Fish gills were chosen for this investigation because they were in direct contact with the aquatic environment and, therefore, could be good indicators of water quality.
Epithelial wrinkling and detachment, hyperplasia and alterations in the shape of microridges are common when the water quality changes. Mallatt (1985) analyzed 130 publications in which morphological changes of the respiratory epithelium due to the action of chemicals were registered. Lesions observed less than 10 times were not considered in his statistical analysis, and the wrinkling of the respiratory epithelium is among these lesions. However, in Metynnis roosevelti, this change appeared in the first hour after contamination, at both concentrations. This indicate probably an initial loss of the osmotic balance in the cells. The regression of the wrinkling afterwards could mean that the epithelium was still able to make some adjustments after an initial aggression.
Mueller et al. (1991), studying the effects of the aluminium on the branchial epithelium, proposed two phases to explain the morphological changes in the respiratory system of fishes. An initial phase that could last from some hours to days, which was characterized by the efflux of ions, the inhibition of the active reception of ions and interference in the gaseous exchanges. In a subsequent phase, there would be a return to the normal morpho-physiological characteristics.
Goss et al. (1992) propose that the squamous cells of the respiratory epithelium of freshwater fish took part in ionic regulation mechanisms. In fact, the ionic change Na+/H+, and the enzymes involved in this process were in the membrane of these cells. It was possible that a sublethal dose of methyl parathion changed the acid-base balance and the absorption of Na+ in this freshwater fish, M. roosevelti, since the wrinkling reached the cell membrane.
It should also be taken into consideration that in the case of methyl parathion, one could observe two subsequent effects. The first effect was a result of the direct contact of the OP with the epithelial cells, including those of the respiratory epithelium, through which it entered the organism. Later, the organophosphorous compound, after having been metabolized in the liver, was distributed to the tissues through the blood flow in the form of methyl paraoxon.
Epithelial detachment in M. roosevelti was even observed in the sublethal concentration of OP. In Mallatt's review (1985), this was the most common branchial change that occurred in freshwater fishes rather than in seawater fish. This could be due to the fact that the first ones were hyperosmotic in relation to the environment, facilitating the influx of water through the epithelium lesion, increasing the volume in the oedema, and consequently the detachment. Abel (1976) repeated this process as being a decrease of the superficial area of the gills what was necessary to maintain the internal osmotic surrouding regarding the functional loss of the epithelial cells. Nowak (1992) found that the respiratory epithelium detachment resulted in the increase of the diffusion distance, affecting the gaseous exchanges. This phenomenon has also been described in another type of environmental contamination such as in acid waters (Kawall, 1993), heavy metals (Oliveira Ribeiro et al., 1994) and salinity (Luvizotto, 1994; Fanta et al., 1995).
In M. roosevelti, the hyperplasia was subsequent to the process of epithelial detachment, which suggested the former was a consequence of the latter. The hyperplasia was characterized by cellular proliferation in the interlamellar region of the repiratory lamellae, decreasing the surface area and making gaseous exchanges more difficult. Magor (1988) repeated that these cells stemed from the epithelium of the filament in the interlamellar space and could act as a barrier impeding the diffusion of harmful substances to the blood of the fish. Khan and Kiceniuk (1984), studying the histopathological effects of raw oil on fishes, found that hyperplasia, together with the mucus secretion, protected the gills against future damages caused by intoxicants. This alteration was extremely intense after contamination with heavy metals (Oliveira Ribeiro et al., 1994).
The branchial responses could be good to hinder the entrance of intoxicants in the blood flow, but they have the undesirable effect of decreasing the oxigenation of tissues, generating a barrier to gaseous exchanges (Mallatt, 1985). This breathing difficulty was confirmed by the behavioural response of M. roosevelti, because when exposed to the 7ppm lethal dose, the animals had strong breathing difficulties. It is possible, therefore, to trace a parallel among the hyperplasia and the behavioural effects of the intoxicated animals.
Nowak (1992) did not detect any significant difference in the diameter of the blood spaces surrounded by the pillar cells in fish exposed to the endosulfan organochloride. However, some studies report damages in the pillar cells caused by pollutants (Schimid and Mann, 1961; Bettex-Galland and Hughes, 1973; Abel, 1976). Such cellular damages are often associated to high doses in which the animals are close to death (Mallatt, 1985).
However, in M. roosevelti a different result was obtained after contamination with OP. At the first hour of exposition to the sublethal concentration, the whole structure of the respiratory lamella of M. roosevelti, including the shape of pillar cells, was altered. This collapse of the pillar cells - progressive along the 96 hours of the experiment - was followed by a loss of shape of the erytrocytes, what can indicate osmotic and ionic alterations.
The action of methyl parathion causes enzymatic inhibition, blocking the acetylcholinesterasis and other enzymes. It is not the methyl parathion that acts but the methyl paraoxon, that results from enzymatic oxidation mainly in the hepatocytes. The cholinesterase inactivation by methyl paraoxon is taken, chemically, as a phosphorilation, leaving the endogenous acetylcholine free (Barberá, 1976). Bettex-Galland and Hughes (1973), suggest that the contraction of pillar cells is facilitated by the acetylcholine in the blood.
Morphologic alterations of the pillar cells can have several secondary consequences. These cells control the blood pressure of the fish, and changes in the blood pressure and flow can affect the number of irrigated lamellae, the distribution of the blood within the lamellae, the permeability of the branchial epithelium and, as a consequence, the osmorregulatory and gaseous exchange mechanisms (Randall, 1982), causing several physiological disorders.
The deformation of erytrocytes was obvious, and has possibly reduced the capacity of oxygen transport, consequently causing a certain level of hypoxia. Consequently, the fish tries to compensate the lower levels of oxygen in its tissue by an increase of the respiratory frequency, as was observed in M. roosevelti. This is observed not only after intoxication with chemicals, but always when there is a change in the respiratory lamellae, caused by any environmental changes (Fanta-Feofiloff et al., 1986; Fanta et al., 1989; 1995).
The microridges are structures commonly found on the branchial surface of the fish (Eiras-Stofella et al., 2001). Their location and functions, however, generate discussions. Datta Munshi and Hughes (1986) studied the breathing surface of the gills of Anabas testudineus and found microridges in the epithelial cells of the respiratory lamella. Hossler et al. (1986) showed a branchial structure similar to the one found in Metynnis roosevelti, through ultrastructural studies. The microridges, called microfolds, are present in the arch and branchial filaments of Morone saxatilis, but they are absent in the respiratory lamellae that are covered by squamous cells with smooth surface. The authors remind that the presence of microridges in the epithelium of the respiratory lamellae has been related to the mucus anchor in order to protect this epithelium against microbial or solid agents in suspension. But they suggest that the absence of such structures in this epithelium can be good to reduce the thickness of the blood/water barrier, what facilitates the gaseous exchanges.
Hughes (1979) proposes different functions for the microridges that were found on the surface of the respiratory lamellae of trout, Salmo gairdneri, such as the increase of breathing surface, the possibility of gaseous exchanges and the production of microturbulence that would facilitate the reception of O2. A possible function, however, would be the occupation of the space among the microridges by mucus, therefore promoting a flat surface for the flow of water on top of the epithelium of the gills.
The microridges in M. roosevelti are concentrically arrangement in the gill arches and branchial filaments. However, under the action of the sublethal concentration of 1ppm OP, a progressive alteration was observed in the shape of the microridges, that disappeared in some cells after 96h of exposition.
Mallatt et al. (1985) showed similar results when they investigated the way of action of a specific poison in the branchial system of lamprey larvae. The authors describe both rare microridges with varied length and forms and alterations in the contact border among underlying cells, that became less evident, after treatment with sublethal doses. The alarming fact in this result is that the substance that was used by the authors was a poison that is specific against the tested organism. If these alterations on the surface of the gills reflect the way of toxic action of the poison, one can assume that 1ppm methyl parathion, even being a sublethal dose, was extremely toxic for M. roosevelti.
Sprage (1973) defines bioassay as a test in which the quality or intensity of a substance is determined by the reaction of an organism exposed to this substance. Some questions, such as "Is that substance toxic?" or still "How toxic is that substance?", can be answered by toxicity tests. However, the question "How do these substances exercise their toxic effects?" cannot be answered by these tests because they are only based on the mortality caused by these substances. Using some tools, such as the cellular biology, the fragility of the use of the single concentration in that 50% of the individuals died (CL 50) as a safe limit for the population, can be noticed. Although the OP methyl parathion has not caused death of any fish after 96h, it generated morphologic alterations in the respiratory system of these animals, that certainly have ecological consequences.
In the present study, the fish were exposed to controlled conditions in laboratory bioassay. However, it is possible to extrapolate the results for natural environmental conditions. Firstly, because some morphologic alterations were registered after a short period of exposition (1h), what can be considered enough time to reach confined fish to some restrict places, for example, small lakes. In addition, the fish did not exhibit escape reaction when exposed to the sublethal concentration. Secondly, the used doses (1ppm and 7ppm) are insignificant if compared to the doses recommended for crops. In order to reduce the plague in the culture of tomato, for example, 110ml is recommended for each 100 liters of water, or 1,100ppm of the organophosphorous compound! Of course, this volume will be quite diluted if it contaminates a waterbody. Even so, reflection on the difference among the concentrations needs to be addressed.
A third point refers to how to use the product and the final label on the packagings. The product can be applied by costal vehicles, bulldozers or airplanes. The manual of the product alerts that, when using airplanes, the wind should be calm or weaker than 8 km/h, in order to avoid the contamination of close atmosphere. It also warns the consumers not to wash the packagings or the tools either in rivers or other waterbodies. Most of our farmers are not preparared to understand these warnings, therefore ignoring the cares with their own health (Bull and Hathaway, 1986).
The generalized use of Brachydanio rerio in toxicity tests all over the world, but also in Brazil, is also debatable, once this species does not belong to the Brazilian ichthyofauna. This problem seems already solved in developed countries. In the manual of U.S. Environmental Protection Agency, we found out that some data regarding the effects of the methyl parathion on aquatic organisms were not used, because such studies were carried out with non-resident species in the USA (EPA, 1986). Sprage (1973) is more demanding when not recommending the use of Pimephales promelas in laboratories of a certain area of the USA, because this species is non-native of this area.
The great difference of sensitivity to methyl parathion, by Brachydanio rerio and Metynnis roosevelti, reinforces the need of toxicity tests with native species for the establishment of "safe concentrations". On the other hand our results show also that further studies on the action of pesticides should be developed, since, even in "safe doses", they can be harmful to vital organs. This, on the other hand, will impair the state of health of individuals or a whole population in a certain region, affecting potentially the whole ecosystem.
The authors are grateful to CAPES/Brazil for funds, supporting the Master Course in Morphology, Departament of Cellular Biology, Universidade Federal do Paraná, and a scholarship to Marcelo Rubens Machado during the course of his study.