Reciprocal Effects of Metal Mixtures on Phytoplankton

Reciprocal Effects of Metal Mixtures on Phytoplankton: Comparison

Please note this is a comparison between Version 1 by Ammara Nawaz and Version 2 by Camila Xu.

Phytoplankton are a key part of marine and freshwater ecosystems as primary producers of the food web. They are exceptional in their role in heavy metals bioremediation. Algae and cyanobacteria when exposed to metals in mixture, show altered responses as compared to the single metal exposure. Algal parameters such as growth, chlorophyll content, photosynthesis, metal uptake and metabolism, or lipid profile are commonly determined to find out the level of stress in algal cells resulting from heavy metals. Phytoplankton have several pathways of metal entry, detoxification and tolerance. It is essential to estimate reciprocal toxicity of metals as in real-time, metals are released in the environment in bulk (reciprocal effects). Phytoplankton can be a powerful tool in such risk assessments.

phytoplankton
reciprocal toxicity
toxic metals
aquatic ecosystem

1. Introduction

Water quality is one of the major factors that affects the health status of all the life forms present in aquatic system. Water contamination is indeed a serious hazard to the world and mankind. Anthropogenic activities are one of the major causes of water pollution, which expel multiple kinds of harmful pollutants into aquatic ecosystems. The contamination of the aquatic environment with heavy metals is a critical issue nowadays due to the potential toxicological risks and accumulative nature of metals in aquatic systems [1]. The properties of the given metal and the given environmental factors naturally influence the distribution of metals in the environment. The mobilisation of heavy metals has increased considerably in natural systems due to human interference. Some metals are essential for the biochemical and physiological processes that occur in living cells in very low or controlled concentrations; however, higher concentrations of these metals above a threshold limit result in cell toxicity and damage on many levels. The indirect effect of heavy metal pollution on the structure and functions of food webs in different ecosystems, including aquatic ones, is observable [2].

2. Phytoplankton as Heavy Metal Pollution Indicators

The phytoplanktonic community is considered to be a basic autotrophic part of any aquatic ecosystem, affecting the assembly and efficiency of the food web of the system. Phytoplankton also affect global biogeochemical cycles, as documented in a recent study on the role of oceanic phytoplankton in the C, P, and N cycles and their distribution ^[3][4][3,4]. These authors demonstrated the importance of particulate N:C and P:C ratios for the regulation of dissolved inorganic matter (dN:P) on the global scale, with the level of marine oxygen being an important control [4]. Their research provides additional information on the potential interdependence of phytoplankton physiology and global climate conditions. Hence, phytoplankton are used as an early warning signal for the health status of water bodies [5]. The variability of metals in the phytoplanktonic community could be used to predict the intensity and potential of heavy metal ecological damage and water quality decline on many levels [6]. Algal parameters such as growth, chlorophyll content, photosynthesis, metal uptake and metabolism, or lipid profile are commonly determined to find out the level of stress in algal cells resulting from water pollutants, including heavy metals ^{[7][8][9][10]}[7,8,9,10]. Lewis [11] mentioned that phytoplankton are good ecotoxicological tools, as they are the main autotrophic component of the water system. Most phytoplankton species exhibit a short life span with a response of very high sensitivity to different environmental fluctuations, including pollutants such as heavy metals [11]. Furthermore, phytoplankton are easily and economically culturable and show rapid growth and cellular turnover with high sensitivity levels for various pollutants. Phytoplankton are being used as bioindicators for heavy metal toxicity studies in aquatic ecosystems, providing valuable results and insights [12]. Phytoplankton can accumulate a certain amount of metals without being damaged, and this process is termed phytoremediation; however, exposure to a high amount of heavy metals usually results in damaging the living cells [13]. Algal species vary in their individual metal sensitivities and interactions of the metal mixture interactions [14], and some studies are focused on studying the resistant strains of freshwater algae [15]. Upon heavy metal exposure, phytoplankton homeostasis disruption can occur. In stress conditions, algal cells are observed to produce excessive reactive oxygen species (ROS), for example, superoxide (O₂∙⁻), hydroxyl radical (∙OH), or hydrogen peroxide (H₂O₂). ROS can be damaging to the proteins, amino acids, nucleic acids, membrane lipids, and DNA of phytoplankton, causing several disorders in the algal cell ^[16][17][16,17]. The antioxidant protection enzymatic and nonenzymatic system, like superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR), and very effective sulfur-rich molecules of phytochelatins/metallothioneins, is released by the photosynthetic cell to decrease the excess ROS induced by metal exposure, to overcome the damage against the given pollutant. Metallothioneins and phytochelatins produced in the cytosol are abundant intracellularly and extracellularly to bind metal ions to the exudate, precipitate, and stabilize them on the cell surface to prevent their cellular entry into microalgae [18]. To decrease cellular oxidative damage induced against the applied metal, the accumulation of free proline in the cell is also noticed in heavy-metal-stressed algal cells [19]. Indeed, higher concentrations of, for example, Cu and Cr, were inhibitory to proline accumulation by Chlorella vulgaris [20]. It is well documented that, in metal-stressed-state algal and phytoplanktonic cells, malondialdehyde (MDA) and TBA-reactive products (TBARS, thiobarbituric acid reactive products) are produced that are frequently used as metal stress indicators in ecotoxicological evaluation studies. Zheng et al. ^[21][22][21,22] studied the geochemical behaviour of heavy metals in the water system. They explained that heavy metals can show three arrangements when released into a water body as, a. Particle form: The metal is adsorbed onto the suspended particles already present in the water body. b. Dissolved form: Metals bond with dissolved organic materials available in the water system. c. Biological form: The metal is taken up by the phytoplankton and algae. It is integrated in the cell and passed on to the food chain. The dynamics of heavy metals in a water body are primarily controlled by these three metal arrangements. Heavy metal bioaccumulation in the food web of aquatic systems is mainly controlled by the amount of metal taken up by the phytoplankton community. This makes phytoplankton a good bioindicator for determining the metal toxicity or health of any aquatic ecosystem and they are used extensively in ecotoxicological studies.

3. Behaviour of Heavy Metals in an Aquatic Ecosystem

Understanding the synergistic effects for metal co-exposure in ecotoxicology is required to evaluate the real-time toxicity of metals, as, in natural ecosystems, pollutants are discharged in the form of a bulk. In nature, organisms in any environment experience exposure to mixtures of metals, while the ecotoxicity of metals alone is different compared to a mixture of different metals ^[23][24][23,24].

Different environmental conditions affect the reciprocal toxicity of heavy metals, together with the different properties of toxic elements, as well as their interactions with organic and inorganic substances outside and inside of living cells [23]. The bioaccumulation of heavy metals in plankton cells depends on multiple factors, including the absorptive ability of individual strains, season, pH, temperature, metal bioavailability, and many others ^[25][26][25,26].

4. Conventional and Novel Methods to Study Reciprocal Metal Mixture Toxicity Used Frequently

There are more articles, including reviews, that are focused on performing a risk assessment and finding the best-fitting modelling calculations of contaminants in aquatic environments, as well as the toxicity of contaminants to aquatic species, including algae, together to determine the relevant important processes and conditions/parameters related to chemical toxicity to organisms ^{[27][28][29][30][31]}[32,33,34,35,36]. Methods as the biotic ligand model (BLM), concentration addition (CA), independent action (IA), sensitivity distribution of EC50 species sensitivity distribution (SSD curves), and others are used to study reciprocal metal toxicity and providing promising results. If the initial assessment shows acute toxicity of a given pollutant (metal, chemical, or any other), a detailed study can be planned to determine the exact nature of, toxicity, and possible damage to this pollutant.

5. Possible Mechanisms of Entry, Toxicity, and Detoxification in Algal Cells

Scientists and environmentalists have been attempting to examine the mixture toxicity of metals in aquatic systems. The objective of such studies is always to determine the possible risk implied by any metal mixture drained into a water body. Phytoplankton are usually used in such assessment tests, as they are a common and reliable indicator organism for the evaluation of aquatic ecosystem pollution. Table 12 summarises the ecotoxicological reciprocal metal assessments made using phytoplankton test species.

Table 1. A summary of reciprocal toxicity assessments made in previous studies using phytoplankton (EE—equivalent effect concentration; NA—not available).

Metal Mixture	Species	pH	Concentrations (mg L⁻¹ or M)	Reciprocal Effect	Reference
Al + Zn	Raphidocelis subcapitata	NA	>0.026 mg L⁻¹ Zn and 0.739 mg L⁻¹ Al; 22.24–37.06 µM Al, 0.08–0.46 µM Zn	Antagonistic	^[32]^[33]
Cu + Cr + Ni	Chlorella pyrenoidosa 251	6.8	0.1–1.0 mg L⁻¹ of Cu, Cr and Ni	Synergistic	^[34]
As + Se	Desmodesmus quadricauda	7.2	29.05 mg L⁻¹ As and 3.65 mg L⁻¹ Se	Synergistic	^[16]
Cd + Co	Chlamydomonas reinhardtii	7	2 × 10⁻⁸ M Cd and Co	Non-interactive	^[35]
Cd + Fe + Mn + Cu	Chlamydomonas reinhardtii	7	2 × 10⁻⁸ M Cd²⁺, 1 × 10⁻¹⁷ M Fe³⁺, 1 × 10⁻⁶ M Mn²⁺, 1 × 10⁻¹³ M Cu²⁺	Non-interactive	^[35]
Cd + Co	Chlorella vulgaris	6.5	0.89 µM Cd and 9.50 µM Co	Antagonistic	^[36]
Cd + Cr	Nile river algal community	NA	0.05–1.00 mg L⁻¹ Cd and 0.25–3.00 mg L⁻¹ Cr	Synergistic	^[37]
Cd + Cu	Chaetoceros gracilis; Isochrysis sp.	NA	0, 0.56, 1.00, 1.80, 3.20, and 5.60 mg L⁻¹ Cd and 0, 0.010, 0.018, 0.032, 0.056, 0.100 mg L⁻¹ Cu	Synergistic	^[38]
Cd + Cu	Chlamydomonas reinhardtii	7.5	40, 60, and 80 nM Cd and Cu	Antagonistic	^[39]
Cd + Cu	Chlamydomonas reinhardtii	8	1 × 10⁻⁶–1 × 10⁻⁵ M Cd, and 1 × 10⁻⁶–1 × 10⁻⁵ M Cu	Synergistic	^[40]
Cd + Cu	Chlamydomonas reinhardtii	6	3.52 × 10⁻⁶ Cu²⁺ M and 3.52 × 10⁻⁶ M Cd²⁺	Antagonistic	^[41]
Cd + Cu	Chlorella pyrenoidosa	8.6	13–25 µM Cu and 6 µM Cd	Synergistic	^[42]
Cd + Cu	Chlorella vulgaris			Antagonistic	^[43]
Cd + Cu	Chlorella vulgaris	NA	1.5 μM Cu and 2.0 μM Cd	Synergistic	^[44]
Cd + Cu	Chlorella vulgaris	6.5	2.80 µM Cu and 0.89 µM Cd	Synergistic	^[36]
Cd + Cu	Chlorella sp.			Synergistic	^[45]
Cd + Cu	Chlorolobion braunii	NA	5 µM Cu and 1 µM Cd	Synergistic	^[46]
Cd + Cu	Dunaliella minuta	7.4	7.57 µM Cu and 0.34 µM Cd	Antagonistic	^[47]
Cd + Cu	Navicula pelliculosa	7	0.42–0.54 µM Cu and 0.50–0.59 µM Cd (EC50 values)	Antagonistic	^[27]
Cd + Cu	Nile river algal community	NA	0.05–1.00 mg L⁻¹ Cd and Cu	Synergistic	^[37]
Cd + Cu	Pseudokirchneriella subcapitata	8.1 (BLM)	0.006–0.046 μM Cu and 0–0.500 μM Cd	Synergistic	^[28]
Cd + Zn	Chlorella vulgaris	6.8	2 × 10⁻⁵ M Zn and 0–8 × 10⁻⁵ M Cd	Antagonistic	^[24]
Cd + Fe	Thalassiosira weissflogii	NA	1 × 10⁻¹⁰ M Cd²⁺ and 1 × 10^−7.8 to 1 × 10^−5.8 M Fe EDTA	Antagonistic	^[48]
Cd + Hg	Anabaena inaequalis	NA		Synergistic	^[49]
Cd + Ni	Anabaena inaequalis	NA		Antagonistic and synergistic depending upon metal conc.	^[49]
Cd + Pb	Scenedesmus obliquus	NA	EE-20 for Cd-Pb synergistic, EE-50 additive	Synergistic	^[50]
Cd + Zn	Chlamydomonas reinhardtii	7	1 × 10⁻⁹ M Zn²⁺, 7 × 10⁻⁹ M Cd²⁺	Antagonistic	^[51]
Cd + Zn	Chlamydomonas reinhardtii	7	7 nM Cd²⁺ and 6 × 10⁻⁹ M	Antagonistic	^[35]
Cd + Zn	Chlorella sp.			Antagonistic	^[45]
Cd + Zn	Skeletonema costatum	7.8 to 9	200–400 μg L⁻¹ Zn 100 μg L⁻¹ Cd	Additive to slight synergistic	^[52]
Cd + Zn	Phaeodactylum tricornutum	7.8 to 9	3000 μg L⁻¹ Cd 4000 μg L⁻¹ Zn	Additive to slight antagonistic	^[52]
Cd + Zn	Scenedesmus obliquus	NA	EE-20 and EE-50 for Cd-Zn additive	Synergistic	^[50]
Co + Cu	Chlorella vulgaris	6.5	9.5 µM Co and 2.8 µM Cu	Synergistic	^[36]
Cu + Fe	Chlamydomonas reinhardtii	6–8	1 × 10⁻¹⁹ M Fe³⁺ and 1 × 10⁻¹³ to 1 × 10^−10.5	Antagonistic	^[53]
Cu + Ni	Pseudokirchneriella subcapitata	6.2–8.2	0.001–2.680 mg L⁻¹ Ni and 0.001–0.659 mg L⁻¹ Cu	Non-interactive	^[54]
Cd + Ca	Micrasterias denticulata	NA	2 mM CaSO₄ and 150 μM CdSO₄	Antagonistic	^[55]
Cu + Pb	Chlamydomonas reinhardtii	7	≤1 mg L⁻¹ of Cu and Pb	Antagonistic	^[56]
Cu + Zn	Chlorella sp.			Antagonistic	^[45]
Cu + Zn	Navicula pelliculosa	7	3.48 µM Zn and 0.51 µM Cu (EC50 values)	Additive	^[27]
Cu + Zn	Phaeodactylum tricornutum	NA	0.25 mg L⁻¹ Cu and 4.00 mg L⁻¹ Zn	Synergistic	^[57]
Cu + Zn	Phaeocystis antarctica; Cryothecomonas armigera	7.9		Antagonistic	^[58]
Cu + Zn	Scenedesmus sp.	7	2.5–40.0 μM CuCl₂.2H₂O and 5–100 μM ZnCl₂	Synergistic	^[19]
Cu + Zn	Pseudokirchneriella subcapitata	8.1 (BLM)	0.20–2.00 μM Zn and 0.006–0.046 μM Cu	Antagonistic	^[28]
Cd + Zn	Pseudokirchneriella subcapitata	8.1 (BLM)	0.20–2.0 μM Zn 0.036–2.100 μM Cd	Antagonistic	^[28]
Cr + Cu	Chlorella vulgaris	NA	0.05, 0.50, 5.00 μM	Additive	^[59]
Hg + Ni	Anabaena inaequalis			Additive	^[49]
Mg + Pb	Chlamydomonas reinhardtii	7	≤1 mg L⁻¹ of M and Pb	Antagonistic	^[56]
Ni + Zn	Navicula pelliculosa	7	0.15–0.19 µM Ni and 3.48–3.71 µM Zn (EC50 values)	Synergistic	^[27]
P + Zn	Raphidocelis subcapitata	NA	0.09 × 10⁻⁶ M to 9.08 × 10⁻⁶ M Zn and 2.3 × 10⁻⁴ M, 2.3 × 10⁻⁶ M and 1.0 × 10⁻⁶ M P	Additive	^[60]
Pb + Zn	Scenedesmus obliquus	NA	EE-20 and EE-50 for Pb-Zn synergistic	Additive	^[50]
As(V) + Cd + Cu + Ni + Pb	Diacronema lutheri	NA	450 µg L⁻¹ As(V), 109 µg L⁻¹ Cd, 34 µg L⁻¹ Cu, 126 µg L⁻¹ Ni, 414 µg L⁻¹ Pb	As(V) had the main toxicity in the mixture	^[61]
Cd + Co	Raphidocelis subcapitata	NA	0.13–0.25 mg L⁻¹ Co, 0.025–0.100 mg L⁻¹ Cd	Synergistic (high Co and low Cd) Antagonistic (low Co and high Cd)	^[62]
Cd + Co + Cu	Chlorella vulgaris	6.5	2.80 µM Cu, 0.89 µM Cd and 9.50 µM Co	Antagonistic	^[48]
Cd + Cr + Cu	Nile river algal community	NA	0.05 mg L⁻¹ Cd and 0.10 mg L⁻¹ Cu, Cr	Antagonistic	^[63]
Cd + Ni + Zn	Nile river algal community	NA	0.05 mg L⁻¹ Cd and 0.10 mg L⁻¹ Cu, Zn	Antagonistic	^[63]
Co + Cu + Zn	Chlorophyceare; Bacilariophyceae; Cyanophyceae	NA	1 × 10⁻⁶ to 1 × 10⁻¹⁰ mg L⁻¹ Cu, Co and Zn	Synergistic	^[64]
Cu + Ni + Zn	Pseudokirchneriella subcapitata	7.2	0.0200 mg L⁻¹ Zn, 0.0010 mg L⁻¹ Ni, 0.0025 mg L⁻¹ Cu	Non-interactive	^[65]
Cu + Ni + Zn	Pseudokirchneriella subcapitata	6.2–8.2	0.001–2.680 mg L⁻¹ Ni, 0.001–0.659 mg L⁻¹ Cu, and 0.001–0.450 mg L⁻¹ Zn	Non-interactive	^[54]
Cu + Pb + Zn	Scenedesmus quadricauda	8	0.1–0.2 mg L⁻¹ Cu, 0.3–0.5 mg L⁻¹ Zn, 0.3–0.6 mg L⁻¹ Pb	Synergistic (growth)	^[66]
Cu + Pb + Zn	Scenedesmus quadricauda	8	0.1–0.2 mg L⁻¹ Cu, 0.3–0.5 mg L⁻¹ Zn, 0.3–0.6 mg L⁻¹ Pb	Antagonistic (photosynthesis)	^[66]
Cu + Ti + Zn (nanoparticles)	Pseudokirchneriella subcapitata	7.5–8	380 mg L⁻¹ TiO, 0.068 mg L⁻¹ ZnO, 6.400 mg L⁻¹ CuO	Non-interactive	^[67]
Cd + Co + Fe + Zn + P	Chlamydomonas reinhardtii	7	1–100 μM P, 5–40 μM CdCl₂	Antagonistic	^[68]
Cd + Cu + Ni + Zn	Nile river algal community	NA	0.05 mg L⁻¹ Cd and 0.10 mg L⁻¹ Cu, Cr, Zn	Synergistic	^[63]
Cd + Cu + Ni + Zn	Pseudokirchneriella subcapitata		0.0200 mg L⁻¹ Zn + 0.0010 mg L⁻¹ Ni + 0.0025 mg L⁻¹ Cu	Non-interactive	^[65]
Cd + Cu + Ni + Pb + Zn	Phaeocystis antarctica; Cryothecomonas armigera	7.9		Synergistic while Zn behaves antagonistic	^[69]
Cd + Cu + Pb + Zn	Pseudokirchneriella subcapitata	NA	30, 60, 120, 250 and 500 mg L⁻¹ for Cd and Zn; and 500, 1000, 2000, 3000, 4000 mg L⁻¹ for Cu and Pb	Exude formation lowers metal toxicity	^[70]
Co + Cu + Fe + Mn + Mo + Ni + Zn	Marine phytoplankton communities	8.1	Various oceanic conc. comparison	Complex interactions with biogeochemical influence of ocean	^[71]
Fe + Cr + Cd	Micrasterias denticulata	Near 7 with added soil with buffering property	600 nM Cd, 10 μM Cr, and 100 μM Fe	Antagonistic	^[72]
Zn + Cd + Cr	Micrasterias denticulata	Near 7 with added soil with buffering property	600 nM Cd, 10 μM Cr, and 300 nM Zn	Antagonistic	^[72]

Conversion of 1 µM (microM) of (semi)metal for the mentioned elements into mg L⁻¹ is following: 1 µM Al = 0.0270 mg L⁻¹ Al; 1 µM As = 0.0749 mg L⁻¹ As; 1 µM Cd = 0.1124 mg L⁻¹ Cd; 1 µM Co = 0.0589 mg L⁻¹ Co; 1 µM Cr = 0.0520 mg L⁻¹ Cr; 1 µM Cu = 0.0636 mg L⁻¹ Cu; 1 µM Fe = 0.0559 mg L⁻¹ Fe; 1 µM Mn = 0.0549 mg L⁻¹ Mn; 1 µM Ni = 0.0587 mg L⁻¹ Ni; 1 µM Pb = 0.2072 mg L⁻¹ Pb; 1 µM Se = 0.0790 mg L⁻¹ Se; 1 µM Ti = 0.0479 mg L⁻¹ Ti; 1 µM Zn = 0.0654 mg L⁻¹ Zn.

Marine and freshwater algae show different toxicity response to heavy metals due to their different living conditions, salinity levels, biology and tolerance etc. The possible mechanism of metal toxicity and the defence system of algal cells is not as studied as in higher plants. It assumes that the entry and detoxification system between plants and algae can be very closely related, as previously described, for the toxicity of Cd, together with the synthesis of phytochelatins and their ability to absorb heavy metal ions in the cytoplasm of algae cells ^[73][74][75][98,99,100]. Biosorption is achieved due to the presence of various biomolecules on the microalgal cell wall, such as proteins, carbohydrates, and lipids, is aided by negatively charged ions such as phosphate (-PO₄)³⁺, carboxyl (-COOH), hydroxyl (-OH), and amino (-NH₂) groups ^[76][101]. In addition to compartmentalisation or efflux, there is another pivotal step in detoxification, namely, the binding of the metal ions with certain peptides and the consequent chelation process ^[77][102]. The cation diffusion facilitator (CDFs) proteins form a family of ubiquitous transporters involved in metal homeostasis and tolerance. These proteins catalyse the efflux of transition metal cations, such as Zn²⁺, Cd²⁺, Co²⁺, Ni²⁺, or Mn²⁺, from the cytoplasm to the outside of the cell or into subcellular compartments ^[78][103]. The gal Irt-like proteins (ZIPs), mainly in the vacuolar and plasma membrane, that regulate Zn and Fe uptake.

Other authors ^[79][104] identified genes encoding multicopper oxidase (CrFOX1) and Fe permease (CrFTR1) in C. reinhardtii that occur in the Fe assimilation pathway. The Fe status in Chlamydomonas is regulated by genes of the ZIP family, such as IRT1 and IRT2 ^[80][105]. These algae species also encode members of the NRAMP family, which are also involved in Fe regulation ^[81][106]. The tolerance of Chlorella sp. to Cr and Pb is probably caused by the reduction in metal influx across the plasma membrane; metal chelation in the cytosol by ligands (e.g., phytochelatins, metallothionein, organic acids, and amino acids); the transport of metal ligand complexes through the tonoplast and accumulation in the vacuole; sequestration in the vacuole by tonoplast transporters; and/or ROS defence mechanisms ^[82][110]. Ferrari et al. ^[83][111] studied H⁺/(SO₄)²⁻ (SULTRs) and Na⁺/(SO₄)²⁻ (SULPs) plasma membrane sulfate transporters and a chloroplast-envelope localised ABC-type holocomplex in Scenedesmus acutus with different Cr(VI) sensitivities, and they observed that the SULTRs’ up-regulation, observed after S-starvation, may directly contribute to enhancing Cr tolerance by limiting Cr(VI) uptake and increasing S availability for the synthesis of S-containing defence molecules.

Current research has indicated that Pb, one of the most toxic metals in nature, causes severe depletion of endogenous cytokinins, auxins, and gibberellin and an increase in abscisic acid content in the green alga Acutodesmus obliquus. Exogenous auxins and cytokinins alleviate Pb toxicity through the regulation of endogenous phytohormone levels ^[84][113]. Cd and Co induced growth and photosynthetic inhibition in Raphidocelis subcapitata ^[62][77]. Antagonism occurs with Cd and Co because they probably compete for the same transport sites on the membrane, since they are bivalent metals ^[62][77]. The tolerance of microalgae to metal toxicity with a high supply of P is probably because microorganisms supplied with this nutrient resist metal toxicity better compared to algal cells under severely limited P conditions ^[60][75].

6. Conclusions

Current toxicological assessments mainly focus on single metal toxicity. To fully understand the potential threat of metals, understanding mixture toxicity in an aquatic ecosystem is vital. The use of phytoplankton can be very useful in ecotoxicological studies, as they make up the first level of any aquatic food web. The recent methodologies used to address this issue have some shortcomings. The EC50 value, the community response, microtox assays, BLM, CA, IA, and other statistical methods provide valuable predictions, but have their shortcomings. In the review, it was noted that most metal toxicity research is conducted with Al, Cd, Co, Cr, Cu, Fe, Ni, Pb, and Zn metals and their reciprocal effects on increased or decreased toxicity. Cu mostly showed antagonistic effects with Cd, Pb, and Zn. The Zn metal ion reduced the toxicity of Al, Cd, Cu, and Ni in many studies. The Cd metal showed variable interactions in different species with Cu and other metals. This suggests that, when studying these metals on phytoplankton, it is important to consider the possible reciprocal interactions of these metals in a mixture. While Zn in the mixture of Cd + Cr + Ni + Pb reacts in the marine algal population as an antagonist, all the mentioned metals can be assessed as synergistic reciprocal effects. Transporters and channels that can regulate metal homeostasis in organelles and cells such as CDF, ZIP, HMA, COPT, MTP, MRP, and NRAMP, regulate and transport essential and non-essential metals through the membranes of algal cells and organelles. A brief introduction to metal cellular pathways and detoxification in the review can help researchers to understand this metal toxicity in phytoplankton for various metal ions, helping future researchers in the field to understand these possible reciprocal toxic metal interactions that affect metal toxicological assessments, and in planning their experiments.