Effects of short- and long-term exposures to copper on lethal and reproductive endpoints of the harpacticoid copepod Tigriopus fulvus

Ecotoxicology and Environmental Safety 147 (2018) 327–333

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Effects of short- and long-term exposures to copper on lethal and reproductive endpoints of the harpacticoid copepod Tigriopus fulvus

MARK

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Francesca Biandolinoa, , Isabella Parlapianoa, Olga Faraponovab, Ermelinda Pratoa a b

CNR-IAMC, Institute for Coastal Marine Environment, Taranto, Italy ISPRA – Institute for Environmental Protection and Research, Rome, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Copepod Tigriopus fulvus Copper Development Reproductive assay Fecundity

The long-term exposure provides a realistic measurement of the effects of toxicants on aquatic organisms. The harpacticoid copepod Tigriopus fulvus has a wide geographical distribution and is considered as an ideal model organism for ecotoxicological studies for its good sensitivity to different toxicants. In this study, acute, sub-chronic and chronic toxicity tests based on lethal and reproductive responses of Tigriopus fulvus to copper were performed. The number of moults during larval development was chosen as an endpoint for sub-chronic test. Sex ratio, inhibitory effect on larval development, hatching time, fecundity, brood number, nauplii/brood, total newborn production, etc, were calculated in the chronic test (28 d). Lethal effect of copper to nauplii showed the LC50-48 h of 310 ± 72 µgCu/L (mean ± sd). It was observed a significant inhibition of larval development at sublethal copper concentrations, after 4 and 7 d. After 4 d, the EC50 value obtained for the endpoint in “moult naupliar reduction” was of 55.8 ± 2.5 µgCu/L (mean ± sd). The EC50 for the inhibition of naupliar development into copepodite stage, was of 21.7 ± 4.4 µgCu/L (mean ± sd), after 7 days. Among the different traits tested, copper did not affect sex ratio and growth, while fecundity and total nauplii production were the most sensitive endpoints. The reproductive endpoints offer the advantage of being detectable at very low pollutant concentrations.

1. Introduction A full evaluation of ecological effects of chemical contaminants on coastal environments should consider toxic effects on population responses of representative benthic species (Bechmann, 1994; Hutchinson et al., 1994a, 1999; Chandler and Green, 2001). Population response is considered as a more ecologically relevant measure of toxicant effect than the other endpoints, because it integrates different individual lifehistory responses and can provide insight into potential effects at higher levels of ecological organization. A limitation to this approach has been the cost and length of time required to generate demographic toxicity data, indeed chronic toxicity data are less available than acute data and the testing procedures less standardized (Forbes et al., 2001). For routine laboratory testing, the reduction of exposure times presents valuable advantages in terms of personnel and resource requirements. Therefore, there is interest in developing short assays that provide adequate data for determining reliably the effects of toxicants on marine organisms, at the community and ecosystem level. Based on the published data, invertebrates, such as harpacticoid copepods with short generation times (3 weeks) and greater toxicant sensitivity offer

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the possibility of examining population effects of contaminants (Bechmann, 1999). Meiobenthic copepods are also intimately associated with all compartments of the sedimentary matrix throughout their lifecycles (i.e., they have no pelagic life cycle stage). The copepod life-cycle assay has been developed and used successfully in several studies with different harpacticoid copepods such as T. japonicas, Amphiascus tenuiremis, Nitocra spinipes, Tisbe battagliai etc. (Brown et al., 2003; Raisuddun et al., 2007; Kwok et al., 2009; Guo et al., 2012). The harpacticoid copepod T. fulvus was selected for this study, as it is widely distributed in the Mediterranean Sea and ecologically relevant. Indeed, it forms an important energy link in marine food webs as lives on micro-algae or bacteria and serves as prey for larger crustaeans, fish larvae and filter feeding bivalves. T. fulvus is an autochthonous, meiobenthic, euryhaline and eurythermal harpacticoid copepod and its biology is well known (Todaro et al., 2001). Since this species is small in size (adult length of around 1 mm), with a short generation time (ca. 21 d) and high fecundity, it has been found very suitable for laboratory rearing and does not present any particular difficulties for maintenance (Faraponova et al., 2003).

Corresponding author. E-mail address: [email protected] (F. Biandolino).

http://dx.doi.org/10.1016/j.ecoenv.2017.08.041 Received 28 February 2017; Received in revised form 24 July 2017; Accepted 17 August 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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Therefore, animals at all developmental stages are available at any time of the year. Previous studies reported T. fulvus as an ideal model organism for ecotoxicological studies for its good sensitivity to different toxicants and good reproducibility of test (Mariani et al., 2006; Faraponova et al., 2005, 2003, 2016; Tornambè et al., 2012; Prato et al., 2011, 2012, 2013, 2015). Moreover, the test with this species is easy-to-use and low-cost, because it requires minimal space and equipment for testing. New standardized procedures for laboratory culturing of T. fulvus and standardized method for acute toxicity were recently published by the UNICHIM in the method 2396:2014 “Water Quality – Determination of the lethal toxicity at 24 h, 48 h and 96 h exposure to the nauplii of T. fulvus (Crustacea: Copepoda). The most recent article reports the results of an interlaboratory comparison involving 11 laboratories in order to validate a new standardized ecotoxicological method on this marine crustacean (Faraponova et al., 2016). Until now, it has been used for evaluating the acute effects of marine contaminants. However, this copepod species shows several advantages for its use in the chronic test (e.g., life-cycle test) including endpoints for development to adult and reproduction. T. fulvus has 12 distinct post-embryonic developmental stages (6 naupliar stages, 5 copepodite stages and an adult stage), dimorphic sex with males slightly smaller than females and with geniculated antennae, and high fecundity of females with multiple broods of eggs developed sequentially after a single mating event (Faraponova et al., 2003). The aim of this study was to exploit the rapid life cycle of T. fulvus to develop sub-chronic and chronic toxicity tests with copper. For this purpose nine individual life-cycle traits of this species, were investigated, including survival, moults number, sex ratio, body lengths, developmental time of nauplius phase, fecundity, hatching time, number of brood and number of nauplii/brood.

Table 1 Nominal and measured concentrations of metals at the beginning (t = 0 h) and after 48 h of the tests, and the percentage of the nominal concentration as measured at each time. Nominal

Measured 0 h

%

Measured 48 h

%

15 30 60 120 250 500 1000

16.4 ± 0.0 31.3 ± 0.8 60.8 ± 0.5 130.4 ± 2.6 257.6 ± 3.8 513.0 ± 4.6 989.0 ± 3.6

109.3 104.3 101.3 108.7 103.0 102.6 98.9

16.36 ± 0.2 31.7 ± 1.2 62.8 ± 0.8 130.5 ± 1.8 238.3 ± 2.8 497.0 ± 2.7 972.0 ± 2.4

109.1 105.7 104.7 108.7 95.3 99.4 97.2

in previous studies (Faraponova et al., 2016). On the same day of the experiment, the stock solution of the test solution and its dilutions were prepared. Moreover, test solutions were analysed to provide an indication of the changes in metal concentrations at beginning (0 h) and after 48-h in static test conditions. Analyses were carried out using an atomic absorption spectrophotometer with a graphite furnace (Perkin Elmer Zeeman 3030). Results of the verifying analyses demonstrated that the percent deviations from the nominal concentrations were always less than 10% (Table 1). 2.3. Test procedures The summary of experimental conditions of all test are given in Table 2. 2.3.1. Acute exposure test (48 h) Nauplii (≤ 24 h-old) of T. fulvus were exposed to increasing concentrations of dissolved copper (60, 120, 250, 500, 1000 µg Cu/L), for 48 h (Prato et al., 2013), in a static acute toxicity test, in which lethality was the only endpoint assessed. Each test was carried out with an array of five dilutions of the toxicant and one control in filtered seawater (FSW). The tests were carried out at the same environmental conditions described for the massive culture. The tests were performed in sterile 12-well suspension cultureplates, flat bottom with low evaporation. Ten nauplii were randomly transferred to each chamber using a pipette to a 5 mL well plate, each containing 3 mL of test solution and a control. Because of the short duration of the tests (48 h), the nauplii were not fed during the experiment. There were three replicates for each concentration and control. The acute test was repeated three times and the LC50 was calculated with 95% confidence limits. Mortality of copepods was checked under a stereomicroscope every 24 h. Mortality criterion was inability to move any external appendage or any internal member in a period of up to 20 s of observation and light stimulation of well solution.

2. Materials and methods 2.1. Test organism T. fulvus used for this study were obtained from massive cultures and kept, for several months, inside the 0.5 L polystyrene tissue culture flasks with filter screw caps. Filtered natural seawater (0.45 µm), at salinity of 38 PSU (most common salinity value in the western Mediterranean Sea) was used. The copepods were maintained at 20 °C ± 2 in 16 h/8 h light/dark photoperiod, at 500–1200 lx luminosity (cool light) (UNICHIM 2396:2014, 2014). The cultures were fed weekly ad libitum with Tetramarin (fish food) or a mix of Tetraselmis suecica and Isochrysis galbana algae. These algae were cultured in a temperature-controlled room with 14:10 h light/dark cycle, using 500 mL flasks filled with autoclaved natural seawater (NSW) collected in an unpolluted area, and filtered through a GF/C Wathman (0.22 µm) filter (Prato et al., 2015). The algae, coming from cultures at the CNR IAMC in Taranto, were cultured using F/2 medium (Guillard, 1975). Toxicity tests were conducted on newborn offspring (nauplii) originating from synchronized cultures (24–48 h). The nauplii were released by ovigerous females selected 24 h prior the test, transferred on an 80 µm mesh plankton net fixed to a Plexiglas tube and fed with a mix of T. suecica and I. galbana algae cultures at 1.5 × 108 and 3.0 × 108 cells/L density, respectively.

2.3.2. Subchronic exposure (4–7 days) The effect of copper on the number of moults, considered as development indicator, was assessed using 4–7 days semi-static toxicity test. Twenty-four nauplii (≤ 24 h-old) of T. fulvus were individually introduced into each chamber of 24-well plates containing 1 mL of test solution. FSW without copper was used as control. Based on the acute toxicity testing, a series of nominal copper concentrations (0, 15, 30, 60 µg Cu/L) lower than the NOEC, was chosen. The test was performed in three replicates and repeated three times. The light and temperature conditions are those described above for culturing. Nauplii were observed on days 4 and 7. In the first 96 h, no food was provided, since this is the longest exposure time that does not require feeding or solution substitution (ISO/FDSI 14669). The test solutions/controls, with food supply (1.5 × 105 cells/mL of the algae I. galbana and T. suecica), were renewed after 96 h; at least

2.2. Test chemicals Stock solution of copper was prepared by dissolving copper (II) sulfate pentahydrate (CuSO4 × 5H2O), in Milli-QTM water to obtain metals concentration of 1 g/L. The stock solution was further diluted in filtered seawater (0.2 µm filter fiber) in volumetric flasks to obtain working solutions at designated nominal concentrations before dosing. Sub-lethal concentrations assayed were chosen based on 48-h LC50 values estimated for T. fulvus 328

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Table 2 Summary of experimental conditions of each toxicity test. Acute test

Sub-chronic test

Chronic test

Test type Stage of development Luminosity Light/dark photoperiod Dilution water

Static Nauplii (≤ 24 h) 500–1200 lx cool light 16 h:8 h Filtered Sea Water (FSW) (0.22 µm)

Semi-static Nauplii (≤ 24 h) 500–1200 lx cool light 16 h:8 h Filtered Sea Water (FSW) (0.22 µm)

Salinity PSU Temperature pH Chamber test Test duration Incubation volume (mL) N° organisms/replicate N° replicates N° run N° concentrations Concentrations (μgCu/L) Solution renewal Feeding

38 ± 2 20 ± 2 8 ± 0.3 12-well plates 48 h 3 10 3 3 5 + control 60–120–250–500–1000 Absent Absent

Endpoint

Mortality rate

Semi-static Nauplii (≤ 24 h) 500–1200 lx cool light 16 h:8 h Filtered Sea Water (FSW) (0.22 µm) 38 ± 2 20 ± 2 8 ± 0.3 24-well plates 4–7 d 1 1 24 3 3 + control 15–30–60 After 96 h I. galbana, T. suecica (1.5 × 105 cell/mL) Moults number

38 ± 2 20 ± 2 8 ± 0.3 12-well plates 28 d 3 10 6 3 3 + control 15–30–60 Every 48 h I. galbana, T. suecica (1 × 105 cell/mL) Survival, larval development, n° broods/female, n° nauplii/brood, n° nauplii/female, sex-ratio, body length, ecc

in isolation until the offspring release. Every day females were inspected and every 48 h were transferred to a new culture plate with fresh solutions; the resulting nauplii were counted under a stereomicroscope. The fecundity (offspring production) was assessed as the number of nauplii per female. The number of broods per female and nauplii per each brood were recorded. Aborted egg sacs were counted to determine the overall hatching success. Effects on growth were investigated by measuring the body length of females from each treatment and control.

90% of the exposure solution was changed, transferring the copepods into new well plates containing fresh treatment solution and food. To evaluate the number of moults, a drop of crystal violet solution was placed in each well, making identification of the exoskeletons under the microscope easy. The seventh day of sub-chronic test, the presence and number of copepodite moults, was determined. The results were expressed as moults percentage reduction at different chemical concentrations compared with the control. 2.3.3. Chronic exposure test (28 days) Chemical concentrations used were the same as sub-chronic toxicity test. To start female copepods with egg sacs were selected and kept in a petri dish (Sterilin, UK) with 25 mL FSW together with food supply before experimentation. After 24 h, newly hatched nauplii (i.e. < 24 h old) were collected in a petri dish with 25 mL FSW. Ten healthy nauplii (i.e. could swim actively), were randomly selected, and transferred to a 5-mL well plates containing 3 mL test solution. Six replicates for each copper concentration were used. At the start of the experiment, algal cells (T. suecica and I. galbana) were added to a density of 1 × 105 cells/mL at each replicate. Temperature was recorded at 24-h intervals. Test solutions with food supply (1 × 105 cells/mL of the algae I. galbana and T. suecica) were renewed every 2 days. Survival of copepods was checked and recorded daily, until animals reached maturity, in each test microwell by using a stereomicroscope. At the same time, the developmental stages were observed to calculate the time of development (i.e., from nauplii to copepodite and from copepodite to adults with egg sacs). Nauplii survival rate calculated as percentage of surviving nauplii, which reached copepodite stage. Adult survival rate calculated as percentage of surviving copepod (male and female) on day 28. After the juveniles period, those individuals that became males were eliminated from the experiment, which continued only with the remaining females. Before male exclusion, male and female were allowed to mate. It has been reported that females of this genus require only one mating event to ensure fertilization of all eggs produced during its life span (Davenport et al., 1997). When a female produced an egg sac, she was transferred to a separate 5 mL culture plate with fresh solutions. Ten ovigerous females per each copper concentration were tested separately as replicates, which allowed each individual to be observed

2.4. Statistical analysis Data are presented as the means ± standard deviation (S.D.) of the three runs. The mortality percentages of T. fulvus were determined for each assay at different times. In order to calculate the LC50 48 h (lethal concentration for 50% of organisms) of the copper to nauplii, the Trimmed Spearman–Karber (TSK) method was used (Hamilton et al., 1977). In all traits, raw data were tested for normality and homogeneity of variances using Shapiro-Wilks and Bartelett's tests. When either assumption was met, data were examined by analysis of variance (oneway ANOVA) and multiple comparison procedure (Tukey test) to find significant variations (p < 0.05) among treatments. When requirements for normality and homogeneity were not met, the non-parametric Kruskal–Wallis test on ranks was applied (p ≤ 0.05). All statistical analyses were conducted using Statgraphics software and package software Past3 (version 1.0).

3. Results 3.1. Acute test Mean percentage survival in the negative controls was > 98% in each test, meeting the acceptability criteria established for the tests with this species. Concerning the acute response of T. fulvus to copper, the mean LC50 value after 48-h f was of 310 ± 72 µgCu/L. The effect of copper on mortality showed not significant differences at the lowest concentration 60 µgCu/L, but significantly increases at 120 µg/L (p < 0.05). 329

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4

120

a CTR

15 µg/L

30µg/L

60µg/L

Nauplii and copepodites %

Mean N° of naupliar and copepodid moults /ind.

F. Biandolino et al.

3

2

Nauplii

Copepodid

100 80 60 40 20 0 CTR

1

15

30

60

Copper µg/L

Fig. 3. Effects (%) of copper on larval development of T. fulvus after exposure of 6 days, during the chronic toxicity test.

0

4d nm

7d nm

7d cm

3.3. Chronic test

Days Fig. 1. Mean number of naupliar and copepodite moults/ individual of the copepod Tigriopus fulvus exposed to sub-lethal concentrations of copper during the sub-chronic toxicity test ( 4 and 7 days). Data are mean ± standard deviation. nm= naupliar moults; cm = copepodite moults; bars with the same letter indicate that the means are not statistically different ( p > 0.05).

After 28 days exposure, the trend of T. fulvus survival decreased as copper concentration increased, from 97% in the control to 87% at 60 μgCu/L, although not significant differences between control and all treatment groups existed (p > 0.05). During the first 6 days, the exposure of nauplii to copper resulted in a significant inhibition of larval development at highest concentrations. At 30 μg/L, the percentage of copepodites was 68% and at 60 μg/L only 38% of nauplii developed to copepodites (Fig. 3). The EC50 value for this endpoint was 43.5 μg/L (30–62). The inhibition rate, i.e. the proportion of inhibiting production of copepodites during 6 days, increased with increasing copper concentration, starting from 30 μg/L (p < 0.05), with an inhibition rate of 5.4 ± 1.0%, until to 21 ± 3.8% at 60 μg/L respect to control (Fig. 4). Although copper slowed down larval development of T. fulvus, all nauplii made it to copepodite stage with a good survivorship. So sublethal copper concentrations did not affect the naupliar and copepodite survival (p > 0.05). Copper did not significantly affect the time to reach sexual maturity, with the appearance of the first precopulatory pairs around the 10th–11th day (p > 0.05). In addition, the mean time for adult female to became ovigerous did not differ among treatments (p > 0.05), with the production of the egg sacs 2–3 days later the pairing. In relation to the number of ovigerous females found at 14thd, the statistical analysis showed significant differences between the control and copper treatments (ANOVA, F = 6.22, P = 0.001), already at the lowest concentration. The percentage of ovigerous females was of 55% in the control and 24%, 27% and 14% at 15, 30 and 60 μg/L, respectively (p < 0.05). As regard sex ratio (i.e., number of female/number of male), there was no significant difference between the control and different treatment groups. The sex ratio was 1.2, 1.4, 1.4, and 1.8 for the control, 15, 30, and 60 µgCu/L, respectively. There was difference in the time required for the offspring release in

3.2. Sub-chronic test After 4 days of nauplii exposure (< 24 h-old) to copper, the EC50 value obtained during larval development for the endpoint in “moult naupliar reduction” was of 55.8 ± 2.5 µgCu/L, showing a significant reduction at 30 and 60 µgCu/L (p < 0.05). The mean number of naupliar moults in the control was 3.6 ± 0.4, while at 30 and 60 µgCu/L it was 2.8 ± 0.3 and 2.0 ± 0.2, respectively (Fig. 1). As regard the inhibition of naupliar development into copepodite stage, the EC50 was 21.7 ± 4.4 µgCu/L after 7 days. For this endpoint, a significant inhibition of larval development occurred already at the lower concentration (p < 0.05). Fig. 2 reports the effect of copper on mean number of copepodite and total moults.

Inhibition rate %

25 20 15 10

5 0 15

30 Copper µg/L

60

Fig. 4. Effect of copper on production of copepodites of T. fulvus during first 6 days of exposure. (Percentages are shown referring to the mean total number of copepodites in the control). Data are mean ± standard deviation. Significant difference from the control treatment is indicated by * p < 0.05.

Fig. 2. Reduction (%), respect to the control, of the mean number of copepoditemoults (A) and mean number of total moults (B), after 7 d, during the sub-chronic toxicity test. Data are mean ± standard deviation. Significant difference from the control treatment is indicated by * p < 0.05.

330

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Brood N°/Female

6

a b

b

5 4

c

3 2 1 0 CTR

15

30

60

Nauplii N°/Brood

Copper µg/L 18 16 14 12 10 8 6 4 2 0

Fig. 6. The cumulative sum of nauplii produced by females (n = 10) of T. fulvus. Data are mean ± standard deviation. Significant difference from the control treatment is indicated by * p < 0.05.

CTR

15

30

However, already on Day 20 a significant reduction in the number of produced total nauplii in the copper treatments was observed. All ovigerous females in the control and test solutions were alive during the experiments. There was statistically significant difference between the sum cumulative of nauplii produced per female in the control and copper concentrations. In particular, it was significantly lower than those in the control already on Day 6 (Fig. 6). Growth of females (body length µm) was not affected by exposition to copper (p > 0.05), ranging from 777.3 ± 77 in the control to 793.33 ± 65 µm at the 15 µgCu/L.

60

Nauplii N°/Female

Copper µg/L 80 70 60 50 40 30 20 10 0

a

b b c

CTR

15

30

60

4. Discussion

Copper µg/L

Aborted sacs %

Copper is an essential metal, although it may become toxic if intracellular concentrations exceed the organism's requirements and its detoxification capability (Viarengo, 1989; Livingstone, 2001). In this study, the impact of Cu on the copepod T. fulvus, by chronic exposure, has been assessed. A longer period can allow us to assess different biological traits and make use of such data to obtain information on the fitness of the population. For T. fulvus no study has provided information on the sensitivity of sublethal endpoints in organisms exposed to copper in a chronic toxicity test. The results reported here showed that by exposing nauplii (< 24 h) of T. fulvus over 28d to Cu it was possible to successfully measure lethal effects on naupliar and adult stages, and non-lethal effects on naupliar development, brood size, fecundity, etc. Early life stages have been found as the most sensitive life-history trait of many crustacean species in many studies (Verriopoulos and Moraıtou-Apostolopoulou, 1982; Forget et al., 1998; O’Brien et al., 1988; Prato and Biandolino, 2005, 2006; Diz et al., 2009). In this study, Cu was very toxic for the early naupliar stages of T. fulvus, in terms of their effect on both survival, in acute toxicity test, and on the reduction in the number of moults, in the subchronic test. The result of LC50 value for Cu was similar to previous studies with this species and other test organisms. Faraponova et al. (2016), in a study of interlaboratory comparisons, reported for T. fulvus, LC50 values at 48 h in a range of 173–443 µgCu/L. Prato et al. (2013), for the same species, reported 48 h LC50 value of 230 µgCu/L. Bao et al. (2013), for T. japonicus exposed to copper for 48 h, reported an LC50 value > 1000 µgCu/L, while Diz et al. (2009) for Tisbe battagliai lower values (83 ± 10 µgCu/L). In general, the LC50 value in organism exposed to copper decrease with exposure time. It is widely accepted that lethality endpoint is not sufficient to assess the effects of pollutants on ecosystems (Cairns, 1992). Sublethal tests at sensitive life-stages have been used to improve prediction of chronic effects compared with acute lethality tests (Hutchinson et al., 1994b). Therefore, a copepod test that takes into account the development

b

20 15 10 5

a a

a

CTR

15

0 30

60

Copper µg/L Fig. 5. Effect of copper on some reproductive traits. Data are mean ± standard deviation. Bars with the same letter indicate that the means are not statistically different (p > 0.05).

the control and copper treatments, that was significantly prolonged at 30 and 60 μg/L (p < 0.05). In particular hatching was observed after 2.49 ± 0.77 d in the control and after 2.94 ± 1.03 d and 3.13 ± 1.06 d in 30 and 60 µgCu/L groups, respectively. Compared with the control, there were significantly fewer broods per female at all copper concentrations and also significantly less broods per female at 60 μgCu/L compared with lower copper exposures (p < 0.05). The broods number per female ranged from 4.67 ± 0.84 in the control to the 2.87 ± 0.94 at 60 μg/L (Fig. 5). Reproductive failure, defined as the percent of broods per females unable to produce viable offspring (aborted egg sacs) was significantly (p < 0.05) increased at 60 μgCu/L, compared with lower copper exposures and controls (p < 0.05). The proportion of broods aborted was 1.36%, 0.79%, 5.03%, and 17.62% in the control and at 15, 30 and 60 μgCu/L, respectively (Fig. 5). No significant effect of copper on mean number of nauplii per brood (P > 0.05) was observed (Fig. 5). However, fecundity (the mean total number of nauplii per female) at the end of the experiments (on day 28) was significantly reduced at all copper treatments (51.7, 46 and 28 at 15, 30 and 60 μgCu/L, respectively), compared with the control (67 mean total nauplii per female) (p < 0.05) (Fig. 5). 331

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of larval stages and the development of nauplii into copepodites may reflect disturbances of molting processes. In this manner, it might be possible to detect disturbances to mechanisms that are necessary for growth and development. Copper has been found to inhibit the larval development causing a reduction in number of naupliar moults (after 4d) and copepodites (after 7d). For naupliar moult reduction (4d), Rinna et al. (2011), obtained EC50 values lower (29 ± 6 µg/L) than those from this study (55.8 ± 2.5 µg/L). Inhibition of larval development by copper occurred at concentrations much lower than the lethal concentrations, over all from nauplii to copepodite stage, showing an EC50 of 21.7 ± 4.4 µg/L. The mechanism for inhibited development of larvae by copper probably is due to disturbance of copper in the activity of more enzymes involved in the biochemical processes associated with glycolysis, Krebs cycle, and electron transport chain (cytochrome c oxidase). It is well established that, in response to contaminant exposure, animals are subject to increased metabolic demands for maintaining homeostasis, for defense and detoxification, causing the redirection of energy from reproduction to other physiological processes (Kwok et al., 2009). Besides, Cu is a superoxide generator, and can induce oxidative stress in tissues of aquatic organisms. Cu has ability to induce the formation of hydrogen peroxide (H2O2), which might be transformed to hydroxyl radical OH (Stenersen, 2004), and OH is extremely reactive and modifies all biomolecules in its vicinity, including lipids, proteins and DNA (Eisler, 1998; Stenersen, 2004). The detailed toxic mechanisms of Cu to copepods have not been well understood. Ki et al. (2009) observed that in T. japonicus the mRNA expression of several genes involved in growth, metabolism, reproduction and hormonal regulation was modulated by exposition to copper. In this study, Cu already at concentration of 30 µg/L induced significant retardation to the development of T. fulvus. Significant differences between copper exposure and control were found, despite literature reports of other crustacean species that show an increase in moults frequency after Cu exposure (Sreenivasa Rao and Anjaneyulu, 2008). This is probably due to the different respiratory protein of Copepoda (haemoglobin), in respect to Malacostraca (haemocyanin) (Terwilliger and Ryan, 2001). Sublethal responses to copper showed a trend in which no or low response at low concentrations occurs, followed by an accelerating negative response as concentrations increase. As expected, the low concentrations of copper did not affect the survival of T. fulvus, however, it decreased with copper increase. For example, 87% of nauplii (< 24 h old) were able to survive at 60 μgCu/L for 28 days, not differing significantly from the control (97%). The implications of exposure to increasing copper concentrations for T. fulvus were apparent from the life-cycle test as these animals showed a slowdown in larval development and in the reproductive success. The larval development of T. fulvus was sensitive to low concentrations of copper and this is evident in the delay to reach copepodite stage. Indeed, as evidenced by % of copepodite moult, already at 30 µgCu/L, the developmental time of nauplii to copepodite stage was significantly lengthened in respect to that of the nauplii exposed to the seawater control. Kwok et al. (2008) and Bao et al. (2013) reported for T. japonicus a significant development slowdown at 10 µg/L. Lee et al. (2008) also found that the nauplius development time was sensitive to different metals and biocides. So, this endpoint that provides results of chronic toxicity in a short time is particularly useful, shortening the exposure to 4 or 7 d. Furthermore, this shorter assay is important in financial terms, since save materials, personnel time and costs. The different EC50 values observed for this endpoint in the subchronic and chronic tests could be due to the fact that in sub-chronic testing, during the first 96 h, copepods have not been fed. It is well known that in small copepods, feeding and reproduction are closely related (Tester and Turner, 1990; Saiz et al., 1997). The slowdown of the larval development did not cause a delay of

sexual maturity and appearance time of ovigerous female, at all concentrations tested. There were, however, effects on the time required for the offspring release that was significantly prolonged at 30 and 60 μg/L (p < 0.05). The mean number of broods and the total number of nauplii per female were the most sensitive endpoints measured for T. fulvus exposed to copper. However, no effect of copper on the number of nauplii produced per brood was observed, suggesting that the metal acts through a direct lethal effect on the current brood (as can be seen by the increase in the % of aborted broods), rather than via a maternal shift in resource allocation reducing the effort put into offspring production. These results are consistent with previous findings that copepod reproductive output is significantly impacted when exposed to metals. Hook and Fisher (2001) observed an effect of dietary Hg and Cd in the reduction of egg production and hatching rate of Acartia tonsa and A. hudsonica. Sunda et al. (1987) stated that the egg-laying rate of A. tonsa was sensitive to Zn and Cu. Kwok et al. (2009) found that the reproductive output of T. japonicus exposed to Cu decreased in a dosedependent manner. Medina et al. (2008) reported for T. angulatus that nauplii production at Cu exposures to 103 and 180 μgCu/L was significantly different from the control. On the contrary, Lee et al. (2008) found that copper did not significantly affected offspring production of T. japonicus at concentrations of 0.1, 1, 10, and 100 μg/L, after 10 days exposure. Bielmyer et al. (2006) reported for A. tonsa exposed to Zn, Cu and Ni a significant reduction of the total number of hatched nauplii. Fecundity and total newborn production have a high predictive capacity for detecting alteration at the population level and assess the long-term effect of the substance in the ecosystem. As stated by Diz et al. (2009), a sublethal effect, such as a gradual decrease in fecundity, would mean a long-term lethal effect because the individual's ability to contribute offspring to the next generation would decrease. Several previous studies have demonstrated the increasing sensitivity of multigenerational exposure to different pollutants, both inorganic and organic (Pennington and Scott, 2001; Pane et al., 2004; Massarin et al., 2010). On the other hand, Kwok et al. (2009) also found that Cu resistance of T. japonicus increased even after one-generational acclimatization to 100 μg/L. Endpoints based on reproductive performance, as shown in this study, offer the advantage of being detectable at very low pollutant concentrations, much below the acute toxic concentrations of the chemical to be assessed. Moreover, due to their high sensitivity, they can be used to predict the impact prior to occurrence of contamination or to monitor the actual effect of this (Calow, 1989). The other life traits quantified in the present study, including body length and sex ratio, were not significantly affected by copper exposure. Although the sex ratio resulted in a rise of the female-to-male ratio in the copepod exposed to copper. On the contrary, Lee et al. (2008) reported a lowering of the female-to-male ratio with 3.4 and 1.7 at 10 and 100 μg/L of copper, respectively. Voordouw and Anholt (2002), in a previous work on copepods, have shown that sex determination during larval development could be affected by physiological state of the parents and disturbed by environmental stressors. 5. Conclusion The results of this study provide further evidence to support the use of T. fulvus in ecotoxicology studies. Naupliar development of T. fulvus (such as larvae that have developed into copepodites in a given time) might be able to detect toxicants at low concentrations. The effect of chronic exposure to Cu on sub-lethal endpoints was consistent. Fecundity appears to be the most sensitive endpoint tested. Therefore, copper could seriously influence the populations in areas where the concentrations of this pollutant are in the high range. Given the key role of early developmental stages on the population dynamics 332

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