Investigation of Metal Toxicity on Microalgae Phaeodactylum tricornutum, Hipersaline Zooplankter Artemia salina, and Jellyfish Aurelia aurita

The escalating global anthropogenic activities associated with industrial development have led to the increased introduction of heavy metals (HMs) into marine environments through effluents. This study aimed to assess the toxicity of three HMs (Cr, Cu, and Cd) on organisms spanning different trophic levels: Phaeodactylum tricornutum (a primary producer), Artemia salina (a primary consumer), and Aurelia aurita (a secondary consumer). The EC50 values obtained revealed varying relative toxicities for the tested organisms. Phaeodactylum tricornutum exhibited the highest sensitivity to Cu, followed by Cd and Cr, while Artemia salina displayed the highest sensitivity to Cr, followed by Cu and Cd. A. aurita, on the other hand, demonstrated the highest sensitivity to Cu, followed by Cr and Cd. This experimental investigation further supported previous studies that have suggested A. aurita as a suitable model organism for ecotoxicity testing. Our experiments encompassed sublethal endpoints, such as pulsation frequency, acute effects, and mortality, highlighting different levels of sensitivity among the organisms.


Introduction
Nowadays, thousands of pollutants reach the marine environment and exert different types of stress and damage on organisms, resulting in negative changes in water quality and ecosystems [1]. Most of them are discharged in the marine environment as a result of countless anthropogenic activities affecting the environment [2,3]. These waste types can be different in nature and include, among others, heavy metals (HMs), detergents, microfibers, or (micro)plastics, which all contribute to the current aquatic pollution problems [4].
In this sense, marine ecosystems are one of the systems most affected by pollution because humans have used them as a dumping ground for their waste, disregarding their complexity and dynamics. Among the pollutants, the accumulation of HMs in marine ecosystems is of vital importance because they can have devastating effects on the ecological balance of the environment and biodiversity [5,6]. It would be beneficial to clarify that, among the metals, some are essential elements that play biological roles but can be toxic at high concentrations, while others are non-essential and do not have known biological functions. It is well-known that chronic HM exposure can have serious long-term health effects [5,7,8].
Aquatic pollution by HMs is related to high levels of Cd, Cr, Cu, Hg, Ni, Pb, and Zn, among others. Of these HMs, Cd, Cu, Hg, and Zn, jointly the metalloid As, are the five elements with the strongest potential impact because of their high toxicity and persistence in all aquatic ecosystems. High concentrations of these pollutants enter the environment largely depend on location, with some biomes showing negligible Cu release and others being major regional sources [30].
To assess the toxicity of HMs, common approaches involve utilizing bio-monitoring methods with bio-indicators. These indicators, encompassing species, species groups, or biological communities, offer insights into contamination through real ecosystem observations. Alternatively, laboratory toxicity tests provide an indirect means of inferring environmental quality. Selecting the right model organism for toxicity tests is critical in applying the bioindicator model, given that certain organisms are more suitable than others for specific tests, as seen in toxicological impact studies on marine organisms like sea urchins [20,31]. Sea urchin gamete models offer advantages a priori, such as their wide geographical distribution, abundance, and easy collection and maintenance [32]. Sea urchin bio-indicators present challenges, including the requirement of a sufficient number of individuals to ensure both male and female specimens with viable gametes, preventing unintended spawning due to temperature changes during transport, and necessitating a fertilization success rate of at least 90% to proceed with the process [31,32], being impractical for toxicity tests.
To gain a comprehensive understanding of HMs' toxicity, Phaeodactylum tricornutum and the crustacean Artemia salina serve as established model organisms, representing primary producers and consumers, respectively. While these models offer valuable insights, there is a lack of secondary consumer models. Addressing this gap, recent research has spotlighted cnidarians as potential indicators of marine environmental conditions owing to their unique sensitivity to stress and swift responsiveness to disturbances [33]. These organisms form part of the gelatinous zooplankton, which includes approximately 2000 widely recognized species [34] as key members of ocean ecosystems. They also play an important role in the organization of marine food webs [35][36][37] as an energy source in both pelagic and deep-sea food webs by supporting C trophic transfer from surface waters to euphotic environments [38,39]. These gelatinous organisms are also active predators that forage on a wide range of prey, from mesozooplankton and ichthyoplankton to microplankton [40][41][42], gelatinous species, and emergent zooplankton [43]. Therefore, jellyfish exert both the top-down and bottom-up control of zooplankton and, indirectly, of phytoplankton communities by cascading effects [44].
Of cnidarians, the jellyfish Aurelia aurita is a promising model organism in the ecotoxicology field because it can be used to predict the effects of chemicals and other stressors on the marine environment, such as oil organic chemicals and HMs [45][46][47][48]. It is one of the most abundant and commonest gelatinous zooplankton species in the world. It is an epipelagic scyphozoan with a cosmopolitan distribution that is located in the waters along the neritic zone [47]. Its biological cycle is complex because it combines a sessile asexual polyp phase and a free-living sexual medusa phase [49]. Worldwide A. aurita populations are defined by their high diversity, characteristic life cycle, abundance, growth, strobilation timing and periodicity, time and size upon sexual maturation, and jellyfish longevity [50]. In nature, strobilation, the process by which the free-living phase is generated, is a seasonal process that starts in winter or early in spring [50,51]. In winter, water temperature lowers and acts as an environmental signal, which is perceived by polyps. A single colony of polyps can asexually produce genetically identical male or female jellyfish [52,53], which is a huge advantage when conducting studies because it avoids intra-and interpopulation variation problems [54].
Of all mentioned above, A. aurita stands out as a valuable model organism thanks to its ease of laboratory handling, with the ability to readily produce ephyrae from polyps through a simple heat shock method involving controlled temperature changes over a brief period, alongside uncomplicated maintenance requirements.
Therefore, to broaden our knowledge about the effects of Cd, Cu, and Cr pollution at different marine trophic levels and specifically in organisms for which their effects have not yet been tested, such as A. aurita, the present study poses the following hypotheses: This work also attempts to support the ephyra of the scyphomedusa A. aurita as a model for marine ecotoxicological assays to make progress in the study of substances introduced into the natural environment and to understand the consequences of this.

Materials and Methods
In order to study organisms' resilience to the introduction of pollutants, three bioassays were performed based on aquatic organisms' reaction to a wide range of pollutants [55].
The toxic reference salts selected to study the effects of Cd, Cu, and Cr were cadmium nitrate Cd(NO 3 ) 2 , copper nitrate Cu(NO 3 ) 2 , and potassium dichromate K 2 Cr 2 O 7 , respectively. All salts (analytical grade) were supplied by Sigma-Aldrich™ (Darmstadt, Germany). This study was carried out with three marine species used as bioindicators of marine toxicity of HMs. The test protocol carried out on each is provided below.

Toxicity Test with Phaeodactylum tricornutum
The microalgae test was carried out according to standardized protocol Algaltoxkit M™ (Marine Toxicity Test with Microalgae) developed by Microbiotests Inc. (Gent, Belgium). The kit follows Standard ISO 10253:2017. This is an algal growth inhibition test performed in vials with the marine diatom Phaeodactylum tricornutum. The concentrations were 0.01, 0.03, 0.06, 0.12, and 0.25 mg/L and they were incubated for 3 days at 20 • C (±2 • C) with constant and uniform lighting provided by cool white fluorescent lamps. Lighting was 10,000 lux for the side of the long cell or 3000-4000 lux for the bottom lighting. Growth was monitored by optical density (OD) measurements in a spectrophotometer equipped with a 670 nm filter and a 10 cm cell holder. ODs were converted into algal numbers with the help of the "Optical Density/Number of Algae" (OD/N) sheet included in each Algaltoxkit M™. After culturing, microalgae growth measurements were monitored for 24 h, 48 h, and 72 h. For each treated concentration and all of the used toxicants (cadmium nitrate, copper nitrate, and potassium dichromate), tests were independently conducted in triplicate.

Toxicity Test with Artemia salina
The toxicity test was carried out according to standardized protocol Artoxkit M™ (Artemia Toxicity Screening Test for Estuarine and Marine Waters) developed by Microbiotests Inc. (Gent, Belgium). Cysts were allowed to hatch during incubation under aerated conditions with constant light (light source of 3000-4000 lux) at 25 • C in standard seawater. Hatching started after about 18-20 h. After 30 h, most larvae had moulted in the instar II-III stage. Briefly, 2 mL of filtered seawater and 10 Artemia larvae were added to each well. The mixture concentrations were 6.25, 12.5, 25, 50, and 100 mg/L for each employed toxicant. After the 24 h and 48 h treatments at each metal concentration, the numbers of living and dead Artemia nauplii were counted. Dead individuals were determined if no movement of appendages was observed within 10 s. For every treated concentration, and for all of the employed toxicants (cadmium nitrate, copper nitrate, and potassium dichromate), tests were conducted in four replicates.

Toxicity Test with Aurelia aurita
This assay followed the guidelines of the test previously carried out by Faimali et al. [48]. Toxicity tests were prepared using the A. aurita ephyra collected immediately after strobilation (0 days of age). The ephyra used in these experiments were obtained directly from the polyps reared in the Oceanogràfic de València laboratories (València, Spain).
Two end-points were evaluated: one was frequency of pulsation, defined as the number of pulsations performed by ephyra within a defined time unit (30 s), measured as the % alteration of pulsations (compared with the control). The second end-point was to measure the % mortality of ephyra for each concentration (compared with the control). Ephyra were placed three by three in a multiwell plate containing 10 mL of solution. The applied concentrations were 1.9, 3.75, 7.5, 15, and 30 mg/L for each utilized salt (the corresponding Cd, Cu, and Cr concentrations found in each salt appear in Table 1). Plates were sealed and left in the thermostatic room at 20 • C in the dark. After 24 h and 48 h, the beats per minute of all of the ephyra and % mortality were evaluated. For each treated concentration, and for all the used toxicants (cadmium nitrate, copper nitrate, and potassium dichromate), tests were conducted in five replicates.

Statistical Analysis
For the three organisms used in the tests, EC 50 (half maximal effective concentration) was obtained using a Probit analysis using the SPSS™ S statistical package (v20, IBM). EC 50 was calculated after 24 h and 48 h for A. salina and A. aurita and after 72 h for P. tricornutum. For the three tested organisms, the statistical significance of the differences between means and groups (p < 0.05) was estimated based on a one-way ANOVA and a Student's t-test using SPSS™ (v20, IBM, Armonk, NY, USA) and MS Excel™ (v17.0. Microsoft Inc., Redmond, WA, USA).

Phaeodactylum tricornutum (Primary Producer Model)
In order to observe the effect of Cu, Cr, and Cd on P. tricornutum growth, algae were exposed to several concentrations of salts and their growth rate was assessed ( Figure 1). The P. tricornutum samples received 0.03, 0.06, 0.12, 0.25, and 0.5 mg/L of each salt (Cd(NO 3 ) 2 , Cu(NO 3 ) 2 , and K 2 Cr 2 O 7 ). Growth was determined after 72 h (the corresponding Cd, Cu, and Cr concentrations on each salt are shown in Table 1). The results of the study revealed significant inhibitory effects of different concentrations of cadmium nitrate, potassium dichromate, and copper nitrate on the growth of the algae. When compared with the control group, a concentration of 0.03 of cadmium nitrate exhibited a growth inhibition of 1.22%, while the concentration of 0.03 of potassium dichromate showed an inhibition of 5.3%. Notably, the inhibition increased with higher concentrations, with the concentration of 0.06 of cadmium nitrate resulting in a growth inhibition of 17% and the concentration of 0.06 of potassium dichromate showing an inhibition of 7.54%. Moreover, the concentration of 0.25 of cadmium nitrate exhibited a substantial inhibition of 34.5%, whereas the concentration of 0.25 of potassium dichromate displayed a remarkable inhibition of 50.6%. The highest inhibitory effects were observed at the concentration of 0.5, with cadmium nitrate inhibiting growth by 49.7% and potassium dichromate showing an inhibition of 64.6%. Comparatively, copper nitrate exhibited relatively lower inhibitory effects, with the concentration of 0.03 resulting in a growth inhibition of 2.05% and the concentration of 0.06 showing an inhibition Toxics 2023, 11, 716 6 of 18 of 2.18%. However, as the concentration increased, so did the inhibitory effects, with the concentration of 0.12 resulting in a growth inhibition of 5.39%, the concentration of 0.25 exhibiting an inhibition of 13.59%, and the concentration of 0.5 demonstrating the highest inhibition of 91.62%. These findings indicate that, among the three substances tested, cadmium nitrate and potassium dichromate exerted stronger inhibitory effects on growth compared with copper nitrate Therefore, among the three HMs tested, the most toxic HMs for P. tricornutum were Cr and Cd followed by Cu at 72 h and the highest concentration.
concentration of 0.25 of cadmium nitrate exhibited a substantial inhibition of 34.5%, whereas the concentration of 0.25 of potassium dichromate displayed a remarkable inhibition of 50.6%. The highest inhibitory effects were observed at the concentration of 0.5, with cadmium nitrate inhibiting growth by 49.7% and potassium dichromate showing an inhibition of 64.6%. Comparatively, copper nitrate exhibited relatively lower inhibitory effects, with the concentration of 0.03 resulting in a growth inhibition of 2.05% and the concentration of 0.06 showing an inhibition of 2.18%. However, as the concentration increased, so did the inhibitory effects, with the concentration of 0.12 resulting in a growth inhibition of 5.39%, the concentration of 0.25 exhibiting an inhibition of 13.59%, and the concentration of 0.5 demonstrating the highest inhibition of 91.62%. These findings indicate that, among the three substances tested, cadmium nitrate and potassium dichromate exerted stronger inhibitory effects on growth compared with copper nitrate Therefore, among the three HMs tested, the most toxic HMs for P. tricornutum were Cr and Cd followed by Cu at 72 h and the highest concentration. The results of the study revealed the EC50 values for the algae P. tricornutum, indicating the concentration at which 50% inhibition of growth occurred. EC50 for potassium dichromate was determined to be 15.3 mg/L. In contrast, cadmium nitrate exhibited a lower EC50 value of 2.49 mg/L, suggesting a higher toxicity to the algae. Similarly, copper nitrate demonstrated a relatively lower EC50 value of 1.205 mg/L, indicating a strong inhibitory effect on the growth of P. tricornutum. These findings highlight the varying toxicities of The results of the study revealed the EC 50 values for the algae P. tricornutum, indicating the concentration at which 50% inhibition of growth occurred. EC 50 for potassium dichromate was determined to be 15.3 mg/L. In contrast, cadmium nitrate exhibited a lower EC 50 value of 2.49 mg/L, suggesting a higher toxicity to the algae. Similarly, copper nitrate demonstrated a relatively lower EC 50 value of 1.205 mg/L, indicating a strong inhibitory effect on the growth of P. tricornutum. These findings highlight the varying toxicities of the tested compounds, with cadmium nitrate and copper nitrate showing lower toxicity compared with potassium dichromate towards P. tricornutum (Table 2).

Artemia salina (Primary Consumer Model)
To investigate the impact of Cu, Cr, and Cd on the mortality of A. salina, the crustaceans were exposed to different concentrations of these HM salts, and their mortality rates were assessed ( Figure 2). The concentrations of each salt used for the larvae ranged from 6.25 to 100 mg/L. Mortality was evaluated after 24 h and 48 h of exposure. the tested compounds, with cadmium nitrate and copper nitrate showing lower toxicity compared with potassium dichromate towards P. tricornutum (Table 2).

Artemia salina (Primary Consumer Model)
To investigate the impact of Cu, Cr, and Cd on the mortality of A. salina, the crustaceans were exposed to different concentrations of these HM salts, and their mortality rates were assessed ( Figure 2). The concentrations of each salt used for the larvae ranged from 6.25 to 100 mg/L. Mortality was evaluated after 24 h and 48 h of exposure. Based on the results obtained after 24 h, Artemia larvae showed low sensitivity to Cr and Cd concentrations up to 25 mg/L, as well as to Cu concentrations up to 12.5 mg/L. The highest mortality rate during de first 24 h was 60% for K2Cr2O7, followed by 37.5% for Cu(NO3)2 and 27.5% for Cd(NO3)2 (t-test, p < 0.05). The calculated EC50 values for Cu(NO3)2, K2Cr2O7, and Cd(NO3)2 after 24 h were 95.773, 91.359, and 150.167 mg/L, respectively (Table 2).
After 48 h of exposure, significant increases in Artemia mortality were observed at concentrations as low as 6.25 mg/L for Cr and 12.5 mg/L for the other two toxicants. The mortality rate for Cd(NO3)2 remained consistent at both exposure times. The highest mortality rate (100%) was recorded for A. salina, indicating that Cr was the most toxic metal, followed by 67.5% for Cu and 30% for Cd (t-test, p < 0.05). The calculated EC50 values for Based on the results obtained after 24 h, Artemia larvae showed low sensitivity to Cr and Cd concentrations up to 25 mg/L, as well as to Cu concentrations up to 12.5 mg/L. The highest mortality rate during de first 24 h was 60% for K 2 Cr 2 O 7 , followed by 37

Aurelia aurita (Secondary Consumer Model)
In order to note the effect of Cu, Cr, and Cd on A. aurita mortality, ephyras were treated with five concentrations of the studied toxicants, and their mortality rate was assessed ( Figure 3). For the A. aurita ephyra, the ranges of concentrations of each toxicant were 1.9, 3.75, 7.5, 15, and 30 mg/L. After that, the results at 24 h revealed that the ephyras were not affected by K 2 Cr 2 O 7 and Cd(NO 3 ) 2 at concentrations up to 7.5 mg/L, but mortality was 100% at the lowest concentration (1.9 mg/L) with Cu(NO 3 ) 2 . At the highest exposure concentration, 100% mortality was caused by Cu and Cd, followed by 93.33% mortality by Cr (t-test, p < 0.05). The calculated EC 50 values of Cu(NO 3 ) 2 , K 2 Cr 2 O 7 , and Cd(NO 3 ) 2 at 24 h were 0.283, 16.571, and 19.880 mg/L, respectively ( Table 2). The results at 48 h of exposure showed that the A. aurita ephyras exposed to Cu(NO 3 ) 2 followed the same pattern as the measurements taken at 24 h with 100% mortality at all concentrations. With K 2 Cr 2 O 7 , mortality started to significantly increase from 3.75 mg/L, and from 7.5 mg/L for Cd(NO 3

Aurelia aurita (Secondary Consumer Model)
In order to note the effect of Cu, Cr, and Cd on A. aurita mortality, ephyras were treated with five concentrations of the studied toxicants, and their mortality rate was assessed ( Figure 3). For the A. aurita ephyra, the ranges of concentrations of each toxicant were 1.9, 3.75, 7.5, 15, and 30 mg/L. After that, the results at 24 h revealed that the ephyras were not affected by K2Cr2O7 and Cd(NO3)2 at concentrations up to 7.5 mg/L, but mortality was 100% at the lowest concentration (1.9 mg/L) with Cu(NO3)2. At the highest exposure concentration, 100% mortality was caused by Cu and Cd, followed by 93.33% mortality by Cr (t-test, p < 0.05). The calculated EC50 values of Cu(NO3)2, K2Cr2O7, and Cd(NO3)2 at 24 h were 0.283, 16.571, and 19.880 mg/L, respectively ( Table 2). The results at 48 h of exposure showed that the A. aurita ephyras exposed to Cu(NO3)2 followed the same pattern as the measurements taken at 24 h with 100% mortality at all concentrations. With K2Cr2O7, mortality started to significantly increase from 3.75 mg/L, and from 7.5 mg/L for Cd(NO3)2. Mortality at 48 h for the highest concentration was 100% for all three studied toxicants (t-test, p < 0.05). The EC50 values calculated for Cu(NO3)2, K2Cr2O7, and Cd(NO3)2 at 48 h were 0.283, 6.726, and 12.343 mg/L, respectively ( Table 2). This study also involved toxicity screening with the three different reference toxic substances. Two end-points were considered: frequency of pulsation (Fp) and mortality (M); the percentage of alteration to Fp (%Fp) and the percentage of mortality (%M) were calculated at the 24 h and 48 h exposure times, and were compared to the untreated control. The results obtained for exposing the 0-day-old ephyra to different concentrations of potassium dichromate are reported in Figure 4. This compound had a significant effect on both end-points. %Fp for both 24 h and 48 h showed an inverse correlation with a rising toxicant concentration, and was 0% at the highest concentration (30 mg/L). Mortality at 24 h was 93.33%, with 100%M at 48 h for the high concentration. Regarding the values obtained for %M at 24 h, the LOEC (lowest observed effect concentration) was 15 mg/L. For %M at 48 h, an incongruent value was observed because the LOEC was 0 mg/L in the untreated control. The same percentage of mortality as the control was obtained at the lowest K2Cr2O7 concentration, and only 1 ephyra of the 15 exposed ones was death. No mortality took place at a concentration of 3.75 mg/L, but at the next concentration (7.5 mg/L), mortality was 100% compared with 0%M at the same concentration after 24 h. M% at 48 h at the remaining concentrations (15 mg/L and 30 mg/L) was 80% and 100%, respectively. In this assay, Fp was the most sensitive end-point (in magnitude of response and data reliability terms) compared with mortality. This study also involved toxicity screening with the three different reference toxic substances. Two end-points were considered: frequency of pulsation (Fp) and mortality (M); the percentage of alteration to Fp (%Fp) and the percentage of mortality (%M) were calculated at the 24 h and 48 h exposure times, and were compared to the untreated control. The results obtained for exposing the 0-day-old ephyra to different concentrations of potassium dichromate are reported in Figure 4. This compound had a significant effect on both end-points. %Fp for both 24 h and 48 h showed an inverse correlation with a rising toxicant concentration, and was 0% at the highest concentration (30 mg/L). Mortality at 24 h was 93.33%, with 100%M at 48 h for the high concentration. Regarding the values obtained for %M at 24 h, the LOEC (lowest observed effect concentration) was 15 mg/L. For %M at 48 h, an incongruent value was observed because the LOEC was 0 mg/L in the untreated control. The same percentage of mortality as the control was obtained at the lowest K 2 Cr 2 O 7 concentration, and only 1 ephyra of the 15 exposed ones was death. No mortality took place at a concentration of 3.75 mg/L, but at the next concentration (7.5 mg/L), mortality was 100% compared with 0%M at the same concentration after 24 h. M% at 48 h at the remaining concentrations (15 mg/L and 30 mg/L) was 80% and 100%, respectively. In this assay, Fp was the most sensitive end-point (in magnitude of response and data reliability terms) compared with mortality. The results of exposing ephyra to different cadmium nitrate concentrations are shown in Figure 5. Like Cr, this compound also had a significant effect on both end-points. %Fp at both 24 h and 48 h lowered when the toxicant concentration rose, and was 100% at 0 mg/L and 0% at 30 mg/L. This also implies, as we mentioned above, an inverse correlation between %Fp and %M, which was observed at 15 mg/L and at both 24 h and 48 h. Mortality increased and, therefore, %Fp decreased (24 h: 33.33%M and 44.79%Fp; 48 h: 86.66%M and 6.17%Fp). Regarding the LOEC value for %M, it is 15 mg/L at both exposure times, with 33.33%M and 86.66%M at 24 h and 48 h, respectively. At 30 mg/L, mortality was 100% at both exposure times. The results obtained for exposing ephyra to different copper nitrate concentrations are shown in Figure 6. This graph depicts a different situation to those previously described. For Fp, the values were 100% at 24 h in the untreated control and 0% at the five different reference toxic concentrations chosen for the test. In mortality terms, the mortality of the exposed A. aurita ephyras was 0% in the control, but 100% at the different toxic concentrations. The obtained results showed that Cu significantly affected ephyras from the lowest tested Cu(NO3)2 concentration (1.9 mg/L) to the highest one (30 mg/L).   The results of exposing ephyra to different cadmium nitrate concentrations are shown in Figure 5. Like Cr, this compound also had a significant effect on both end-points. %Fp at both 24 h and 48 h lowered when the toxicant concentration rose, and was 100% at 0 mg/L and 0% at 30 mg/L. This also implies, as we mentioned above, an inverse correlation between %Fp and %M, which was observed at 15 mg/L and at both 24 h and 48 h. Mortality increased and, therefore, %Fp decreased (24 h: 33.33%M and 44.79%Fp; 48 h: 86.66%M and 6.17%Fp). Regarding the LOEC value for %M, it is 15 mg/L at both exposure times, with 33.33%M and 86.66%M at 24 h and 48 h, respectively. At 30 mg/L, mortality was 100% at both exposure times. The results obtained for exposing ephyra to different copper nitrate concentrations are shown in Figure 6. This graph depicts a different situation to those previously described. For Fp, the values were 100% at 24 h in the untreated control and 0% at the five different reference toxic concentrations chosen for the test. In mortality terms, the mortality of the exposed A. aurita ephyras was 0% in the control, but 100% at the different toxic concentrations. The obtained results showed that Cu significantly affected ephyras from the lowest tested Cu(NO 3 ) 2 concentration (1.9 mg/L) to the highest one (30 mg/L).  The results of exposing ephyra to different cadmium nitrate concentrations are shown in Figure 5. Like Cr, this compound also had a significant effect on both end-points. %Fp at both 24 h and 48 h lowered when the toxicant concentration rose, and was 100% at 0 mg/L and 0% at 30 mg/L. This also implies, as we mentioned above, an inverse correlation between %Fp and %M, which was observed at 15 mg/L and at both 24 h and 48 h. Mortality increased and, therefore, %Fp decreased (24 h: 33.33%M and 44.79%Fp; 48 h: 86.66%M and 6.17%Fp). Regarding the LOEC value for %M, it is 15 mg/L at both exposure times, with 33.33%M and 86.66%M at 24 h and 48 h, respectively. At 30 mg/L, mortality was 100% at both exposure times. The results obtained for exposing ephyra to different copper nitrate concentrations are shown in Figure 6. This graph depicts a different situation to those previously described. For Fp, the values were 100% at 24 h in the untreated control and 0% at the five different reference toxic concentrations chosen for the test. In mortality terms, the mortality of the exposed A. aurita ephyras was 0% in the control, but 100% at the different toxic concentrations. The obtained results showed that Cu significantly affected ephyras from the lowest tested Cu(NO3)2 concentration (1.9 mg/L) to the highest one (30 mg/L).

Discussion
Regarding Cr toxicity for the marine algae, our results ( Table 2) showed an EC50 ± SD of 15.378 ± 7.081 mg/L for potassium dichromate at 72 h. However, other authors report different values (see Figure 7). In the study of Uba [56], K2Cr2O7 obtained an EC50 value of 8.07 ± 0.03 mg/L, while the R 2 value was 0.99. Other authors have studied how species' sensitiveness to the same chemicals can vastly vary [57]. They characterized the nonstandardized diatom Chaetoceros tenuissimus by growth inhibition, biochemical, and infrared-spectroscopy (FT-IR) tests to compare the results to the standardized diatom Phaeodactylum tricornutum. The two species were exposed for 72 h to four chemicals: nanoparticles (n-TiO2, n-ZnO), potassium dichromate, and surfactant (polyethylene glycol, PEG). The obtained EC50 ± SD (mg/L) for P. tricornutum and C. tenuissimus were 22.97 ± 1.34 and 19.84 ± 1.45, respectively [57]. Other authors have evaluated the impacts of 16 different leachates of plastic-made packaging on marine species from different trophic levels (bacteria, algae, and echinoderms) [58]. The results obtained in that study evidenced that the tested doses were unable to significantly affect bacteria (Vibrio fischeri) and algae (P. tricornutum). Algae responses were measured by K2Cr2O7 (EC50 = 16.21 ± 1.72 mg/L). Another ecotoxicity test [59] has studied the toxicity of metal aqueous suspensions to microcrustaceans Daphnia magna (72 h exposure), algae P. tricornutum (72 h growth inhibition), and rotifer Brachionus plicatilis (48 h exposure). EC50 calculated for algae at 72 h was 8.11 mg/L. Pastorino et al. [57] has demonstrated that EC50 for P. tricornutum after 72 h of exposure to K2Cr2O7 fell within the 16.76-20.84 mg/L interval. According to our results, EC50 obtained for K2Cr2O7 came quite close to the reference values of other papers, which indicates that this alga resisted Cr more than other HMs.

Discussion
Regarding Cr toxicity for the marine algae, our results ( Table 2) showed an EC 50 ± SD of 15.378 ± 7.081 mg/L for potassium dichromate at 72 h. However, other authors report different values (see Figure 7). In the study of Uba [56], K 2 Cr 2 O 7 obtained an EC 50 value of 8.07 ± 0.03 mg/L, while the R 2 value was 0.99. Other authors have studied how species' sensitiveness to the same chemicals can vastly vary [57]. They characterized the non-standardized diatom Chaetoceros tenuissimus by growth inhibition, biochemical, and infrared-spectroscopy (FT-IR) tests to compare the results to the standardized diatom Phaeodactylum tricornutum. The two species were exposed for 72 h to four chemicals: nanoparticles (n-TiO 2 , n-ZnO), potassium dichromate, and surfactant (polyethylene glycol, PEG). The obtained EC 50 ± SD (mg/L) for P. tricornutum and C. tenuissimus were 22.97 ± 1.34 and 19.84 ± 1.45, respectively [57]. Other authors have evaluated the impacts of 16 different leachates of plastic-made packaging on marine species from different trophic levels (bacteria, algae, and echinoderms) [58]. The results obtained in that study evidenced that the tested doses were unable to significantly affect bacteria (Vibrio fischeri) and algae (P. tricornutum). Algae responses were measured by K 2 Cr 2 O 7 (EC 50 = 16.21 ± 1.72 mg/L). Another ecotoxicity test [59] has studied the toxicity of metal aqueous suspensions to microcrustaceans Daphnia magna (72 h exposure), algae P. tricornutum (72 h growth inhibition), and rotifer Brachionus plicatilis (48 h exposure). EC 50 calculated for algae at 72 h was 8.11 mg/L. Pastorino et al. [57] has demonstrated that EC 50 for P. tricornutum after 72 h of exposure to K 2 Cr 2 O 7 fell within the 16.76-20.84 mg/L interval. According to our results, EC 50 obtained for K 2 Cr 2 O 7 came quite close to the reference values of other papers, which indicates that this alga resisted Cr more than other HMs.
On Cd toxicity for the marine algae, our results for cadmium nitrate indicated an EC 50 ± SD (mg/L) of 2.494 ± 2.494 at 72 h (Table 2). Some authors reported that, at 72 h, the Cd EC 50 value for P. tricornutum was as high as 22.39 mg/L, which reveals its excellent tolerance to Cd. Compared with other microalgae species, they reported lower EC 50 values: 1.87 µg/L of Cd for Scenedesmus quadricauda, 2.13 µg/L for Aulacoseira granulate, and 1.8 mg/L for Teraselmis gracilis [60,61]. According to our results, EC 50 for Cd(NO 3 ) 2 was farremoved from the reference values of the aforementioned papers. Hence, further research into exposing P. tricornutum to Cd must be conducted. On Cd toxicity for the marine algae, our results for cadmium nitrate indicated an EC50 ± SD (mg/L) of 2.494 ± 2.494 at 72 h (Table 2). Some authors reported that, at 72 h, the Cd EC50 value for P. tricornutum was as high as 22.39 mg/L, which reveals its excellent tolerance to Cd. Compared with other microalgae species, they reported lower EC50 values: 1.87 µg/L of Cd for Scenedesmus quadricauda, 2.13 µg/L for Aulacoseira granulate, and 1.8 mg/L for Teraselmis gracilis [60,61]. According to our results, EC50 for Cd(NO3)2 was farremoved from the reference values of the aforementioned papers. Hence, further research into exposing P. tricornutum to Cd must be conducted.
On Cu toxicity for the marine algae P. tricornutum, we obtained an EC50 ± SD (mg/L) of 1.205 ± 0.322 at 72 h for Cu(NO3)2 ( Table 2). Population growth started to be affected at a concentration of 0.06 mg/L (Figure 1). Wang and Zheng [62] observed that, at the Cu 2+ concentration of 0.32 µg/mL, P. tricornutum cell density was significantly lower than that in the control (t-test, p > 0.05). EC50 for Cu 2+ at 72 h for this alga was calculated using a regression analysis and was 0.565 µg/mL. In that study, Cu at lower concentrations (<0.2 µg/mL) did not have any obvious adverse effect on P. tricornutum population reproduction, but Cu significantly inhibited this diatom's reproduction at >0.32 µg/mL. This finding indicates that >0.32 µg/mL Cu 2+ concentrations exceed the safety concentration level for this alga. In previous studies, Jung et al. [63] exposed marine algae Nitzschia pungens to several antifouling biocides, including copper pyrithione. EC50 (µg/L) of this compound recorded at 96 h was 0.319 ± 0.016. Franklin et al. [64] studied Cu toxicity in P. tricornutum.
The EC50 values at 48 h and 72 h were 142 ± 47 and 158 ± 63 nmol/L, respectively, and complete growth inhibition occurred at 11.8 µmol/L. The cell light scatter properties of P. tricornutum depended on cell size and intracellular granularity. Franklin et al. [64] noted how Cu brought about an increase in cell size after 24 h of exposure, with 50% of cells being larger than the controls at 3.15 µmol/L, and similar increases in cell size observed after 48 h and 72 h at 157.5 nmol/L and an EC50 value of 126 ± 47 nmol /L. Similar changes in cell size and granularity were also reflected in side-angle light scatter changes, with an EC50 value of 189 nmol/L after 48 h and 72 h of Cu exposure [65]. In line with our EC50 On Cu toxicity for the marine algae P. tricornutum, we obtained an EC 50 ± SD (mg/L) of 1.205 ± 0.322 at 72 h for Cu(NO 3 ) 2 ( Table 2). Population growth started to be affected at a concentration of 0.06 mg/L (Figure 1). Wang and Zheng [62] observed that, at the Cu 2+ concentration of 0.32 µg/mL, P. tricornutum cell density was significantly lower than that in the control (t-test, p > 0.05). EC 50 for Cu 2+ at 72 h for this alga was calculated using a regression analysis and was 0.565 µg/mL. In that study, Cu at lower concentrations (<0.2 µg/mL) did not have any obvious adverse effect on P. tricornutum population reproduction, but Cu significantly inhibited this diatom's reproduction at >0.32 µg/mL. This finding indicates that >0.32 µg/mL Cu 2+ concentrations exceed the safety concentration level for this alga. In previous studies, Jung et al. [63] exposed marine algae Nitzschia pungens to several antifouling biocides, including copper pyrithione. EC 50 (µg/L) of this compound recorded at 96 h was 0.319 ± 0.016. Franklin et al. [64] studied Cu toxicity in P. tricornutum. The EC 50 values at 48 h and 72 h were 142 ± 47 and 158 ± 63 nmol/L, respectively, and complete growth inhibition occurred at 11.8 µmol/L. The cell light scatter properties of P. tricornutum depended on cell size and intracellular granularity. Franklin et al. [64] noted how Cu brought about an increase in cell size after 24 h of exposure, with 50% of cells being larger than the controls at 3.15 µmol/L, and similar increases in cell size observed after 48 h and 72 h at 157.5 nmol/L and an EC 50 value of 126 ± 47 nmol /L. Similar changes in cell size and granularity were also reflected in side-angle light scatter changes, with an EC 50 value of 189 nmol/L after 48 h and 72 h of Cu exposure [65]. In line with our EC 50 results, these values differ from other studies (Figure 7). Hence, more research is required to collect more reliable data. Therefore, according to our EC 50 results for each salt (Table 2) and the corresponding concentration of each metal on its respective salt (Table 1), we conclude that the relative toxicity for P. tricornutum is Cu > Cd > Cr.
The brine shrimp A. salina is a suggested organism for bioassays because it functions like other zooplankton crustaceans, which accumulate trace elements and then transfer them to a higher trophic level [66]. Artemia has been used to study metal toxicity in other studies [67], some of which have demonstrated that brine shrimp is moderately sensitive to a wide range of metals [31,[68][69][70].
On Cr toxicity for the crustacean A. salina, our results for K 2 Cr 2 O 7 showed an EC 50 ± SD (mg/L) of 91.359 ± 6.746 at 24 h and a value of 23.554 ± 2.383 at 48 h ( Table 2). Kalčíková et al. [71] tested A. nauplii immediately after hatching, called the first instar, and the mean 24 h EC 50 of K 2 Cr 2 O 7 was 39.7 mg/L (n = 8) with SD = 10.2 mg/L. For the second and third instars, which were tested 24 h after hatching, the mean 24 h EC 50 of K 2 Cr 2 O 7 was lower (27.9 mg/L (n = 8)) and SD was 5.1 mg/L, which revealed the test's lower variability.
The test using the second and third Artemia instars was assessed because, the greater the sensitivity, the lower the variability. It was, therefore, used for other toxicity testing. In another study carried out in 2012 by Umarani et al. [72], acute Cr toxicity at 96 h to adult and subadult Artemia exposed to different salinity conditions was tested: at 40, 60, and 80 ppt salinity, the EC 50 values for subadult Artemia were 0.519, 0.784, and 1.192 mg/L, respectively, while the EC 50 values for adult Artemia were 1.031, 0.413, and 0.887 mg/L, respectively. In another study, Eduardo et al. [73] determined that EC 50 was variable in different development stages. In the first 24 h after hatching, EC 50 was one of the highest found in that study (21 µg/mL); it then decreased to 15 µg/mL in the 2nd stage and remained unchanged until the 5th stage. There were no significant differences. In the 6th and 7th stages, EC 50 increased to 21 µg/mL, with a further rise to 25 µg/mL in the 8th stage, which represented the peak EC 50 value in that study. A lowering EC 50 trend occurred in the 10th and 11th stages, with values of 21 µg/mL and 16 µg/mL, respectively. From the 12th to the 15th stage, the EC 50 values did not significantly differ from one another. In another paper, acute K 2 Cr 2 O 7 toxicity in A. salina larvae was studied as an alternative method to be applied to ecotoxicology. In this versatile method, 24 h nauplii were exposed to different concentrations of the compound, and EC 50 was 12.5 mg/L [26]. According to the published papers on Artemia toxicity to K 2 Cr 2 O 7 , the EC 50 values moderately differed from those obtained herein (Figure 7). However, they all showed the toxicity of this compound for Artemia larvae.
On Cd toxicity for the crustacean, our results for Cd(NO 3 ) 2 gave an EC 50 ± SD (mg/L) of 150.167 ± 27.496 at 24 h and a value of 153.840 ± 65.674 at 48 h (Table 2). Previous studies showed that Artemia was among those crustaceans that were most tolerant to Cd toxicity, as shown by the 24 h EC 50 values corresponding to the different studied species and populations. They ranged from 98 mg/L to 286 mg/L compared with the 48 h EC 50 (0.5-17 mg/L) reported for other crustaceans [70,74]. This tolerance can be partly explained by the marked effectiveness of Cd for metallothionein induction in Artemia [70]. Hadjispyrou et al. [24] reported that the EC 50 value for cadmium chloride to cause 50%M in Artemia was 155.5 mg/L at 24 h, with a 95% confidence interval (95% CI) of 148.8-162.5 mg/L. According to the bibliography and the obtained EC 50 values (Figure 8), we determined that A. salina was well tolerant to cadmium nitrate because the EC 50  Our results showed that Artemia quite well tolerated copper nitrate up to concentrations of around 60 mg/L ( Figure 8). Therefore, according to our EC 50 results for each salt (Table 2) and the corresponding concentration of each metal on its respective salt (Table 1), we conclude that the relative toxicity for A. salina is Cr > Cu > Cd.  Figure 8). Therefore, according to our EC50 results for each salt (Table 2) and the corresponding concentration of each metal on its respective salt (Table 1), we conclude that the relative toxicity for A. salina is Cr > Cu > Cd.
Regarding Cd toxicity for the A. aurita ephyra, our results for Cd(NO3)2 gave an EC50 ± SD (mg/L) of 19.880 ± 5.519 at 24 h and a value of 12.343 ± 2.588 at 48 h (Table 2). According to the Fp results ( Figure 5), at both 24 h and 48 h, when mortality increased, %Fp dropped (24 h: 33.33%M and 44.79%Fp; 48 h: 86.66%M and 6.17% Fp). The LOEC value for %M is 15 mg/L at both exposure times. In the study conducted by Faimali et al. [48], after 24 h of exposure, cadmium nitrate had an effect on both end-points (acute and sublethal), as evidenced by the 0.5 mg/L concentration for immobilization and that of 0.1 mg/L for Fp. After 48 h, the same effects were caused by the lower concentration of 0.05 mg/L for both end-points. It should be highlighted that both end-points showed 100% response at 1 mg/L after 24 h of exposure. From the obtained data (EC50) on the effect of cadmium nitrate on A. aurita, we obtained 0.07 mg/L at 24 h and 0.13 mg/L at 48 h. The comparison of EC50 with A. aurita showed that the new biological model appeared to be the most sensitive of the considered model organisms [48]. Costa et al. [45] exposed Aurelia sp. ephyras to different 1-4 µm microplastics (MPs). A relatively and slightly significant difference in effect terms (immobility, Fp) in treatments was observed. A difference in sensitivity in the end-points for LOEC, Fp, and EC50 after 24 h was noted. These results showed that the behavioral end-point (Fp) was more sensitive than the acute one (immobility) (LOEC Fpn = 0.01 mg/L versus LOEC immobility = 0.1 mg/L) for all of the exposure conditions. Conversely, after 48 h, MPs had significantly affected (p < 0.05) both end-points at the lowest tested concentration. A toxic effect was also observed, but only for immobility in EC50 terms at both exposure times and independently of the exposure conditions. The EC50 values at 24 h for immobility and Fp were 0.40 and 0.13 mg/L, respectively. Overall, the behavioral end-point (Fp) was very sensitive because a significant effect was noted at the lowest tested concentration (0.01 mg/L) [44]. In another study, Gambardella et al. [32] investigated the potential toxicity of Ag-NPs (silver nanoparticles) for the marine ecosystem by analyzing effects on several organisms belonging to different trophic levels. Algae (Dunaliella tertiolecta and Skeletonema costatum), cnidaria (A. aurita jellyfish), crustaceans (Amphibalanus amphitrite and Artemia salina), and echinoderms (Paracentrotus lividus) were exposed to Ag-NPs and different end-points were evaluated. The results showed that all of the end-points were able to underline a dose-dependent effect. Jellyfish were the most   [48], after 24 h of exposure, cadmium nitrate had an effect on both end-points (acute and sublethal), as evidenced by the 0.5 mg/L concentration for immobilization and that of 0.1 mg/L for Fp. After 48 h, the same effects were caused by the lower concentration of 0.05 mg/L for both end-points. It should be highlighted that both end-points showed 100% response at 1 mg/L after 24 h of exposure. From the obtained data (EC 50 ) on the effect of cadmium nitrate on A. aurita, we obtained 0.07 mg/L at 24 h and 0.13 mg/L at 48 h. The comparison of EC 50 with A. aurita showed that the new biological model appeared to be the most sensitive of the considered model organisms [48]. Costa et al. [45] exposed Aurelia sp. ephyras to different 1-4 µm microplastics (MPs). A relatively and slightly significant difference in effect terms (immobility, Fp) in treatments was observed. A difference in sensitivity in the end-points for LOEC, Fp, and EC 50 after 24 h was noted. These results showed that the behavioral end-point (Fp) was more sensitive than the acute one (immobility) (LOEC Fpn = 0.01 mg/L versus LOEC immobility = 0.1 mg/L) for all of the exposure conditions. Conversely, after 48 h, MPs had significantly affected (p < 0.05) both end-points at the lowest tested concentration. A toxic effect was also observed, but only for immobility in EC 50 terms at both exposure times and independently of the exposure conditions. The EC 50 values at 24 h for immobility and Fp were 0.40 and 0.13 mg/L, respectively. Overall, the behavioral end-point (Fp) was very sensitive because a significant effect was noted at the lowest tested concentration (0.01 mg/L) [44]. In another study, Gambardella et al. [32] investigated the potential toxicity of Ag-NPs (silver nanoparticles) for the marine ecosystem by analyzing effects on several organisms belonging to different trophic levels. Algae (Dunaliella tertiolecta and Skeletonema costatum), cnidaria (A. aurita jellyfish), crustaceans (Amphibalanus amphitrite and Artemia salina), and echinoderms (Paracentrotus lividus) were exposed to Ag-NPs and different end-points were evaluated. The results showed that all of the end-points were able to underline a dosedependent effect. Jellyfish were the most sensitive species, followed by barnacles, sea urchins, green algae, diatoms, and brine shrimps. The comparison of EC 50 to the selected species highlighted that jellyfish appeared to be the most sensitive model organisms of all of those investigated: EC 50 was 0.09 with 0.15 mg/L of cadmium nitrate at 24 h and 48 h, respectively. When considering previous ephyra studies [48], we find that the EC 50 and LOEC values for the mortality and Fp of A. aurita are lower than those obtained herein ( Figure 9). However, a correlation appeared between the mortality and Fp end-points, and ephyra responded efficiently to the selected range of toxicant concentrations. On Cu toxicity for the A. aurita ephyra, our results for Cu(NO 3 ) 2 gave an EC 50 ± SD (mg/L) of 0.283 ± 0 t 24 h and a value of 0.283 ± 0 at 48 h (Table 2). In mortality terms, the exposed A. aurita ephyras presented 0%M in the control, but 100%M at the different toxic concentrations ( Figure 6). Lucas and Horton [76] studied the short-term effects of HMs (including Cu) on the polyps of the common jellyfish A. aurita. They examined the independent effects of Cu on polyp condition aspects, including budding, strobilation, deformities, and mortality. The results showed that 200 µg Cu/L exceeded polyps' tolerance to this metal and rapidly led to mortality. For all of the treatments with a high Cu concentration (200 µg/L), polyp mortality was 89.5 ± 8.3% by day 4, reaching 100%M by day 17. In another study, Karntanut and Pascoe [77] examined the comparative sensitivity of three Hydra species to three important metal pollutants: Cu, Cd, and Zn. The selected species were Hydra vulgaris, Hydra viridissima, and Hydra oligactis. The acute toxicity data indicated a similar response of all of the species to each metal, with Cu being the most toxic and Zn being the least toxic. The range of 96 h EC 50 for Cu, Cd, and Zn for all of the Hydra species was 0.025-0.084 mg/L, 0.16-0.52 mg/L, and 11-14 mg/L, respectively. According to our data and the data observed in the above-cited studies (Figure 9), the conclusion is that Cu, and thus Cu(NO 3 ) 2 , is highly toxic for cnidarians at very low concentrations.
As for Cr toxicity for the A. aurita ephyra, our results for K 2 Cr 2 O 7 showed an EC 50 ± SD (mg/L) of 16.571 ± 4.246 at 24 h and a value of 6.726 ± 2.004 at 48 h ( Table 2). %Fp at both 24 h and 48 h showed an inverse correlation with an increasing toxicant concentration, with 0% at the highest concentration (30 mg/L), 93.33%M at 24 h, and 100%M at 48 h. According to the values obtained for %M at 24 h, the LOEC was 15 mg/L. For %M at 48 h, an incongruent value was observed because the LOEC was 0 mg/L in the untreated control. Unfortunately, we did not find any paper in which cnidarians were exposed to Cr to compare them. However, we noted that %M and %Fp followed a similar pattern to that of Cd(NO 3 ) 2 (Figure 8), which demonstrates that A. aurita is very sensitive to these metals. Therefore, according to our EC 50 results for each salt (Table 2) and the corresponding concentration of every metal on its respective salt (Table 2), it can be concluded that the relative toxicity for A. aurita is Cu > Cr > Cd.

Conclusions
The global increase in anthropogenic activities in relation to industrial development implies that ever-growing quantities of HMs enter the marine environment through effluents. This calls for continuous monitoring to control or limit their emission levels. For this purpose, the toxicity of and impact on different marine trophic levels need to be considered and deduced. The present study is a good start in the risk assessment and marine environmental conservation quest. This experimental work also allowed us to support other studies that have proposed A. aurita as a model for ecotoxicity tests, because our experiments enabled us to identify two end-points (sublethal and acute) with different sensitivity levels. The comparison of the EC 50 values obtained herein for the three reference toxicants indicates that jellyfish are a very promising model organism for ecotoxicological research purposes. Given the hypotheses posed in this study, and having performed the ecotoxicological test for three organisms, it can be assumed that the toxicity of the three elements is different at the trophic levels they affect and all of the toxicants cause damage during acute exposure. However, algae together with A. aurita ephyra have a lower EC 50 . Thus, they could support the jellyfish as a proper model for marine ecotoxicological assays in order to better evaluate the toxicity in higher trophic levels. Data Availability Statement: Oscar Andreu-Sánchez, PhD, is responsible for the manuscript entitled "Investigation of metal toxicity on microalgae Phaeodactylum tricornutum, hipersaline zooplankter Artemia salina and jellyfish Aurelia aurita". On behalf of the rest of the coauthors, with this document, I warrantee and sign that the datasets generated and used during the current study are available from the corresponding author upon reasonable request.