Ferns, amphibians, and fish are groups of nontarget organisms affected by many types of pesticides that end up in the environment. This chapter aims to approach the following themes: the influence of different pesticides on the spore germination process and on the differentiation of their gametophyte; aspects regarding the impact of some pesticides on breathing in fish (physiology and histopathology at the branchial level), as well as a series of effects at the hematological and biochemical levels; and changes of some hematological, biochemical, and structural parameters in amphibians. Species that are not directly targeted by the action of the pesticide in the environment, ferns can be used in their gametophyte stage, young or mature sporophyte in different biotests to evaluate the risk associated with these substances. The biochemical, hemathological, and histopathological changes recorded in both fish and amphibians can be considered biomarkers of pesticide pollution.
Contamination of the natural environment, due to natural factors and human action, has been subject of numerous studies. In many ecosystems, human activity has led to changes of natural biogeochemical cycles, resulting in the accumulation of some substances.
Chemical contaminants are found everywhere in nature, and ecologists are the ones who assess their impact on natural communities of organisms. Among these, pesticides are the most common type of aquatic ecosystem contaminant.
Considering the physical and chemical properties (vapor expansion, volatility, evaporation capacity from water—codistillation) that transform pesticides, their bioaccumulative stability and capacity as well as the publication of ecotoxicological data on the action of different pesticides have become indispensable to continually monitor changes in the environment.
Different research on morphology, physiology, and biochemistry of gametophyte and sporophyte of fern species has shown that pesticides affect these processes, depending on species sensitivity, concentration, and exposure time.
Fish assimilates pesticides through gills or contaminated food. Gills are the main channel of pesticide penetration, which is why any disease at this level will have a great influence on the adaptive changes in the fish body. The effects of pesticides on fish are numerous and varied: they cause mortality both directly and indirectly, by starvation (destroying the organisms they feed on), affects hatching, growth rate, can lead to malformations, affects reproduction rate, modifies enzyme activity, and cause histopathological changes in organs, genetic effects, etc. Although under experimental conditions fish can survive pesticides in different concentrations, under natural conditions they are more vulnerable to disease, predators, no longer competitive, and no longer dealing with stress caused by changing seasonal temperature, reproduction season or temporary starvation.
Amphibians are organisms that populate aquatic ecosystems, being involved in aquatic trophic chains both by eating food and being food for predators. Over the last 20 years, scientists have reported the global decline of amphibian populations. Therefore, in 1996, the International Union for Conservation of Nature registered 156 species of amphibians in the Red List, and recent data show that 1856 species (about 32.5% of all amphibian species) are currently registered.
2. Influence of pesticides on spore germination and gametophyte differentiation in ferns
2.1 Gametophyte of ferns
Ferns and lycophytes (pteridophytes) represent 4% of Terra’s vascular plants , numbering about 11,000 species in 40 families and 300 genres . Different species of pteridophytes are used as medicinal, food, horticultural, and agricultural plants  and in the last decade as organisms in acute  and chronic phytotoxicity tests .
The life cycle of pteridophytes is characterized by an alternation of generations between a well-developed sporophyte and a reduced gametophyte, independent of the sporophyte . The gametophytic generation of ferns begins with the formation of spores (meiospores), from which, a gametophyte or multilcellular prothallium is differentiated by germination. The gametophyte of leptosporangiate ferns is above ground, photosynthetic, short lived, and heart shaped . From the spore to the mature gametophyte stage with gamentagia, the differentiation of the gametophyte involves the passage through the prothallium filament and spatula-shaped prothallium lamella .
The spores germinate on the ground, the first division resulting in a smaller base cell (the initial cell of the rhizoid) and a larger apical cell (the initial cell of the prothallium). The initial cell of the rhizoid elongates and forms a hyaline rhizoid having no chloroplasts. The initial cell of the prothallium will form a multicellular prothallium filament, consisting of a variable number of cells with numerous chloroplasts. One or more rhizoids are formed on the cells at the base of the prothallium filament. Lamella formation (prothallium plate) is initiated by the longitudinal division of the end cell of the prothallium filament. One of the daughter cells divides through a slanted wall resulting in an apical (initial) wedge-shaped cell. The fore side of the prothallium then extends to a width of 3–4 cells and becomes spatula shaped. When the apex of the prothallium is heart shaped, the initial cell is replaced by a flat multicellular meristem. The activity of the lateral zones of this multicellular meristem helps the formation of the prothallium wings. The cells behind the apical meristem divide through a plan parallel to the underlayer, leading to the formation of a multi-layered medial crest. On the underside of the prothallium, there are antheridia, archegonia, and rhizoids. Prothallium differentiates different types of prothallium trichomes in some species.
The processes of spore germination and differentiation of gametophyte in ferns are influenced by a series of endogenous and exogenous factors, with environmental pesticides in the latter category.
2.2 Changes in the fern gametophyte caused by pesticides
Over the time, many researchers have highlighted the advantages of using spores and gametophytes of ferns in their experimental research  and ecotoxicology in recent years [3, 7]. The most advantages include: (1) the formation of a large number of spores/individual, (2) spores can be preserved in the laboratory and can thus be available throughout a year (3) are small and light, requiring reduced storage space, (4) spore germination and gametophyte differentiation may be obtained and monitored
The spore germination process can be completely inhibited or delayed by pesticides in the environment. The glyphosate herbicide in concentrations of 0.48–19.20 mg/l significantly inhibited the macrospores germination process of
A fungicide containing 50% metal copper applied in a concentration of 0.1–0.3 g/100 ml Knop solution significantly inhibited the spore germination process in
A bifenthrin-based insecticide applied in a concentration of 0.01–0.04 ml/100 ml of Knop solution to the two above-mentioned species resulted in a decrease in spore germination rates, and at the highest concentration in both species, the gametophyte was in the form of a three-dimensional cell mass, whereas the gametophyte in the control variant was in cordate prothallium stage .
Acetamiprid-based insecticides (20%) dramatically affect spore germination in
The evaluation of glyphosate herbicide impact on
Also, the fern sporophytes are affected by pesticides. The relative growth rate, the amount of chlorophyll pigments, and the photosynthetic activity of
Saturn herbicide in
The paraquat herbicide applied in the culture medium of
The mature sporophyte of
3. Influence of pesticides on histological and biochemical parameters in fish
As a result of penetration into the aquatic environment, pesticides affect a wide range of nontarget organisms such as invertebrates and fish . Aquatic ecosystems are the “final destination” of pesticides used in agriculture . Early life stages are the most sensitive. Many toxicity tests are focused on the study of hatching and the occurrence of deformities in hatched larvae, decreased mobility or even immobility, lack of coordination in swimming movements; for example, deltamethrin in concentrations >0.005 ppb decreases hatching rate in common carp ; bifenthrin in concentrations of 50–200 ppb determines the lack of coordination in swimming movements, malformations of axial development in
Acute exposure to pesticide resulted in reduced fish populations and increased mortality [21, 22]. Chronic exposure to small amounts of pesticide increased the incidence of disease, stress, and behavioral disorders . Pesticide bioaccumulation causes a major danger—bioaccumulation factor of cypermethrin in fish is 1200× . Acute tests of toxicity are used to evaluate pesticide toxicity and get rapid consistent results at least within certain limits, regarding the concentration-response relationship. These tests allow the determination of LC50 values for different periods of time, NOEC values, providing a very rich database on the toxic effect of many pesticides on different fish species.
Considering the large amount of data in the specialized studies on the effects of pesticides on fish, the data in this paper refer in particular to the pesticide groups on which we have conducted our own experiments. Many authors have established concentrations or lethal doses for different pesticides and species. For example, the toxicological values for chlorpyrifos are between 1.3 μg l−1 (96-h LC50) for
As regards their action, pesticides block a certain metabolic process; the practical way in which this is done is sometimes difficult and in many cases is unknown or only partially clarified. The use of high sensitivity physiological indices is a tendency of recent years in aquatic toxicology. The most used physiological method is to determine pesticide action on fish breathing. Specialists can determine the concentrations in which breathing disorders begin to occur by recording the frequency of breathing movements and determining the oxygen consumption of fish kept in sublethal solutions of toxic substances (in state of rest or activity). The fish alters their energy metabolism in terms of spending a greater amount of energy to alleviate toxic stress , which results in improved oxygen use under hypoxia and anoxia conditions . Determination and monitoring of oxygen consumption in aquatic organisms can be considered a better method to assess a substance toxicity than doing acute toxicological tests, because it has good results even in low concentrations of toxic substance . One of the early symptoms of fish poisoning is breathing difficulties. Therefore, decreases in oxygen consumption in the gills were found by Cebrián et al. , even 24 h after exposure of
Hematological parameters reflect fish state faster than determining other parameters because they change extremely rapidly under modified environmental conditions. For this reason, they are widely used to describe the state of fish health. Fish-nucleated red cells can be an ideal model to understand the harmful action of pesticides in different cell compartments; mitochondria and the nucleus of fish red blood cells are sensors in the programmed cell death mechanism .
Svobodova et al. reported significantly lower values of erythrocyte and hemoglobin in carp after acute exposure to deltamethrin (as a result of changes in hematopoiesis process), while the number of leukocytes did not change significantly . Research carried out by Sopinska and Guz on permethrin poisoned carp revealed a decrease in the number of white blood cells, granulocytes in particular . Anemia resulting from carp exposure to cypermethrin was also reported by Doruncu and Girgin .
Jayaprakash and Shettu evaluate the changes in some hematological parameters of the freshwater fish
Decrease in the number of erythrocytes in carp poisoned with pyrethroid insecticides (permethrin and cypermethrin), due to dysfunction of hematopoiesis [37, 39]. Exposure to Talstar EC 10 in a concentration of 57.5 μg l−1 had no effect on the number of erytrocytes in common carp (
Galloway and Handy studied the effect of organophosphorus pesticides (parathion, chlorpyrifos, malathion, and diazinon) on the immune system in invertebrates, fish, and other vertebrates . Studies on fish have demonstrated an immunosuppressive effect to the action of these pesticides .
Physiological stress indicators, such as plasma cortisol and glucose level are commonly used to assess whether fish is stressed and whether the hypothalamo-pituitary-interrenal system works properly . The intensive use of lactic acid in gill oxidation during stress caused by exposure to pesticides is mentioned in the literature  and is due to the increase in their energy demand  which is only covered by exogenous glucose. Glycogen mobilization is linked to the increase of energy demand; during pesticide-induced stress, a large amount of glycogen can be synthesized and stored in the muscles  which may later be in the form of glucose into blood tissues .
Due to the fact that fish live in a carbohydrate-deficient but protein-rich environment, they can use lipids and proteins as sources of energy more efficiently than carbohydrates . Anaerobic glycolysis, recycling, and use of lactic acid preserve the energy potential of fish, thus ensuring better adaptation of fish to the polluted environment. Increases in the amount of energy (glucose) associated with lactate production have been reported in fish poisoned with pyrethroids .
High levels of lactic acid and strongly stimulated LDH activity in various tissues suggest the great importance of anaerobic glycolysis in adapting fish to stress caused by pyrethroid pesticides . Velisek et al. examined the biochemical profile of carp (
Velisek et al. showed an increase in blood glucose in trout and prussian carp as a result of metabolic stress induced by the action of Talstar 10 EC insecticide . Jee et al. recorded an increase of blood glucose level in
Acetylcholinesterase is a highly used biomarker in aquatic ecotoxicology research , sensitive to low concentrations of organophosphates. Determination of acetylcholinesterase activity in erythrocytes is used to assess the degree of intoxication with acetylcholinesterase inhibitors. Recovery of acetylcholinesterase activity affected by exposure to organophosphorus pesticides can be carried out by dephosphorilation or AchE synthesis, which is a very slow process in fish; the disappearance of the inhibitory effect on acetylcholinesterase in
Costin et al. found a decrease of the specific activity of catalase, glutathione peroxidase, and glutathione reductase in prussian carps exposed to a sublethal concentration of deltamethrin (2 μg/l water), while glutathione-S-transferase activity increased . Examination of the biochemical profile after 96 h of carp exposure to bifenthrin in a concentration of 57.5 μg l−1 resulted in significant increases in glucose, ammonia, aspartate aminotransferase, creatine kinase, and monocytes .
Bifenthrin inhibits the production of ATPase , which explains the stronger effect of bifenthrin on aquatic organisms compared to terrestrial ones (maintaining the critical concentration of ions against the concentration gradient, in the much diluted aquatic environment, requires intensive ionic transport processes, the necessary energy being supplied by ATPase; the decrease in production of this enzyme leads to the death of organisms). Li et al., following the study of propiconazole action on
Fish gills are the main place of ion exchange with the environment and, at the same time, the main channel of pesticide penetration. They are in constant contact with water, and any change in its composition may affect the gills’ permeability and their osmoregulatory functions. Cengiz found histopathological changes in the carp gills following acute exposure to deltamethrin in concentrations of 0.029 and 0.041 mg/l−1 (exfoliation, necrosis, edema, hyperplasia, fusion of secondary lamellae, etc.) ; similar changes caused by deltamethrin action were identified in
Fusion of secondary lamellae as a result of exposure to pesticides appears to have a protective role in diminishing the affected gill surface; this response slows down the penetration of toxic and may result in fish choking [64, 65]. The toxic effect of pyrethroids on fish increases with increasing liposolubility ; because of their strong lipophilic character, the pyrethroids are well absorbed by the gills, even in low-pesticide waters, thus decreasing the availability of oxygen to the tissues of the internal organs .
Histological changes due to exposure to pesticides also occur in other organs. Melo et al. observed hepatic tissue alterations in
Much of the research on toxicological aspects of pesticide poisoning in fish refers to oxidative stress, causing the activity of antioxidant enzymes, proposed as biomarkers of pesticide intoxications [71, 72, 73]. It has been demonstrated that pyrethroid metabolism generates oxygen reactive species in poisoned fish . The occurrence of oxidative stress in different fish tissues as a result of exposure to deltamethrin is supposed to be the main cause of product toxicity . Chlorpyrifos toxicity is also manifested by the induction of oxidative stress . It seems that oxidative stress contributes to the development and severity of syndromes in acute intoxication with these insecticides . Mancozeb stimulated oxidative stress, while the amount of antioxidant serum enzymes decreased significantly; Fabra et al. demonstrated that this fungicide caused significant biochemical changes in cell membranes .
Exposure to pesticides may bring about behavioral changes such as: hyperactivity, loss of balance, and impossibility of maintaining the normal position in trout exposed to cypermethrin . Symptoms of fish exposed to pyrethroid pesticides are: swimming near water surface, hyperactivity, loss of balance, increase of branchial mucus secretion, etc. Levine et al. found a decrease of swimming intensity in
Toxicity of different pesticides on fish and other aquatic organisms can be influenced by various factors such as: pH, temperature, dissolved oxygen, water hardness, etc. Mauck et al. found that bifenthrin is more toxic at low temperatures, and its toxicity is only slightly influenced by water hardness . Macek et al. recorded 96-h LC50 values of 7.1, 15, and 51 μg l−1 in
Although the data on histochemical, biochemical, hematological, and behavioral changes in poisoned fish are quite numerous, they are little related to
As a contribution to the study of pesticide effect on fish, we will briefly present some of our results for these species exposed to the action of six pesticides—3 insecticides (Talstar One—in concentrations of 0.05, 0.1, 0.2 and 0.4 μl bifenthrin/l water, Reldan 40 EC—0.4, 0.8, 1.6, and 3.2 mg chlorpyrifos/l water, and Actara 25 WG—0.016, 0.032, 0.064 and 0.128 mg thiamethoxam/water) and 3 fungicides (Tilt 250 EC—1.06, 2.12, 4.24, and 3.2 mg chlorpyrifos/l water, Dithane M-45—2, 4, 8, and 16 mg mancozeb/l water, and Tiradin 70 PUS—0.007, 0.014, 0.028, and 0.56 mg thiram/l water).
The analyzed pesticides reduce energy metabolism and breathing rhythm in crucian carp, bass, and bleak [83, 84, 85, 86, 87, 88, 89, 90, 91, 92]. In many of the variants, the immediate response to the toxic action is a breathing rate stimulation of variable duration, in inverse ratio to the pesticide concentration, with high decrease in oxygen consumption, followed by the “stabilization phase” to the new conditions, with small fluctuations of the mentioned indices around an average value [85, 86, 87, 88, 89, 90, 91, 92]. The advantage of determining energy metabolism and breathing rate is the rapidity of this response to the action of the stressor (pesticide) on the one hand and their noninvasive character on the other.
The number of erythrocytes increases after exposure to Reldan 40 EC, Tilt 250 EC, and Tiradin 70 PUS [86, 87, 88, 89, 92]; decreases of this hematologic parameter are recorded under the action of Actara 25 WG and Talstar One [84, 90, 92]. Blood glucose levels in fish increase for 2 weeks under the action of Talstar One, Actara 25 WG, and Tilt 250 EC [84, 85, 86, 90, 92]. Dithane M-45 fungicide and Reldan 40 EC insecticide have a hypoglycemic effect [83, 86, 92]. Tiradin does not change blood glucose level in crucian carp and bleak intoxicated with a concentration of 0.02 ml/l for 2 weeks [91, 92].
Behavioral changes occur in crucian carp and perch exposed to pesticides: hyperactivity in the first 24–48 h, disordered movements, frequent rising at the surface of water in the first hours after exposure to toxic, and apathy under the action of insecticides and fungicides [85, 86, 87, 88, 89, 90, 91, 92]. Increases in mucus production occur especially in crucian carp under the action of fungicides [83, 87, 88, 89, 91, 92].
There are different results as regards the sensitivity of the species: the prussian carp was more sensitive under the action of Reldan 40 EC and Actara 25 WG [86, 92] as well as Tiradin 70 PUS and Tilt 250 EC fungicides [87, 88, 91, 92]; the bleak was more sensitive to Talstar One and Dithane M-45 . In some experimental variants, there were no significant differences in the reactivity of the species under the action of the six pesticides.
Among the physiological parameters, blood glucose values and the number of red blood cells give a clear signal of fish stress, which is why we recommend using these parameters as biomarkers for pesticide-induced toxic stress. The study of oxygen consumption and breathing rate combined with the number of erythrocytes and blood glucose as well as the main behavioral changes allow the formation of a symptomatological picture of fish poisoned with pesticides.
4. Influence of pesticides on histological and biochemical parameters in amphibians
The impact of pollutants on humans in their environment is particularly complex and explains the lack of action or late and often confusing reactions regarding the protection measures. The degree of exposure depends on the simultaneous presence of some essential factors (nature and concentration of pollutants) and modifiers that ultimately influence the absorbed quantity.
Amphibians are some of the most representative species of vertebrates in aquatic and agricultural ecosystems, because they are the natural enemies of many pests of crop plants. Due to their high sensitivity to the changes occurring in their natural environment and because their larval development occurs exclusively in the aquatic environment, amphibians have been considered bioindicators for the aquatic and agricultural ecosystems [93, 94, 95, 96] and have been used as test specimens to study the action of different chemicals in these ecosystems.
Bridges  conducted numerous studies on the effects of long-term exposure to pesticides. The presence of contaminants in the environment may alter the predator-prey interaction among aquatic species by changing the predator or prey levels of activity or behavioral change of the predator. The effect of predator-prey meeting may be dependent on the fact that both species are exposed simultaneously to a contaminant/pollutant or exposure occurs only to one of the two species.
Due to the short period of exposure to nonpersistent pollutants, it is important to examine the long-term effects of short-term exposure on amphibians and the sensitivities at a certain stage in their development cycle. Any delay in metamorphosis or any decrease in size during metamorphosis may have an impact on the evolution of the amphibian populations, leading to its decline or local extinction.
Sampath et al.  studied the toxic effect of two pesticides (carbaryl and methyl-parathion) on the excretory system of
Tomizawa and Casida [100, 101] demonstrated that neonicotinoid insecticides acted selectively on the nicotine-acetylcholine receptor in insects and mammals, making them the safest insecticides (as a mode of action). They described the mechanism of selective toxicity of neonicotinoid insecticides.
Gendron et al.  published the conclusions of a study on the effects of the leopard frogs’ exposure to a mixture of pesticides. The hypothesis shows that the exposure of
Greulich and Pflugmacher  suggested a number of factors for the recent decrease in amphibian populations, one of them being exposure to pesticides. Specialists studied the absorption and effects of the pyrethroid cypermethrin insecticide in relevant concentrations from the environment for two different amphibian species,
Some studies show that glyphosate has a harmful effect on the environment, especially on amphibians. Howe et al.  studied the effect of glyphosate on four North American amphibian species:
Chen et al.  conducted studies on the effect of glyphosate on fauna in wetlands, having the zooplankton,
Relya  studied how four commercial compounds (diazinon, carbaryl, malathion, and glyphosate) affected the survival and larval growth rate of five species of amphibians (
The toxicity of pesticides in general and their genotoxicity on nontarget organisms in particular was of special interest to researchers. Shaolong et al.  studied the toxicity and genotoxicity of two pesticides (imidacloprid and RH-5849), used in China since 1992, for two species of amphibians. RH-5849 insecticide did not prove to be toxic to tadpoles even if they were kept for 96 h in a saturated solution. Two techniques were used to study the genotoxicity of the two insecticides: micronucleus test and single cell gel electrophoresis. These tests showed significant changes in the DNA of amphibian erythrocytes.
Relya  studied six North American species of amphibian larvae (
The values range from 0.55 to 2.52 mg of active ingredient (IA)/L, considerably lower than those used in previous studies. Stress increased by predators made Roundup two times more toxic to one of the six species (
Relyea et al.  also studied the effect of pesticides (glyphosate and malation) on the natural environment of amphibians in the presence of zooplankton and phytoplankton (algae). Three species of amphibian larvae (
Toxicological investigations on amphibian larvae have mainly focused on the effects of heavy metals and pesticides on their growth, development, and behaviour, and only a few data refer to the bioaccumulation of toxins under natural conditions. Hofer et al.  studied the accumulation of inorganic (Pb and Cd) and organic (organochlorine and polyaromatic hydrocarbon pesticides) toxic in the body of
Seifert and Stollberg  investigated the interaction of the neonicotinoid imidacloprid insecticide with the nicotine-acetylcholine receptor (nAcChR) in the frog embryonic muscle cells. The response of the muscle cell to the action of acetylcholine, nicotine, and imidacloprid was recorded as a contraction. The contractions of acetylcholine or nicotine are inhibited by α-bungarotoxin. Imidacloprid does not lead to the contraction of the muscle cell, but it can attenuate the contractions produced by acetylcholine or nicotine. They have found that imidacloprid is an antagonist of nAcChR receptor in the muscle cell and an agonist in its toxic action on insect nerve receptors.
Honda et al.  studied how to activate or inactivate 11 nicotinic insecticides in a two-step system of metabolic coupling and receptor binding. The authors incubated neonicotinoid insecticides with CYP3A4 and NADPH or AOX on a cosubstrate of N-methyl-nicotinamide for metabolism. They used ketoconazole or menadione for inhibition of subsequent conversions. They also used
Neurotoxicity of pesticides has been studied by many researchers on the sciatic nerve of
Casco et al.  demonstrated high toxicity of cypermethrin in
In 2006, Relya  published an article on the effects of pesticides, pH, and stress due to predation on amphibians. It was shown that pH and stress due to predation and a single application of the insecticide (carbaryl) affected the survival and growth of
The application of auxin herbicides of 2,4-dichlorophenoxyacetic acid (2,4-D) type in deciduous forests is mainly used in reafforested areas. The absorption of this herbicide in the target plants is reduced due to its low solubility in water. Because of this, the acid is esterified to increase its liposolubility and to diffuse into the plant-bearing vessels. But the esterification of auxin herbicides increases their absorption in other organisms, such as amphibians .
Numerous researches have shown that the gelatinous shell of amphibian embryos has a protective role against various environmental pollutants. Edginton et al.  studied the rate of absorption, metabolism, and excretion of butoxyethyl ester for 2,4-dichlorophenoxyacetic acid (2,4-D BEE) in
Vonesh and Buck  have shown that pesticides influence the process of laying eggs in amphibians. Four experimental batches were used to examine the effect of the pesticide marketed under the name of Sevin® and the active substance, carbaryl, on the method of laying eggs in
Gurushankara et al.  investigated the effect of malathion in various concentrations on the survival, growth, and food consumption in
Bioaccumulation of malathion and its impact on
The amount of xenobiotic substances was determined by gas chromatography in liver, muscle, skin, and bones. Malathion was higher in muscle, skin, and bones after 1 day of exposure and in viscera, after 2 days of exposure. To determine the biological impact of malathion on
Aquatic organisms are exposed to repeated pesticide applications over time. Repeated and long-term (79 days) administration of low concentrations of malathion (10–250 μg/l)—the most widely used insecticide, affects the entire aquatic community of zooplankton, phytoplankton, periphyton, and amphibian larvae . This caused the decline of zooplankton followed by an increase in the biomass of the phytoplankton and the decline of periphyton after repeated treatments. The decline of the periphyton has had little influence on the development of
Toxicity caused by endosulfan (an organochlorine chemical compound used as an insecticide in vegetable, fruit, cotton, and coffee) was studied on
Similar changes have also been reported in intoxication of larvae and
Relyea and Jones  set LC50 after 96 h for glyphosate (Roundup) for various North American amphibian species. There were used nine species of larval anura Groser 25 belonging to
Endosulfan insecticide appears to be highly toxic in low doses for amphibian populations and also exhibits long-term toxic effects. Toxicological studies conducted by Jones et al.  established LC50 in 4 days (LC504-d) for endosulfan insecticide on tadpoles of nine amphibian species belonging to three families (
Atrazine is a widely used herbicide for weed control in corn crops. Its residues could be detected in the soil 1 year after application. Numerous studies have tracked the effect of atrazine on embryonic development of amphibians in the environment and reported its ability to induce an enzyme—aromatase, which appears to convert testosterone to estrogen [126, 127, 128, 129, 130, 131, 132].
Some studies followed the effect of atrazine on juvenile and adult amphibians. Storrs et al.  followed the effect of atrazine administered directly on the soil on the behaviour of
Small quantities of herbicides used in agriculture often affect the surface of water and can become a stress factor for aquatic organisms. Williams and Semlitsch  studied the effect of 4 herbicides (the active substance atrazine, S-metolachlor and glyphosate) on the amphibian larvae of 3 species:
Chronic exposure to atrazine and S-metolachlor has no side effects on survival, metamorphosis, or larval period in the organisms. These results show the importance of adjuvants in the preparation of pesticides and their different effects on organisms even in the case of similar products.
Budischak et al.  followed how some parasites of amphibians can influence the toxicity of some pesticides. They used two batches of
The influence of six pesticides (Reldan 40EC, Actara 25WG, Tilt 250EC, Champion 50WG, Fusilade Forte, and Dual Gold 960EC) on physiological indices of
The fact that some species of amphibians survive in agroecosystems is due to their special plasticity. Thus, irrigation canals represent new habitats that amphibians colonize, and in the absence of predators, which are less mobile (fish), they become metapopulations of altered landscapes [137, 138, 139].
As nontarget species for pesticides in the environment, ferns can be used in their gametophyte stage, young or mature sporophyte in different biotests to evaluate the risk associated with these substances. The biochemical, hemathological, and histopathological changes recorded in both fish and amphibians can be considered biomarkers of pesticide pollution.
This work was supported by a grant of the Romanian Ministery of Research and Innovation, CCCDI-UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0332”/Project 3 “Development of plant extracts and innovative phytosynthesized nanostructured mixtures with phytotherapeutical applications aimed at eliminating the biocenotic stress in horticultural crops”, within PNCDI III.