Environmental concentrations of metals (Cu and Zn) differently affect the life history traits of a model freshwater ostracod

Abstract Zinc (Zn) and copper (Cu) are common contaminants of inland water bodies under severe anthropogenic pressure. The main aim of this study was to test the influence of environmental aqueous concentrations of Zn and Cu on the fitness-related life history traits of the cosmopolitan freshwater ostracod Heterocypris incongruens. Overall, eight subchronic laboratory experiments were conducted to test the effects of exposure to two Cu (260 and 460 µg Cu L−1) and Zn (230 and 410 µg Zn L−1) concentrations compared to the control conditions using asexual individuals originating from a wild population and from a commercial toxicity test (Ostracodtoxkit f). The response of ostracods to exposure to Zn and Cu differed considerably. The Cu treatments significantly reduced the total hatching success and hatching dynamics of resting eggs, increased the mortality of juveniles and reduced the survival of adults, dramatically decreasing the fitness of ostracods. On the other hand, the Zn treatments were less harmful and did not affect adult survival or the timing of juvenile hatching from subitaneous eggs but extended the timing of laying eggs at lower concentrations and stimulated hatching dynamics and success at higher concentrations. It seems that the increased Zn body burden did not strongly impact the fitness of the studied specimens. Moreover, the different responses of the laboratory and wild populations in the Zn experiments may suggest a genetic variation in the tolerance of ostracods to metals. Therefore, experiments involving laboratory H. incongruens as analogues of wild ostracods should be cautiously interpreted. We concluded that the major toxicant in our study was Cu, while Zn could be considered a micronutrient even supporting ostracod fitness, although its higher levels are likely to exert strong toxic effects.


Introduction
Heavy metal contamination has recently become one of the most important ecological problems in aquatic environments (e.g., Kapoor & Singh 2021).Metal contamination may originate from natural processes (e.g., volcanic eruptions, rock weathering), but more often, it is a result of human-mediated activities (e.g., agricultural runoff, untreated industrial effluents) (e.g., Bagul et al. 2015;Hange & Awofolu 2017).In the environment metals are not degradable; therefore, they contribute to metal cycling (e.g., Kumar et al. 2021).In water bodies organisms are exposed to metals in the bottom sediment, in the overlying water column and from dietary sources (Rainbow 2002).Metal ions in a free hydrated form are highly available for organisms (e.g., Engel & Fowler 1979;Sanders & Jenkins 1984).An increase in the concentration of free metal ions in solution leads to an increased rate of metal uptake in aquatic organisms (Rainbow 1995).Since heavy metals tend to accumulate in the bodies of living organisms (bioaccumulation), metal concentrations increase in tissue at successively higher levels in the trophic chain (biomagnification).Thus, progressively greater concentrations of metals are incorporated, showing the lowest levels in producers and primary consumers and reaching the highest values in tissues of the top predators (e.g., Ali & Khan 2018).
Aquatic organisms accumulate metals in their body tissues, whether they are metabolically essential or not.When the level of a given metal in an organism exceeds metabolic needs, possible toxic effects may occur.For that reason, organisms regulate the uptake of metals from the environment to meet metabolic needs but also to prevent the negative consequences of exceeding the toxicity threshold.Within the body of an aquatic organism, excess metal can be excreted or bound to specific macromolecules and thus detoxified (Mason & Jenkins 1995).The detoxification process involves the binding of metallothioneins to insoluble granules (e.g., Mason & Jenkins 1995;Langston et al. 1998).Various invertebrates are characterized by different patterns of metal accumulation; therefore, taxa living in the same habitat can show different metal concentrations in their bodies (Rainbow 2002;Iglikowska et al. 2023a,b).Furthermore, differences in metal uptake can be related to the specific tissue or body part within an organism (Temara et al. 1997;Iglikowska et al. 2018).
Zinc (Zn) and copper (Cu) are common and widespread water contaminants, especially in environments severely impacted by anthropogenic actions (e.g., Rogula-Kozłowska et al. 2021).Both of these compounds are considered essential for metabolic processes; for instance, Zn is a key component of many enzymes, such as carbonic anhydrase.Copper is involved in the respiration process as a haemocyanin in molluscs and malacostracan crustaceans (Taylor & Anstiss 1999).However, for both metals at higher concentrations, harmful effects can be observed (e.g., Brown et al. 2004).In aquatic environments, Zn is the most deleterious to organisms under conditions of elevated temperature, low dissolved oxygen, and low water pH (Eisler 1993).Most Zn introduced into water bodies is readily sorbed and partitioned into sediment particles, and its bioavailability depends on sediment chemical traits (pH, salinity, oxygen concentration, etc.) (Eisler 1993).The exposure of aquatic organisms to elevated Zn concentrations may affect survival (e.g., Sevilla et al. 2014: in Heterocypris incongruens (Ramdohr)), growth (e.g., Nedrich & Burton 2017: in Hyalella azteca (Saussure)) and reproduction (De Schamphelaere et al. 2004: in Daphnia magna (Straus)).Copper in freshwater occurs mainly in the form of cupric ions (Cu 2+ ).Copper salts are highly soluble in water, and their solubility is strongly affected by reducing conditions, and further modified by water temperature, hardness, pH, and other factors (Davis & Leckie 1979).The concentration of Cu in the bottom sediment is directly related to the Cu concentrations in the overlying water.Sediment-bound Cu is highly available to benthic deposit feeders (Bryan & Langston 1992).The lethal effect of Cu in aquatic invertebrates is related to metabolic disturbance (e.g., disruption of osmoregulation, disturbed transport via the epithelium, and a reduction in enzyme activity) (Cheng 1979;Hansen et al. 1992).Because the problem of environmental contamination with Zn and Cu has recently increased worldwide, the toxicity of both metals is commonly investigated with the use of direct exposure biotests involving aquatic invertebrates (e.g., Sevilla et al. 2013Sevilla et al. , 2014;;Muna et al. 2018).
Direct contact biotests involve a variety of model benthic invertebrates, such as midge larvae (Chironomus riparius (Meigen)), amphipods (Hyalella azteca) and ostracods (Heterocypris incongruens).The Ostracodtoxkit f (subchronic H. incongruens exposure test) is a popular, standardized bioassay for routine monitoring of sediment toxicity under laboratory conditions (ISO).The indisputable advantages of this test are its low cost, high level of reproducibility, simple performance, and small laboratory scale.Ostracod microbiotests have been used to evaluate the toxicity of soils (e.g., Chial & Persoone 2003), contaminated sediments in rivers (e.g., Watanabe et al. 2008), and road dust runoff (e.g., Watanabe et al. 2011), among others.Experiments based on microbiotests are regarded in environmental studies as analogues of toxic exposures observed in natural environments.These methods enable the quantification of the toxic effects of individual contaminants on a single species under laboratory control.However, it is not fully understood whether the reactions of individuals of a given biotest species, which were bred for generations under laboratory conditions, properly reflect the response to toxic compounds observed in natural populations of this species.Moreover, Heterocypris incongruens is known to be sensitive to elevated metal concentrations (e.g., Jośko et al. 2016).However, earlier reports focused on the influence of metal contamination on the survival and growth of freshly hatched juveniles using the Ostracodtoxkit f procedure (e.g., Kudłak et al. 2011;Sevilla et al. 2013Sevilla et al. , 2014;;Muna et al. 2018), whereas studies dealing with the influence of environmental concentrations of heavy metals on other fitnessrelated life traits, including hatching success or the survival of adult individuals originating from

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A. Iglikowska et al. natural populations are extremely rare (e.g., Hiki et al. 2017).To the best of our knowledge, there are also no data on the bioaccumulation of heavy metals in nonmarine ostracods exposed to metals dissolved in ambient water.Therefore, the main objective of the present study was to assess the influence of different concentrations of Zn and Cu in fresh waters on several life history traits of H. incongruens, such as the survival of adults and juveniles, the time of egg laying and hatching, and hatching success.Furthermore, we aimed to compare the response to aqueous metal exposure in a test population from Ostracodtoxkit f with that in a wild population of H. incongruens originating from a natural water body that is impacted by the anthropogenic activity.Finally, the body concentrations of Zn and Cu were measured in relation to different background levels of heavy metals in ambient water.In this study, we intended to apply experimental conditions imitating those in the natural environment.By testing the concentrations of metals naturally occurring in water bodies, we wanted to assess limits for the occurrence of H. incongruens, a model species that is a common inhabitant of freshwater under strong anthropogenic pressure.The performed experiments enabled a risk assessment for different traits of the ostracod life history to reveal those life stages that are particularly sensitive to the toxic effects of Zn and Cu exposure.

Test species
Heterocypris incongruens is a morphospecies that displays considerable morphological and genetic variation and occurs nearly worldwide as a frequent inhabitant of various freshwater and slightly brackish-water lentic ecosystems, including small humanmade bodies of water.Over most of its geographical range, the species is represented by parthenogenetic females, while bisexual populations are found mainly around the Mediterranean Sea (Karan-Žnidaršič et al. 2018;Kilikowska et al. 2022).Ecologically, H. incongruens is a benthic organism, and being an omnivorous is a primary and secondary consumer (Meisch 2000).Because H. incongruens is a prey for other invertebrates and fish (Vandekerkhove et al. 2012), it contributes to the trophic transfer of metals within the food chain.After hatching, the body size of neonates is approximately 200 µm, whereas adults are approximately 1.5 mm in length.The species is adapted to inhabit temporary pools and survives a dry season by producing diapausing dormant eggs (Angell & Hancock 1989).At the beginning of the wet season, when pools fill with water, most drought-resistant eggs hatch, but some of them remain dormant for a longer time period.This bet-hedging strategy can reduce the risk of local population extinction in highly unpredictable environments (Rossi et al. 2016).

Test specimens, culture conditions, and experimental design
Experiments were performed using asexual individuals of the ostracod H. incongruens originating from both a wild population and a commercial toxicity test (hereafter "laboratory individuals or population").Specimens of the wild population were collected from a small temporary concrete pool of the municipal drainage system in Gdańsk, Poland (N 54.38491, E 18.57672) Copper and zinc in freshwater ostracod Cu260 and Cu460, respectively).The selected metal concentrations were taken from Sevilla et al. (2014) and satisfied the criteria to be high enough to affect the studied ostracods but also to be low enough to maintain the majority of the examined population alive to carry out subchronic tests.Additionally, all mentioned Zn and Cu concentrations were observed in natural water bodies (e.g., Takamura et al. 1989;Seker & Kutlu 2014;Chen et al. 2019;Chueh et al. 2021) thus they can be considered environmental concentrations.In each experimental setup, a control sample containing pure EPA medium without added metal was included.
To ensure trace metal purity under laboratory conditions, all tools, vessels, and laboratory ware (exclusively plastic ones) were digested for 24 h in 3 M HNO 3 (Merck, Suprapur) solution, rinsed for another 24 h in Milli-Q water, and finally dried in a drying chamber for 5-6 h at 40°C.Aqueous exposure experiments were carried out without sediment.The compounds contained in sediment can capture or react with studied metals; therefore, the lack of sediment prevented the binding of metal ions by sediment particles and ensured constant aqueous concentrations of the studied metals during the course of the experiments.
Overall, eight subchronic (7-18-day-long) experiments (Experiments I-VIII) were conducted to test the effects of exposure to two environmental concentrations of Cu (Cu260 and Cu460) and two environmental concentrations of Zn (Zn230 and Zn410) compared to those of the control (CTRL) on seven fitness-related life history traits (Traits 1-7) at various developmental stages (resting and subitaneous eggs, juveniles, and adult parthenogenetic females) of the wild and laboratory populations of H. incongruens: 1) overall survival of adults (of 1st and 2nd successive generations) (Experiments I-IV), 2) adult survivorship (survival curves) (Experiments V-VI), 3) timing of laying first eggs and 4) timing of juvenile hatching from subitaneous eggs (Experiment VII), 5) total hatching success, 6) hatching dynamics (shape of cumulative hatching curves) and 7) juvenile mortality within 24 h after emergence (Experiment VIII).Finally, concentrations of both metals were measured in the bodies of adult ostracods exposed to environmental concentrations of Cu and Zn compared to those of specimens from the control conditions (Experiment IX).In total, 4890 individuals were used for the entire experimental plan.

Adult survival subchronic experiments (Experiments I-IV)
Adult survival experiments were conducted in a laboratory incubator under acclimatized conditions mimicking natural conditions at the temperature of 20°C and photoperiod of 12:12 (light:dark).
During the experiment, the medium was changed, and ostracods were fed every four days.The medium was changed to maintain the constant concentration of examined metals in solution as well as to remove the decaying food and faeces (Vandekerkhove et al. 2012).For H. incongruens from the wild population, the exposure test was repeated for two successive generations (separately for Zn and Cu).
To test the effects of 1) the laboratory vs. wild population and 2) the first (I) vs. second (II) generations on the survival rate of adult females of H. incongruens exposed to environmental Cu and Zn concentrations compared to the control conditions, the animals were randomly divided into 70 dm 3 containers, each containing 100 individuals.Then, the containers were randomly assigned to one of the six combinations of the three experimental metal treatments (control (CTRL) with pure EPA medium without metals, lower metal concentration (Cu260 or Zn230) or higher metal concentration (Cu460 or Zn410)) and to one of the other two additional effects (laboratory or wild population and generation I or II).Each combination was replicated 3-5 times depending on the availability of animals, and four 7-11 day-long experiments were performed.
Experiment I tested the effect of the laboratory vs. wild population on survival at different Zn concentrations (with six treatment combinations: wild population CTRL, wild population Zn230, wild population Zn410 and laboratory population CTRL, laboratory population Zn230, laboratory population Zn410).
Experiment II tested the effect of the laboratory vs. wild population on survival under different Cu concentrations (with six treatment combinations: wild population CTRL, wild population Cu260, wild population Cu460 and laboratory population CTRL, laboratory population Cu260, laboratory population Cu460).
Experiment III tested the effect of the I vs. II generations on the survival of the wild population at different Cu concentrations (with six treatment combinations: generation I CTRL, generation I Cu260, generation I Cu460 and generation II CTRL, generation II Cu260, generation II Cu460).
Experiment IV tested the effect of the Zn concentration on the survival of wild population in the I vs. II generations (with six treatment combinations: generation I CTRL, generation I Zn230, generation I Zn410 and generation II CTRL, generation II Zn230, generation II Zn410).
The number of surviving individuals was checked each time the experiments terminated.Mortality was confirmed by examining passive individuals under a binocular microscope (at magnification of 25×) because of the absence of valve and appendage movement.
Differences in the survival rate between control individuals and those exposed to the stress metal treatments were tested for each combination of the above four experiments either in the balanced (Experiments III and IV) or unbalanced (Experiments I and II with unequal numbers of replicates within each factor level) design by two-way permutational multivariate analysis of variance (PERMANOVA) with the Euclidean distance as a resemblance measure and 9999 permutations, followed by pairwise tests if the main PERMANOVA test was statistically significant.Statistical analyses were conducted using PRIMER 7 software (Clarke & Gorley 2015) with the PERMANOVA+ add-on package (Anderson et al. 2008).

Analysis of adult survival curves (Experiments V-VI)
In addition to the tests of global adult survival (Experiments I-IV), adult survival curves were compared between the Cu treatments (Cu260 and Cu460) and the CTRL separately for the wild and laboratory populations in the 18-day-long Experiment V and the 17-day-long Experiment VI, respectively.Both of these experiments were conducted under the same conditions as those of Experiments I-IV, with 100 adult females at the beginning of the exposure test.The mortality of the test specimens and assessment of the survival percentage were recorded every 3-4 days when the medium and food were renewed.Survival curves were calculated by averaging the fraction of survivors over the replicates as a function of time.The averaged curves were compared between the Cu treatments and the CTRL with the Gehan-Breslow test and pairwise multiple comparison procedure with the Holm-Sidak method using SigmaPlot ver.11.0 software (SigmaPlot 2008).

Timing of the laying of first eggs and immediate juvenile hatching (Experiment VII)
This experiment was performed in 6-well (replicate) polystyrene plates filled with 10 cm 3 of two Zn solutions, Zn230 and Zn410, and one control (EPA medium without metal addition).At the beginning of the experiment, five adult females from the laboratory population were placed in one well and immersed in one of the three solutions.The experiment was carried out for 14 days at 20°C under a photoperiod of 12:12, and ostracods were fed every four days with spinach leaves (Vandekerkhove et al. 2012).Observations were performed every other day, and the time of laying the first eggs and the time of hatching the first juveniles (the number of days between inundation of females and the appearance of first eggs and emergence of juveniles, respectively) were monitored.
The differences in the timing of laying and hatching of eggs between the Zn treatments and the CTRL were tested by one-way PERMANOVA using PRIMER 7 software (Clarke & Gorley 2015) with the PERMANOVA+ add-on package (Anderson et al. 2008).

Hatching dynamics, hatching success, and juvenile mortality experiment (Experiment VIII)
For the experiment on hatching dynamics, 6 six-well (replicate) plates and resting eggs (cysts) of H. incongruens from Ostracodtoxkit f were used.One plate -serving as a control -was filled with EPA medium without metal.The other four plates were subjected to metal treatments (Zn230, Zn410, Cu260, and Cu460), while the remaining four plates were filled with a mixture of two metals in four different combinations (Zn230 + Cu260, Zn230 + Cu460, Zn410 + Cu260 and Zn410 + Cu460).At the beginning of the experiment, ca.40 cysts were inundated in each well, and then the experiment was conducted for 14 days under a photoperiod of 12:12 and at 20°C.Hatching was observed every day at the same time (11:00 AM), and hatching and mortality were recorded.Each day, freshly hatched neonates were counted and immediately removed from the experimental wells.The number of live and dead juveniles in each replicate was calculated, and the ratio of the number of juveniles that died within 24 h to the total number of juveniles that hatched was assessed.We tested three fitness-related life history traits: a) the dynamics of hatchability (i.e., shape of the cumulative hatching curves), b) the total hatching success at the end of the experiment (proportion of resting eggs that hatched), and c) the mortality rate of juveniles within 24 h after emergence (the ratio of the number of juveniles that died within 24 h to the total number of juveniles that hatched).Differences in these life history traits between the control eggs or juveniles and those exposed to the metal stress treatments were tested Copper and zinc in freshwater ostracod by one-way PERMANOVA (with 9999 permutations and the Euclidean distance as a resemblance measure in the case of hatching success and juvenile mortality and with the maximum distance in the case of hatchability curves) followed by pairwise tests using PRIMER 7 software (Clarke & Gorley 2015) with the PERMANOVA+ add-on package (Anderson et al. 2008).

Chemical analyses of the bioconcentrations (Experiment IX)
The body concentration of metal was measured at the end of the exposure mortality experiments (I-VI) in all specimens (dead and alive) exposed to specific concentration of a particular metal.Ostracods were dried in a drying chamber at 40°C and then digested using 1.5 mL of concentrated (69%) HNO 3 (Merck, Suprapur) and 0.3 mL of 30% H 2 O 2 (hydrogen peroxide, Merck, Suprapur).Afterward, the digested samples were diluted with Milli-Q water to 15 mL and then weighed.The final solution was measured for Zn (in the Zn control, Zn230, and Zn410 treatments) and for Cu (in the Cu control, Cu260, and Cu460 treatments) using inductively coupled plasma mass spectrometry (ICP MS, Elan 9000, Perkin Elmer).To ensure quality control, a blank control and standards were analysed, and the precision (2.8% for Cu and 1.7% for Zn) and recovery (97-103% based on 40 measurements of 6 reference materials) were satisfactory.For testing differences among measured body concentrations of Zn and Cu in specific treatments, a randomized block design of PERMANOVA was used with no replicates.Two factors were examined in this analysis: "population" with two levels (wild and laboratory) and "treatment" with three levels (CTRL, Zn230, Zn410 or CTRL, Cu260, and Cu460) based on Euclidean distance matrices.

Effect of Zn and Cu exposure on ostracod survival (Experiments I-IV)
In the Zn exposure Experiment I conducted on adult specimens originating from natural and laboratory populations, no clear relationships were found between the concentrations of Zn and the survival of ostracods, although the differences between populations were statistically significant (two-way PERMANOVA pseudo-F = 12.54; p = 0.003, Table I).Survival was significantly lower in the wild specimens than in to the laboratory ostracods and this pattern was observed in the control (mean survival  and Zn410), nor the combination of these two effects had a significant effect on ostracod survival (Table I).The experiments did not confirm the negative effects of the examined Zn concentrations on the survival of adults originating from wild and laboratory populations of H. incongruens.Contrary results were found in Experiments II and IV, which examined Cu exposure.In Experiment II, which included wild and laboratory specimens, the survival of both populations differed significantly among treatments (i.e., Cu concentrations) (two-way PERMANOVA pseudo-F = 7.243; p = 0.010, Table I).Post-hoc pairwise comparisons revealed differences between the control treatment and both Cu treatments but not between the Cu260 and Cu460 treatments (Table II, Figure 1).No differences were found in the survival of wild or laboratory H. incongruens exposed to aqueous Cu concentrations.For comparison of two different wild generations, statistically significant differences were noted for generation (first and second), treatment (CTRL, Cu260, and Cu460), and the interaction between those two factors.Additionally, pairwise comparisons confirmed differences between the control and Cu treatments but not between the Cu260 and Cu460 treatments (Table II).

Effect of Cu exposure on ostracod survival curves (Experiments V-VI)
Since significant differences in overall adult survival were observed among the treatments in the Cu exposure Experiments II and IV, a more detailed analysis of the survival curves was performed in subsequent Experiments V-VI.We found statistically significant differences among survival curves for both laboratory H. incongruens (Gehan-Breslow 30.455; p < 0.001) and wild specimens (170.272,p < 0.001).The survival curves showed similar trends in both experiments, with Cu460 treatment having an evidently stronger impact, although the shape and scale of the curves differed between the wild and laboratory populations (Figure 2).The results of pairwise multiple comparisons between survival curves of the laboratory and wild 692 A. Iglikowska et al. populations using the Holm-Sidak method are presented in Table III.

Effect of Zn exposure on the timing of egg laying and juvenile hatching (Experiment VII)
To test the influence of the two environmental Zn concentrations on the time of laying first eggs and hatching juveniles from subitaneous eggs, adult females from the laboratory population were used.We found significant differences in the time of egg laying among treatments (one-way PERMANOVA pseudo-F = 5.27; p = 0.024).The mean time ± SD for the CTRL and Zn410 treatments was 2.67 ± 1.03 days and 2.00 ± 0.00 days, respectively, whereas for Zn230, the first eggs were laid on average at 4.33 ± 1.97 days after inundation.When considering the timing of juvenile hatching, under the control conditions the first neonates appeared on average 4.00 ± 1.79 days after inundation, while in the Zn230 and Zn410 treatments the juveniles appeared after 5.67 ± 0.82 and 5.00 ± 1.09 days, respectively.Copper and zinc in freshwater ostracod 693 However, differences in the timing of juvenile hatching from subitaneous eggs were not statistically significant (one-way PERMANOVA pseudo-F = 2.5; p = 0.149).

Effect of Zn and Cu exposure on total hatching success, hatching dynamics, and juvenile mortality (Experiment VIII)
Hatching of the resting eggs was monitored daily in aqueous concentrations of Zn and Cu separately as well as in solutions containing four combinations of both metals at different concentration proportions (Zn230 + Cu260, Zn230 + Cu460, Zn410 + Cu260 and Zn410 + Cu460).The mean proportion of resting eggs that successfully hatched varied among treatments from 18.8% to 88.3% (Figure 3) and differed considerably (PERMANOVA Pseudo-F = 11.404;p < 0.001).Although overall hatching success was highest in the Zn treatments (78.8-88.3%),lowest in the Cu treatments (18.8-22.1%)and moderate in the combinations of both metal treatments (32.5-59.6%)(Figure 3), only two Cu treatments (Cu260 and Cu460) and one combination of both metals (both with the highest concentrations of Zn410 + Cu460) significantly affected the total hatching success (Table IV: t = 3.741-6.261;p-(perm) = 0.002-0.006),which was 2-3 times lower than that in the control (CTRL) conditions, where on average 60.8% ± 16.33% (SD) of the eggs hatched (Figure 3).Furthermore, we analysed the hatching curves as a function of time for the metal treatments compared to the CTRL (Figure 4).In the Cu treatments, most of the eggs hatched in the first 3-4 days (Figure 4(b)), whereas in the experiments with Zn addition, the hatchlings were appearing gradually until 8-9 days of the experiment, when the hatching curve reached a plateau (Figure 4(a)).
Although hatching success was greater in both Zn treatments higher than in the CTRL treatment beginning on the 4th day of the experiment, only the Zn410 concentration significantly affected egg   The mean proportion of juveniles that died within 24 h after emergence varied greatly among treatments (from 3.6% to 85.0%) and differed significantly (PERMANOVA Pseudo-F = 15.393;p < 0.001).Compared with the CTRL treatment, each metal treatment significantly increased the mean juvenile mortality rate by 3.64% (±3.18%SD) (Figure 5: t = 2.804-5.228;p(perm) = 0.002-0.034),and the Cu (Cu260 and Cu460) and Zn410 + Cu260 treatments had the most deleterious effects, as 81.3-85.0% of the neonates died after hatching (Figure 5).

Bioconcentration of Zn and Cu after exposure tests (Experiment IX)
After termination of Experiments I-IV, which tested the effects of metal exposure on adult H. incongruens survival, all dead and living specimens were used to measure the concentrations of Zn and Cu in the biomass.A positive relationship between the aqueous and body Zn concentrations was found in both the wild and laboratory ostracods.In wild H. incongruens the lowest Zn concentration (369 µg g −1 ) was detected in ostracods under the control conditions, a moderate value (529 µg g −1 ) was noted in the 230 µg Zn L −1 water concentrations, and the highest Zn body concentration (1038 µg g −1 ) was found in the 410 µg Zn L −1 treatment group (Figure 6, Table V).In the control laboratory, ostracods had a lower body Zn concentration (180 µg g −1 ) than did the control wild population (Table V), although in the laboratory, the Zn bioconcentration of H. incongruens exposed to Zn stress increased dramatically, reaching 1246 µg g −1 in the 230 µg Zn L −1 and 2250 µg g −1 treatment groups at 410 µg Zn L −1 water concentrations (Table V, Figure 6).
Similarly, for Cu exposure experiments, a positive relationship between environmental and body Cu concentrations was recorded; however, generally, bioconcentrations of Cu were clearly lower than those of Zn (Figure 6).In the Cu control for the wild and laboratory ostracods, low values of 6.5 and 32.2 µg Cu g −1 were noted, respectively.At lower (260 µg Cu L −1 ) aqueous concentrations, 404 µg g −1 was found in the bodies of wild ostracods, whereas slightly lower values, i.e., 346 µg g −1 , were observed in laboratory H. incongruens.In the wild population exposed to the higher (460 µg Cu L −1 ) Cu environmental concentration 815 µg Cu g −1 body level was recorded, and a lower value, 690 µg Cu g −1 , for the laboratory population was noted (Table V).The PERMANOVA test did not confirm significant differences among the observed Zn and Cu body levels considering either the population factor (i.e., wild vs. laboratory ostracods) or the treatment factor (i.e., CTRL vs. metal stress treatments: Cu260 and Cu460 or Zn230 and Zn410) (Table VI).

Effect of Zn and Cu exposure on adult ostracod survival
We found that both concentrations of Cu (260 and 460 µg Cu L −1 ) significantly decreased the survival of adults in both the wild and laboratory populations.In contrast, we found no effect of the environmental concentrations of Zn used (230 and 410 µg Zn L −1 ) on the survival of H. incongruens adults, which may suggest that such water concentrations of Zn were too low to exert toxic effects on ostracods.Indeed, in earlier studies on H. incongruens, the median lethal concentration (LC 50 ) of ZnSO 4 ranged from 360 to 1200 µg Zn L −1 (e.g., Kudłak et al. 2011;Sevilla et al. 2013;Muna et al. 2018) depending on the experimental conditions (type of medium: distilled water/EPA medium/lake water, presence or absence of sediment, and others), and most of the Zn concentrations used by other authors were higher than those in the present experiment.Surprisingly, Sevilla et al. (2013) found Zn to be more toxic to H. incongruens than Cu, which was confirmed by specific LC 50 values assessed for Zn (715 µg Zn L −1 ) and Cu (1240 µg Cu L −1 ).However, in most previous reports, Cu was more toxic than Zn in H. incongruens (e.g., Kudłak et al. 2011;Sevilla et al. 2014), which was also confirmed for other aquatic invertebrates, such as the cladoceran Daphnia magna (Burba 1999) and brachyuran crab (Chasmagnathus granulatus Dana) embryos (Lavolpe et al. 2004).This is Copper and zinc in freshwater ostracod in agreement with our results since we found decreased survival rates at higher (i.e., Cu460) CuSO 4 concentrations.Importantly, most Zn and Cu exposure tests using Ostracodtoxkit f were performed according to the protocol of a 6-day experiment testing the mortality of neonates, whereas our results provide new data on the H. incongruens response in the adult stage.In certain experimental units we observed lower survival rates in the control samples than in the treatments containing Zn.This can be interpreted as indicating that Zn addition may serve as a booster for ostracod survival and fitness.Jośko et al. (2016) reported the stimulated growth of H. incongruens exposed to ZnO nanoparticles in sediment.Furthermore, Hiki et al. (2017) reported that H. incongruens reared in media supplemented with 107 or 239 µg Zn L −1 exhibited an increased lifetime of egg production and a longer overall lifespan.A stimulating effect on biological processes induced by low doses of toxic metals (i.e., a hormesis effect) was observed in aquatic invertebrates, including crustaceans (e.g., Wang et al. 2022).In this study, the hormesis effect is one of possible interpretations of the observed stimulating effect of lower Zn levels; however, this finding requires further study.In fact, Zn is an essential micronutrient for the functioning of aquatic organisms (e.g., Sikorski 1990;Jośko & Oleszczuk 2013).Although a certain amount of Zn was likely to be delivered in our experiments with food, the EPA medium, which was used in the control in this study, does not contain Zn (Muna et al. 2018); therefore, the lack of this microelement could be a limiting factor for ostracod functioning under control conditions.The Zn concentrations used in the present study can be considered to meet the physiological requirements of adult ostracods although higher Zn levels most likely have toxic effects (Rainbow 2002).
In the present exposure tests the response of wild H. incongruens was compared to that of the laboratory population.In the Zn treatments, ostracods from the laboratory population showed significantly greater survival rates than specimens originating from the wild population.Interestingly, in the Ostracodtoxkit f stock population, in which the ostracods were cultured according to the original test procedure until adulthood, we observed delayed development and a relatively high mortality rate in older juveniles (most of whom died before reaching maturity) despite the lack of experimental stressors (e.g., no metal addition).Afterward, under the experimental conditions with Zn addition, laboratory adult ostracods showed greater fitness than did adults from the natural population.It is possible that delayed development and lower juvenile survival in the Figure 5. Average mortality of juveniles (%) within 24 hrs after emergence from resting eggs of the laboratory Heterocypris incongruens in control conditions (CTRL) and those exposed to different combinations of metal treatments (Experiment VIII).Symbols show averages of the means over replicates and whiskers represent 1 × SD (standard deviation).Statistical significance symbols denote differences between metal treatments compared to CTRL: * = statistically significant differences at 0.01 < p(perm) ≤ 0.05; ** = statistically significant differences at 0.001 < p(perm) ≤ 0.01.
Copper and zinc in freshwater ostracod stock culture of the laboratory individuals in the EPA medium was a result of the deficiency of essential elements (such as Zn and possibly Cu).Admittedly, Muyssen and Janssen (2001) reported that after rearing in zero Zn concentration (e.g., in EPA medium), decreased Zn tolerance may appear, although this was not observed in our experiments.There are also other biological factors that may explain the observed variability.The ostracods used in this study reproduce parthenogenetically, and clones may vary genetically in

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A. Iglikowska et al. sensitivity/tolerance to heavy metals and other physiological traits.Thus, the observed differences between laboratory and wild populations can be ascribed to interclonal variations related to different genotypes.Generally, the parthenogenetic (i.e., asexual) reproduction mode can be considered an advantage in ecotoxicological investigations since the use of the offspring of one female excludes genetic variation from the experiment.However, our results indicating differences in the adult survival rate between the wild and laboratory populations of Ostracodtoxkit f in the Zn treatments may suggest that the use of H. incongruens from Ostracodtoxkit f as a wild population analogue in ecotoxicological studies has certain limitations and requires careful interpretation of the obtained results.

Effect of Zn exposure on the time of egg laying and juvenile hatching from subitaneous eggs
We found significant differences in the time of egg laying among the examined Zn treatments, although differences in the time of juvenile hatching from subitaneous eggs appeared to be insignificant.Surprisingly, under the control conditions and in the Zn410 treatment group, eggs were laid on average on the second day, whereas in the Zn230 treatment group, eggs were laid on the fifth day of the experiment.
The observed variability in the timing of egg laying is difficult to interpret.Considering the delay in egg laying as a symptom of stress caused by the presence of Zn ions, it is not clear why such a delay was recorded at a lower Zn concentration (Zn230) but was absent at higher Zn concentrations (Zn410).Havel and Talbott (1995) examined the life history traits of H. incongruens originating from a natural population but further reared under laboratory conditions without any metal stressors.These authors found that each adult female laid from one to five clutches with 1-36 eggs, and the time period between egg laying was 1-4 days.Furthermore, in their experiment, high variability among individuals in terms of fecundity, egg development and hatching time was revealed (Havel & Talbott 1995).This result suggested that the variability in time of egg laying and hatching observed in our study may not be a consequence of exposure to Zn ions but rather a manifestation of the natural variability of reproductive dynamics (Angell & Hancock 1989).Importantly, the observed high variability in the time of egg laying and hatching can be a critical drawback in using these life traits in ecotoxicological studies since it is not clear whether the recorded differences are caused by the examined toxicant or are a result of natural intraspecific variability.Therefore, further studies involving a wider range of Zn concentrations in experimental solutions are needed.Copper and zinc in freshwater ostracod 701

Effect of Zn and Cu exposure on overall hatching success, hatching dynamics, and juvenile mortality
Our experiments on hatching success and dynamics performed on laboratory ostracods showed a clear significant negative impact of both examined Cu concentrations and a generally insignificant influence of the Zn concentrations (only the Zn410 treatment positively affected hatching dynamics).Both metals, however, significantly increased the mortality rate of juveniles, although Cu had a much stronger effect than Zn.When testing the combined effects of two metals in solution, juvenile ostracods also exhibited significantly greater mortality than did the control ostracods, although the effects of the combined metal treatments were generally less pronounced than those of the single Cu treatments but distinctly more pronounced than those of the single Zn treatments.This may suggest that the negative effects of Cu can be masked to some extent by the positive influence of Zn ions on some metabolic processes.These observations confirm that the major toxicant in our experiments was Cu, while Zn ions could serve as micronutrients to support ostracod development and fitness.
Numerous studies (e.g., Brown & Ahsanullah 1971;Lavolpe et al. 2004) have reported that neonates and early larval stage crustaceans are particularly vulnerable to the negative effects of heavy metal exposure.In particular, a strong negative effect of Cu ions on posthatching success was recorded for different crustacean taxa.López Greco et al. (2002) reported greater mortality of neonates, eye and appendage setae abnormalities in embryos, and delayed development of embryos in palaemonid shrimps (Palaemon pugio (Holthuis)) exposed to 1000 µg Cu L −1 .Zapata et al. (2001) reported negative effects on the viability of neonates of a crab (C.granulata) exposed to Cu concentrations between 50-500 µg Cu L −1 .Moreover, these authors (Zapata et al. 2001) reported reduced egg production and larval abnormalities (e.g., atrophy of dorsal spines and maxillipeds, hypertrophy of melanophores in the cephalothorax, hypopigmented eyes, and others) in a media supplemented with 500 µg L −1 Cu.Liu and Chen (1987) reported that Cu ions disturb the hatching process in crustaceans through effects on the enzyme system.Indeed, in shrimp Artemia salina (Linnaeus) the activity of trypsin and amylase was disrupted during Cu exposure at 2000 µg Cu L −1 (Alayse- Danet et al. 1979).Interestingly, Zapata et al. (2001) noticed that acute lethal toxicity to crustaceans was greater for Cu than for Zn, although the effect was less pronounced under chronic exposure.The morphological abnormalities of crab juveniles were also recorded in Zn exposure experiments, although most of these abnormalities occurred at concentrations as high as 1000-10,000 µg Zn L −1 (Lavolpe et al. 2004).Unfortunately, most data on the effects of heavy metal toxicity on hatching dynamics and success have been obtained for marine crustaceans thus far, and reports on freshwater taxa are particularly scarce.It is possible that certain aspects of metal toxicity act differently in freshwater environments.This highlights the relevance and urgency of further investigations on the influence of heavy metals on different aspects of freshwater crustacean life history traits to assess the risk and limits for this ecologically important group.

Bioconcentration of Zn and Cu
A positive relationship between ambient water metal concentrations and body concentrations of those metals was observed in the present study.This may indicate that adult H. incongruens females accumulate rather than eliminate heavy metals from their bodies.Differences between the bioconcentration of Zn in the wild and laboratory ostracods were found, although these differences were not statistically significant.Our wild population originated from a municipal temporary pool likely contaminated with heavy metals (including Zn), whereas the laboratory specimens were reared in pure EPA medium that does not contain Zn (Muna et al. 2018).Body Zn levels increased dramatically in ostracods (especially in the laboratory specimens) exposed to Zn230 and even more to Zn410, confirming the high accumulation potential of these compounds.Interestingly, H. incongruens retained a high fitness and ability to reproduce despite high body Zn concentrations.This observation strongly suggests the involvement of a highly effective mechanism to neutralize Zn toxicity, possibly through processes such as metallothionein-mediated detoxification.Metallothioneins (MTs) are cysteine-rich proteins that are capable of binding heavy metals through the thiol group of cysteine residues to carry metals in detoxified form (Amiard et al. 2006).Thus, MTs serve as a protector against metal toxicity and play a key role in regulating the body concentration of metals by binding and releasing them depending on the organism's needs (Wang & Rainbow 2010).In our study, we detected a lower Cu bioconcentration than the measured Zn levels.Copper exposure likely has a greater toxic effect on ostracods, which was confirmed in this study by the significantly higher juvenile mortality rates and lower adult survival at both moderate
(Cu260) and higher (Cu460) environmental aqueous concentrations.This suggests that a greater Cu body burden can induce toxic effects in H. incongruens.The observed positive relationship between environmental and body Cu concentrations indicates the potential for Cu bioaccumulation and the likely involvement of MT proteins in detoxification (Brown et al. 2004;Le Pabic et al. 2015).On the other hand, relatively low Cu levels in the body may suggest a low Cu uptake rate or the contribution of physiological mechanisms to the partial removal of Cu ions from the body.Various crustacean taxa use different physiological mechanisms to eliminate or detoxify heavy metals (Rainbow 2002).For instance, the marine decapod Palaemon elegans accumulates Zn in its body to meet metabolic needs until a threshold is reached, after which excess Zn is excreted to avoid toxic effects.A different strategy is employed by cirripeds; when the Zn concentration in body tissues exceeds the toxic threshold, Zn is stored in a detoxified form without any excretion.The third pattern combines both mechanisms and is used by Palaemon elegans Rathke for Cu regulation.Thus, excess Cu in P. elegans is partially excreted from the body and is partially bound and stored within the body in a detoxified form (Rainbow 2002).The consequences of the abovementioned mechanisms are reflected by different bioconcentration patterns (Table VII).Taxa excreting metals show low body levels, species that tend to store metals in detoxified forms can exhibit extremely high body concentrations, and crustaceans using both excretion and detoxification are characterized by moderate metal bioconcentrations.The ostracod body levels of Cu and Zn found in the present study are similar to those of cirripeds, which accumulate heavy metals without excretion; therefore, this mechanism is also probable for H. incongruens.This finding reveals that H. incongruens is a potentially effective bioindicator Table VII.Selected data on bioconcentrations of Zn and Cu (µg g −1 ) in marine and freshwater crustacean species.Zn and Cu concentrations detected at the following water levels (if available): 1 maximum value at 320 µg Zn L −1 , 2 maximum value at 56 µg Cu L −1 , 3 maximum value at 373 µg Zn L −1 , 4 maximum value at 240 µg Cu L −1 , 5 maximum values at <4 µg Zn L −1 , 6 at 250 µg Zn L −1 , 7 maximum value at 2456 µg Zn L −1 , 8 maximum value at 127 µg Cu L −1 , 9 value at 410 µg Zn L −1 , 10 value at 460 µg Cu L −1 .

Marine crustaceans
Copepoda Anomalocera patersoni Copper and zinc in freshwater ostracod 703 of Cu and Zn contamination in freshwater environments and can be successfully used in field ecotoxicological surveys.

Conclusions
The results of the experiments demonstrated that waterborne exposure to Zn and Cu differently affects ostracod adult survival, neonatal mortality, resting egg hatching dynamics and overall hatching success.We found a positive relationship between waterborne environmental Cu concentrations and reduced hatching success of resting eggs, increased mortality of juveniles and reduced survival of adults, dramatically decreasing the fitness of H. incongruens.
In contrast, the studied environmental Zn concentrations did not significantly influence the total hatching success or adult survival, though they did increase juvenile mortality.Although the addition of Zn ions to the experimental medium seemed to stimulate hatching dynamics and, consequently, the fitness of the studied ostracods H. incongruens, previous studies reported that adverse biological effects may occur at higher Zn levels.Additionally, in experiments combining the influence of Zn and Cu in the same treatment we observed that the presence of Zn ions partially neutralized the negative effects of Cu reducing the mortality of neonates.
Our study revealed that the life stage of freshly hatched juveniles was most sensitive life stage to both Cu and Zn exposure.Differences in the response of the laboratory and wild populations in terms of adult survival in our Zn exposure tests were recorded, which can be ascribed to genetic interclonal variability influencing tolerance/resistance to metals and other physiological adaptations.For this reason, the use of laboratory H. incongruens as an analogue of wild ostracods should be treated with great caution.
Positive relationship between Zn and Cu aqueous concentrations and ostracod body concentrations was observed.Importantly, we found increased metal body burden without associated fitness costs, which was particularly noticeable regarding Zn bioconcentrations.It seems that H. incongruens has evolved to tolerate Zn exposure which is a robust adaptation in species commonly inhabiting urbanized water bodies characterized by potentially elevated Zn contamination.Due to its high bioaccumulation potential, H. incongruens contributes to the transfer of heavy metals to higher trophic levels.On the other hand, the ability to accumulate Cu and Zn metals confirms that H. incongruens can be a useful tool in ecosystem biomonitoring for the assessment of toxicity in environmental samples.Copper and zinc in freshwater ostracod 707

Figure 1 .
Figure 1.Comparison of the total survival (%) of the wild and laboratory (Lab) adult Heterocypris incongruens ostracods exposed to the environmental concentrations of Cu (Cu260 and Cu460) compared to the control conditions (CTRL) in the Experiment II.Bars show averages over replicates and whiskers indicate 1 × SE (standard error).Statistical significance symbols denote differences between all treatments: * = statistically significant differences at 0.01 < p(perm) ≤ 0.05, ** = statistically significant differences at 0.001 < p(perm) ≤ 0.01, n.s.= non-significant.

Figure 2 .
Figure 2. Survival curves of the wild (panel A, Experiment V) and laboratory (panel B, Experiment VI) adult individuals of Heterocypris incongruens exposed to the environmental concentrations of Cu (Cu260 and Cu460) compared to the control conditions (CTRL).The curves show averages over replicates and whiskers display 1 × SE (standard error).Statistical significance symbols denote differences between metal treatments compared to CTRL: *** = statistically significant differences at p(perm) < 0.001.

Figure 3 .
Figure3.Total hatching success (%) within 14 days of inundation of resting eggs of the laboratory Heterocypris incongruens in control conditions (CTRL) and those exposed to different combinations of metal treatments (Experiment VIII).Symbols show averages of the means over replicates and whiskers represent 1 × SD (standard deviation).Statistical significance symbols denote differences between metal treatments compared to CTRL * = statistically significant differences at 0.01 < p(perm) ≤ 0.05, ** = statistically significant differences at 0.001 < p(perm) ≤ 0.01, n.s.= non-significant.

Figure 4 .
Figure 4. Cumulative hatching (%) curves for resting eggs of the laboratory Heterocypris incongruens as function of time in the control conditions (CTRL) and Zn treatments (panel A), Cu treatments (panel B) and the treatments of combination of both metals, Zn and Cu in different concentrations (panel C) (Experiment VIII).Symbols and lines show mean cumulative hatching success of six replicates (± 1 standard error).Statistical significance symbols denote differences between metal treatments compared to CTRL: * = statistically significant differences at 0.01 < p(perm) ≤ 0.05; ** = statistically significant differences at 0.001 < p(perm) ≤ 0.01; n.s.= non-significant.

Figure 6 .
Figure 6.Comparison between wild and laboratory adult females of Heterocypris incongruens in bioconcentration (µg g −1 ) of Zn (panel A) and Cu (panel B) in their bodies in relation to ambient water metal concentration (Zn210, Zn410 and Cu260, Cu460 as well as control conditions CTRL).

Table I .
Results of the two-way unbalanced design PERMANOVA (Experiments I and II) and two-way crossed PERMANOVA (Experiments III and IV) of Zn and Cu exposure experiments on survival of adult Heterocypris incongruens.Factor "treatment" with three levels for Zn (CTRL, Zn230, and Zn410) and three levels for Cu (CTRL, Cu260, and Cu460).Only statistically significant results are bolded and p-values calculated using permutation (perm) test.

Table II .
Results of the post-hoc pairwise comparisons for PERMANOVA testing relationships between each pair of treatments in the Experiments II and IV on the effects of Cu exposure on adult H. incongruens survival.

Table III .
Results of pairwise multiple comparisons between survival curves of the wild (Experiment V) and laboratory (Experiment VI) populations of H. incongruens in the Cu treatments vs. control conditions using Holm-Sidak method.

Table IV .
Results of PERMANOVA post-hoc t test in 14-days Experiment VIII on hatching success.Pairwise comparisons between each combination of metal treatments and the control conditions are displayed.The p-values calculated using permutation (perm) test, only statistically significant p-values are bolded.

Table V .
Bioconcentration of Zn and Cu in a biomass of dead and alive adult specimens of H. incongruens (in µg g −1 ) after exposure to environmental concentrations of Zn and Cu in the survival Experiments I-IV.Control samples -EPA medium without metal addition.