4. Effect on the Reproductive System

The adverse effects of metals on the fish reproductive system are increasing every day, mainly due to increased water pollution and the usage of polluted water for fish culture. Healthy eggs and sperms are essential for the process of successful fertilization. However, the quality of eggs and sperm is affected by induced spawning, gamete storage methods, and more importantly, water pollution. The motility time of spermatozoa is very important for effective fertilization. According to the literature, sperm motility is affected by metallic trace elements. For example, although the sperm morphology of mummichog (Fundulus heteroclitus) was not affected by methylmercury (CH₃Hg), it triggered a significant loss in the motility of sperms ^[74][75][98,99]. Lead, Cd, and Cu caused a significant decrease in the motility of European carp (Cyprinus carpio) spermatozoa ^[76][77][78][100,101,102]. Similarly, Cu toxicity caused adverse effects in the spermatozoa activity in C. carpio ^[79][103], while Sionkowski et al. ^[80][104] showed that the higher concentration of Cu and Pb caused reduced spermatozoa motility in grass carp (Ctenopharyngodon idella). Likewise, the effects of Zn on the sperm motility of some common carp were also explored. Metallic trace elements are also responsible for several endocrine complications among fish. For example, Cd decreased the thyroid hormone level, inhibited the estrogen receptors, and interrupted the expression of growth hormone ^[81][105]. On the other hand, iodine metabolism interruption by Pb was also recorded to inhibit thyroid synthesis ^[82][106]. Prooxidative possessions of the metal ions could also cause oxidative harm to the cell membrane. They can also induce oxidative stress in fish. Lead, Pb, and Cu can also trigger the genotoxic effects on the fish ^[83][84][85][107,108,109]. A tabulated review of different references is provided to show the deteriorating effects of different metals on fish’s reproductive system (Table 3).

Fish Species	Metal Concentration (mg L−1)	Metal Composition	Stage of Exposure	Exposure Duration	Effect Observed on Fish	References
Effect of Cadmium (Cd)

observed significant abnormalities in fathead minnow embryos treated with Hg, including spinal curves, heart damage, and abnormal growth of the heart cavity. Samson and Shenker ^[120][144] reported tissue anomalies in zebrafish (Danio rerio), including abnormalities in fin overlaps and caudal parts. The first 24 h of fish embryonic development are the most vulnerable to metallic trace elements toxicity. A study found that during the first 24 h after insemination in contaminated water, almost 20% of developing embryos died, even in a controlled environment ^[121][145]. The blastula stage had the highest mortality rate (15%), and metal exposure during this stage significantly affected the life span of developing embryos. Embryos exposed to 0.1 mg/L of Cu had significantly decreased survival rates compared to the control group, and at 0.3 mg per litre, all embryos died. Copper exposure caused most fish embryo deaths during the blastula stage (25%), followed by the stage of body division (15%). However, embryo mortality decreased significantly at later developmental stages ^[79][103]. Slominski et al. ^[121][145] also reported that mortality significantly declined during organ formation, the division of the body, and eye coloration phases. Most fetuses (5%) expired during organ formation before the eye coloration phase. Metallic trace elements toxicity also increases the death of fish hatchlings in various species, including rainbow trout (Onchorynchus mykiss), Atlantic salmon, common carp, and grass carp. Freshly inseminated ovum of Oncorhynchus mykiss were more susceptible to Ni than the embryo at the organogenesis stage, while goldfish (Carassius auratus) eggs’ mortality was greater during the blastula stage than at the eyed stage when exposed to Cd or Hg. Rainbow trout (Oncorhynchus mykiss) fetuses were more vulnerable at the eyed stage than newly inseminated eggs when presented with a mixture of metals. The abnormalities caused by different metallic trace elements at the embryonic stages of fish are reviewed in Table 4.

Fish Species	Metal Concentration (mg L−1)	Metal Composition	Stage of Exposure	Exposure Duration	Effect Observed on Fish	References
Effect of Cadmium (Cd)
Heteropneustes fossilis	0.050	CdCl2	Adult	24 h	Decreased ovulation.	[86]
Leuciscus idus	0.1000	CdCl2	[	110	]
Egg, sperm	21 dpf	Reduced larval survival, growth, and delayed development.	[	78	]	[102]	Pimephales promelas
Oryzias latipes	0.005	0.0019CdCl2	CdCl212 months	2H2O21 days	Reduced egg production.	Embryo, larva	20 dpf	Morphological abnormalities were observed.][73]	[51][75]	Cyprinus carpio and Capoeta	Kor River (Fars Province)	Hg, Cd, As, Pb
[	122	]	[	146	]	Blood cells, liver, kidney	Hyperemia, cellular degeneration, and vacuolation.	[26
Pimephales promelas	0.003	Cd(NO3)2	]	[	Adult50]
4 days	Elevated auditory threshold.	[	Oryzias melastigma	50	0.010	CdCl2	5 months][74]	30 days	Decreased gonadal development.	[87][111]	Oreochromis niloticus
Cyprinus carpio	0.06	Challawa River (Kano, Nigeria)
CdCl	2	Eggs	60 dpf	Retardation in the developmental stages of eye pigmentation and spine curvature, lack of tail formation and head.	[	123][147]	Pimephales promelas	0.060Zn, Cd, Fe, Pb	Muscles	Higher bioaccumulation in muscles compared to bioaccumulation factor.	Prochilodus magdalenae	24.90	CdCl2	2 years	7 days	Reduced fertility rate.	[88][112]
Danio rerio	34.8	CdCl2	72 hpf	72 h	[	CdCl227]	Adult[51]
21 days	Decreased vitellogenin gene expression and increased estrogen receptor beta.	[	51	]	[	75]	Neuromast damage, coagulated egg, increased mortality rate.	[124][148]	Clarias gariepinus	Effect of Mercury (Hg)		Lake Maryout (Egypt)	Cd, Pb, Hg, As	Gonads	The ovary exhibits lytic characteristics with oocytes at various stages, a decreased quantity of germinal cells, and an augmented interstitial space in the testes.
Danio rerio	0.112	CdCl2	[		28][52]
0–96 hpf	4 days	Immunotoxicity, behavioural alteration, and oxidative stress.	[	52	]	[	76]
Danio rerio	0.8018	CdCl		2	6 hpf
24 h	Increased apoptotic event and induced cell death in brain of embryo.	[	125	]	[	149]	Auchenoglanis occidentalis	Tiga Dam (Nigeria)	Zn, Cd, Pb, Fe	Gills, liver, kidney	Lesions in the gills, liver, and kidney.	[29
Effect of Mercury (Hg)							Heteropneustes fossilis	0.050	HgCl2	Adult	24 h	Increased germinal vesicle breakdown.	[86][110]
Leuciscus idus	0.1	CdCl2	]	[	Embryonic and larval53]
21 days	Reduced embryonic survival, increased frequency of malformation, and delayed hatching.	[	78	]	[	102]	Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Carassius auratus,	Cyprinus carpioCyprinus carpio, Silurus asotus	Yangtze River	Cd, Cr, Cu, Hg, Pb, Zn	4.990Fish size	Positive and negative relationships were observed between fish size and metal concentration.	[
Diplodus sargus	HgCl	30	]	0.0022	3 years[	12 h54]
HgCl	2	Juvenile	7 days	Increased anxiety, decreased number of optic tectum cells, and altered swim behaviour.	Decreased motility and fertility of sperms, damaged eggs.	[89][
Danio rerio	0.8909	CdCl2	113	Channa striatus, Heteropneustes fossilis	Kali River (India)	Cr, Cd, Pb, Ni	Liver, kidney, gill, muscle, brain	Decreased level of glutathione (GSH), increased oxidative stress.	[31][55]
[	53	]	[	77	]
]
Embryonic and larval	96 hpf	Increased heartbeat rate of larvae and decreased brain size.	[	126	]	[150]	Pimephales promelas	0.720	MeHg	Oncorhynchus mykiss	10.00	HgClAdult	230 days	Decreased levels of dopamine and hyperactivity.	[54]	3 years[	4 h	Reduced motility of sperm.	[90][114]
Leuciscus idus L.	0.1	CdCl2	Embryos and newly hatched larvae	78	2 h	]	Reduced egg swelling, slowed the rate of development (especially body movements), and delayed hatching.	[127][151]	Etroplus maculates, Cirrhinus reba, and Ompok bimaculatus	Bhadra River
Danio rerio	(Karnataka)	Cu, Zn, Cd, Ni, Fe, Pb	Liver, kidney, muscle, gills	Degeneration of the hepatocytes in liver, vacuolar degeneration in the tubular epithelium in kidney.	[32][56]
10	MeHg	Adult	56 days	Mitochondrial dysfunction, and	oxidative phosphorylation.	Danio rerio	0.015	HgCl2	Adult	5 days	Delayed gonadal development, imbalanced sex hormone.[	[91]
Odontesthes bonariensis	0.00025	55	]	CdCl2	[	79]	[115]
Advanced-stage embryos and newly hatched larvae	10 days	Decreased hatching rate and survival of embryo and larvae.	[	103	]	[127]	Oreochromis niloticus, Geophagus brasiliensis, Hoplias malabaricus, Astyanax altiparanae, Rhamdia quelen	Sao Francisco do Sul River (Brazil)	Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Pb	Muscle, liver, and gonads	Metals accumulated in the gonads, liver, and muscle, with chromium levels in the muscle reaching fifty times the maximum limit set by Brazilian legislation.	[33
Danio rerio	0.720	MeHg	]	Adult and embryo	[57]
Danio rerio	0.030	HgCl	30 days	2	AdultDecreased level of dopamine and hyperactivity.	30 days[56]	Decreased testosterone level.[80]	[92][116]
Effect of Mercury (Hg)							Oligosarcus spp., Chyphocharax voga	Sinos River (Brazil)	Al, As, Cd, Co, Cr, Cu, Fe, Mn, Zn, Pb	Clarias gariepinus	0.119Liver	HgCl2Detritivores species accumulated more metals than carnivorous species.	[34][58]
Danio rerio	0.027	HgCl2	5–72 hpf	~3 days	AdultHyperactivity causing mortality.	30 days[57]	Disruptive effect on gamete development.[81]	[93][117]	Salminus franciscanus	Paraopeba River (Brazil)	Cu, Pb, Cd, Zn, Cr, Hg, Fe	Liver, spleen, and muscle
Danio rerio	0.016	Hepatocytes exhibited fat accumulation along with pigmented macrophages in the liver. Fibrosis was observed in the spleen, and contaminated fish showed decreased oocyte diameter and increased follicular atresia.	[	35	][59]
Effect of Lead (Pb)						HgCl	2	Adult	Pseudoplatystoma corruscans
Effect of Lead (Pb)		168 hpf	Decreased hatching rate, increased mortality, increased malformation rate in larvae.	[	92	]	[116]	Paraopeba River (Brazil)	Hg, Cd, Zn, Cr, Pb
		Danio rerio	0.010	Pb(CH3COO)2Liver, muscle, and spleen	The liver and spleen showed higher concentrations of metals compared to the muscle. Additionally, liver fibrosis was observed.	[	0–72 hpf36	3 days][60]
			10 Gene expression changes in 89 genes associated with nervous system development.	[
Heteropneustes fossilis	0.050	(Pb(NO3)2	58	]	Adult	[82]	96 h	Increased germinal vesicle breakdown.
Cyprinus carpio	0.00001	HgCl2	[	86	]	[110]	Embryo	96 h	SOD and GPx reduced up to 85%.	[129][153]	Bryconamericus iheringii	Ilha River (Brazil)	Al, Cd, Mn, Ni, Fe, Pb, Cr, Zn	Blood—micronucleus analysis, gills, and muscle	In rural areas, a higher frequency of micronuclei, nuclear abnormalities, and mucous cells was detected. Conversely, urban areas exhibited a lower condition factor, higher frequencies of lamellar alterations, and higher concentrations of chromium (Cr) and nickel (Ni) in muscle.	[
Danio rerio	0.020	Pb(CH3
Clarias gariepinus	COO)	2	140.0	Pb(C2H3O2)2	Adult37]	0–144 hpf[61]
96 days	Reduced sperm motility.	[	94	]	[	118]	Prochilodus magdalenae, Pimelodus blochii	Magdalena River (Colombia)	Cd, Pb, Ni	Gills, liver, and muscle	Pimelodus Blochii showed a higher accumulation of metals, particularly an increased concentration of cadmium (Cd) in the liver.	[38
Oryzias melastigma	]	[	62	]
Aequidens metae, Astyanax bimaculatus	Ocoa River (Colombia)	Hg, Cd	Blood and liver	There was a decrease in the number of erythrocytes, lymphocytes, and neutrophils, as well as a decrease in hemoglobin concentration and hematocrit percentage.	[39][63]

3. Effect on the Nervous System

Deposition of various metallic trace elements in fish can cause serious damage to the nervous system, affecting behaviour, response to stimuli, and recognition patterns among fish ^[40][64]. Mercury is known to cause numerous disorders, primarily on the biochemical level in the central nervous system of fish. For example, exposure to HgCl caused a significant increase in lipid peroxidation and depletion of total lipids in the brain of catfish (Heteropneustes fossilis) ^[41][65]. Copper-induced morphological abrasions are evident in the sensory organs of fish ^[40][64]. Copper is a vital metal and a fundamental component of many enzymes, but it can be extremely toxic to fish when its concentration exceeds normal levels ^[42][66], especially in freshwater due to the high ionic copper content ^[43][67]. Increased Cu concentration in cellular membranes reduces the antioxidative capacity of lipids, causing lipid peroxidation and severe damage to cellular membranes ^[44][68]. As the formation of free radicals and lipid peroxidation increases, they can cause serious cellular trauma. In Cu-exposed marbled electric ray (Torpedo marmorata), ultrastructural analysis of neurons in the central nervous system showed an increased number of lipofuscin granules erosion of mitochondria ^[45][69] and a reduction in Golgi apparatus as well ^[46][70]. Long-term exposure to Pb can cause neurochemical changes in the brain of walking catfish (Clarias bathrachus). For instance, Pb increases the histamine and serotonin levels while decreasing the gamma-aminobutyric acid (GABA), monoamine oxidase (MAO), and acetylcholinesterase (AChE) contents. Furthermore, cholesterol, brain lipid, and protein contents are also decreased ^[47][71]. The adverse effects of different metals on the nervous system (CNS and peripheral) from various studies are compared (Table 2).

Fish Species	Metal Concentration (mg L−1)
6 days
Decreased axon length and decreased locomotion (speed).
[
59
]
[
Effect of Lead (Pb)
83
]
Danio rerio	0.100	Pb(CH3COO)2	2–120 hpf	~5 days	Altered color preference (adults).	0.050	PbCl2	5 months
Danio rerio	0.100	Pb (C2H3	30 days	O2)2	Decreased gonadal development.	[60	Adult][84]
[	87	]	[	111	]	30 dpf	Distance moved by juvenile zebra fish decreased, and swimming activity alterations in larvae and juvenile fish.	[130][154]	Danio rerio	0.207	Pb(CH3COO)2	2–24 hpf	~2 days	Decreased learning (adults).	[61][85
Danio rerio	]
Effect of Copper (Cu)							0.005	Pb (CH3COO)2	Adult	144 hpf	Delayed hatching, spinal and tail deformity, pericardial edema, and yolk swelling was observed.	[131][155]	Danio rerio	1.730	Pb(CH3COO)2	0–24 hpf	24 h	Decreased Nrxn2a gene expression.	[62][86]
Danio rerio	0.040	CuSO4	Adult	30 days
Danio rerio	99.885	Pb (C2H3O	Damaged structure of gonads, altered steroid hormone level.	2)2	[	Adult95	72 hpf][119]	Deformed CNS, increased levels of Gamma-aminobutyric acid (primary inhibitory neurotransmitter).	[132][	Effect of Copper (Cu)
156	]	Cyprinus carpio	0.60	Cu
Pimephales promelas	0.075	CuCl2	12 months	21 days	Decreased abundance of post-vitellogenic follicles, increased follicular atresia.
Danio rerio	1.6	[	Pb (NO₃)₂	Embryo	120 hpf	96][120]	Spinal malformation.	[133][157]	Juvenile	96 h	Increases in brain ROS production, lipid peroxidation, and protein oxidation.	[63][87]
Capoeta umbla
Daphnia magna	1.041	CuCl2	Adult	21 days	Reduced rate of reproduction.
Pterophyllum scalar	20	[	97	]	[	121]	PbCl2	Embryo	3 days	Tilt, loss of vision or the lack of effect on growth delay.	[134][158]	3.0	CuSO4∙5H2O	112 ± 5 g	96 h	Induce astroglial response accompanied by modulations of NF-kB and PARP-1 expression.	[
Poecilia reticulata	45	64	CuO	Adult
Effect of Copper (Cu)				Larvae	]	[	88]
96 h		Decreased reproduction success.	[	98	]	[122]	Danio rerio	0.100
Poecilia reticulate	CuSO	4	0.026∙5H	CuSO4 5H2O
Gobiocypris rarus	40.00	NaAsO2	3 months	96 days	Accumulation in testis.
Danio rerio	0.016	HgCl2	Adult	2 hpf	T3 and T4 content in larvae increased.	[128][152]		2O	Adult	10 days	Negatively affect the associative learning capabilities.	[65][89]
2.5–3 months	56 days	Gonadosomatic index, offspring production decreased.	[	99	]	[123]
Leuciscus idus	0.100	CuSO4·5H2O	Egg, sperm	21 dpf	Reduced larval survival, growth, and delayed development.	[78][102]	Oreochromis niloticus	120	CuSO4∙5H2O	Adult
Oryzias latipes	96 h	0.0185	CuCl	Loss of balance and exhaustion.	2 H2O	[66][90]
Effect of Arsenic				Embryo, larva	20 dpf	Percentage of deformed larvae significantly increased.	[122][146]	Effect of Arsenic (As)
Poecilia reticulata	1.50	CuSO4·5H2
O	[	Embryo	15 days	Abnormalities in blastodisc to middle-eyed stages of development.	100	][124]	[135][159]	Danio rerio	15	Na2HAsO4	Adult
Daphnia magna	0.049	96 h	NaAsO2	Alteration in behaviour and ectonucleotidase activities.	Adult	[67]	48 h[91]
Stable reproduction rate.	[
Danio rerio	0.018	CuSO4	101	]	[	125]	72 hpf	72 h	Neuromast damage, coagulated egg, increased mortality rate.	[124][148]	Danio rerio	0.050	As2O3	Juvenile	96 h
Gambusia affinis	0.075	NaAsO
Leuciscus idus	0.10	CuSO4	Antagonistic effects on brain.	2	·5H2OJuvenile	[68	30 days][92]
Lower gonadal-somatic indices.	Embryo and larval	[	102	]	[	126]	21 days	Reduced embryonic survival, increased frequency of malformation.	[78][102]	Danio rerio
Leuciscus idus L.	0.500	As+	Larvae, juvenile and adult	96 h
Effect of Zinc (Zn)		Alteration in motor function		(embryo-adult), effects on associative learning.			[69][93]
		0.10	CuSO4	Embryos and newly hatched larvae	2 h	Reduced egg swelling slowed the rate of development (especially body movements) and delayed hatching.	[127][151]	Clarias batrachus	20	As2O3
Odontesthes bonariensis	0.021	ZnSO	Adult	496 h	Increased body discoloration, excessive mucous secretion, loosening of the skin, and complete loss of skin (head region and fins).	7H2O[70][94]
Adult	10 days	Reduced embryo and larval survivability.	[	103
Odontesthes bonariensis	0.00025	]	CuSO4	Advanced-stage embryos and newly hatched larvae	[	10 days127]	Decreased hatching rate and survival of embryo and larvae.	[103][127]	Effect of Zinc (Zn)
Danio rerio	500	Zn	Adult
4 days	Majority of eggs were dead, larger hatching time.	[	104	]
Carassius auratus	1	[	Cu2−	Embryo	24 h post-hatching	Scoliosis and tail curvatures.128]	[136][160]	Anguilla anguilla	0.12	Zn
Effect of Arsenic (As)				Juvenile	28 days	Cholinergic neurotoxicity did not occurr, only liver GST increased significantly.	[71][95]
Clarias magur	300	Zn(CH3COO)2	Mature	60 days	The highest GSI and fecundity.	[105][129]	Leporinus obtusidens	4.57
Oryzias melastigma	0.010	ZnSO4·7H2O	Adult	30	Irregular oocytes, partly adhesion, empty follicle, and increased follicular atresia, loose follicular lining.	[87][111]

5. Effect on Embryonic Development

The influence of water-borne metals can disrupt the embryonic development of spawners. ^[106][130] found elevated levels of Cd, Zn, and Pb in the female gonads of stone loach when exposed to toxic concentrations of these metals. Ellenberger et al. ^[107][131] investigated the levels of Cu in the reproductive organs of European perch (Perca fluviatilis) exposed to Cu-polluted ponds. White suckers in polluted lakes exhibited higher amounts of Cu and Zn in their testicles and female gonads compared to fish in uncontaminated water ^[108][109][132,133]. Common carp exposed to Cu, Cd, and Pb showed decreased egg swellings in a concentration-dependent manner, contrasting with about 40% expansion in egg width observed in the untreated groups ^[110][134]. Copper and Cd accumulation in the gonads of Mozambique tilapia (Orechromis mossambicus) was found to be elevated when fish were kept in metal-polluted water, and blue tilapia (Oreochromis aureus) exposed to Cd and Pb for seven days showed metal accumulation in the testicles and female gonads, particularly Cd levels in the ovaries ^[111][135]. Metal exposure to spawners can result in the deposition of metallic trace elements accumulated in eggs and sperm, severely affecting the survival of fertilized eggs and the embryonic development of fish ^[79][103]. Metals can also influence the physical characteristics of an egg’s outer surface. Benoit and Holcombe ^[112][136] observed that eggs of Zn-exposed fathead minnow (Pimephales promelas) became sticky and more prone to breakage soon after egg laying. Fathead minnow embryos rapidly absorb Hg from surrounding water sources, with concentrations in juveniles increasing to 2.80 µg per gram humid mass after four days of exposure to 25 µg per cubic decimeter of methylmercury ^[113][137]. Chromium was found to accumulate in the outer protective coatings of Cyprinus carpio eggs at pH 6.3 ^[114][138]. Copper can alter selective membrane permeability, disrupting cation trade between the liquid in the yolk membrane and the outside water ^[115][139]. During the early development of fish eggs in a toxic (metallic trace elements) environment, the outer protective coating of the egg blocks most of the metal concentration, but a significant toxic amount still enters the fluid inside the egg membranes, while only a small amount infiltrates the embryo ^[116][140]. Beattie and Pascoe ^[117][141] found that eggs of Atlantic salmon (Salmo salar) exposed to 10 mg per litre of Cd at 22 h old retained 98% of the metal in the outermost membrane. Similarly, the outer membrane of Japanese rice fish (Oryzias latipes) eggs retained 94.4% of Cd ^[118][142]. In Zn-treated Atlantic herring (Clupea harengus) eggs, 30% to 50% of Zn accumulated in the outermost membrane, while the rest accumulated primarily in the yolk sac and in lower quantities in the embryos. However, even a small amount of metals penetrating the egg can significantly influence fish embryonic growth ^[117][119][141,143]. Devlin ^[113][137]

Danio rerio
360.32
NaAsO
2
Adult
120 h
Tail bud deformation in embryo.
[
137
]
[
161
]
Danio rerio
0.5
NaAsO	2	Adult	120 hpf	No effect on mortality and developmental deformations.	[	138][162]
Labeo rohita	198.18	NaAsO2	Adult	120 hpf	Reduced survival rate with abnormal development.	[139][163]
Danio rerio	0.5	NaAsO2	Embryo	14 dpf	Thinning of the retinal pigmented epithelium (RPE) layer in embryos.	[140][164]
Effect of Zinc (Zn)
Odontesthes bonariensis	0.021	ZnSO4 7H2O	Hatchling	10 days	Cumulative embryo survival was significantly reduced.	[103][127]
Pagrus major	2.5	ZnCl2	2 years	10 days	Low hatching rate, high mortality, abnormal pigmentation, hooked tail, spinal deformity, pericardial edema, and visceral hemorrhage.	[141][165]
Melanotaenia fluviatilis	33.3	Zn	Embryo	2 h	Spinal deformities.	[142][166]