). For example, the exposure to cadmium altered secondary production of benthic communities (Faupel et al., 2012) and cladocerans (Souza et al., 2014), and aquatic insects exposed to high zinc concentrations had significantly different secondary production (Carlisle and Clements, 2003). After maturation, cladocerans invest most of their energy in reproduction (Koivisto and Ketola, 1995), but the increased metabolic costs of coping with toxicant stress can decrease reproduction and growth (De Schamphelaere et al., 2007). In the present study, 100% of adult females in the control and T1 treatment produced eggs, while this was only possible in 90% and 50% of the females in the T2 and T3 treatments, respectively. In the present study, secondary production of D. laevis was different between the treatments, and decreased in a Cu concentration dependent manner (Fig. 6). In all treatments, secondary production (P; Fig. 6C) was determined by reproductive production (Pr; Fig. 6B), i.e. food energy was invested in egg biomass production. This implies that the organisms invested more energy in reproduction than growth, which is in line with the suggestion that Daphnia invests more energy in reproduction (Lynch, 1980). Studies correlating metal exposure and cladoceran secondary production are scarce. Hence, it is difficult to make comparison with our results. However, our results are in agreement with those of Souza et al. (2014), who observed a decrease in secondary production of Simocephalus serrulatus exposed to cadmium. We suggest that changes observed in Daphnia laevis under copper exposure are related with contaminated food and not with genetic diversity, since we used only one D. laevis clone in our study and it is known that occur a loss of sodium in cladocerans in the presence of contaminant, causing an osmotic imbalance and affecting the fitness of organisms (Bianchini and Wood, 2002; Koch et al., 2009). Our results reinforce the impact of dietborne copper on cladocerans. We found that copper changes cladoceran life cycle, reproduction and secondary production. Initially, more profound changes occurred in samples with more copper, affecting algae and cladocerans that fed them. This copper ingested could be transferred and altered the functioning of the higher trophic levels. Although we were not able to analyze the partitioning of metals in algae and cladocerans, as suggested by other authors (Rainbow et al., 2011; Wang, 2013b), it is a very interesting and necessary approach for better understanding of metal exposure routes, intracellular compartmentalization and subsequent toxicity to freshwater organisms. 5. Conclusion In the present study, we observed that increasing copper concentration decreased cell density of the microalgae, and

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zooplankton exposed to contaminated food had lower number of neonates and longevity. In addition, we observed an increase in the concentration of the metal in the algae with increasing Cu concentration, while the concentration of the metal was relatively constant in Daphnia laevis fed with Cu contaminated algae. This suggests that this cladoceran acts as a regulator organism, keeping the metal concentration in the body constant. However, the production of neonates and longevity was altered, suggesting some internalization of the metal, which decreased secondary production of D. laevis. Based on our results and from literature, we suggest that the bioaccumulation of the metal in food (algae) is an important Cu exposure route to D. laevis. Hence, we recommend that chronic exposure assays of toxicants using cladocerans should include the use of secondary production as an ecotoxicological endpoint.

Acknowledgments The authors are grateful to the São Paulo Research Foundation (FAPESP), Brazil for financial support (Grants nos. 2007/02073-4 and 2007/02680-8).

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