In Argentina, from the late 1990s to 2013, the area cultivated with soybeans increased from 9 million hectares to more than 20 million. As a consequence, the application of herbicides and pesticides increased from 127 thousand tons in 1999 to more than 280 thousand in 2013. Taking into account that these potentially toxic substances end up in the lagoons and rivers, it is reasonable to think that the fish that inhabit these bodies of water can be affected.
Indeed, if silverside (Odontesthes bonariensis) is exposed to a glyphosate formulation, its energy metabolism is affected, which indicates that the presence of the herbicide generates stress, according to Renata Menéndez Helman, a researcher who developed her doctorate at INQUIMAE (Institute of Physical Chemistry of Materials, Environment and Energy) of Exactas UBA. Likewise, effects of the active principle were observed on the central nervous system of another native species widely distributed in the region: the little mother (Cnesterodon decemmaculatus).
"The objective was to evaluate sublethal effects, that is, that they did not cause death but rather served as early signs of exposure to pollutants," explains Menéndez Helman. She is in charge of studying energy metabolism, that is to say, the synthesis and degradation reactions of energy molecules inside the cell in different tissues (liver, brain, muscle) of the silverside. These experiments were part of his doctoral thesis, directed by María dos Santos Afonso (researcher at INQUIMAE) and Alfredo Salibián, from the National University of Luján, and were carried out in collaboration with Leandro Miranda from INTECH Chascomús.
The researchers measured the levels of ATP molecules, "which is the energy currency of the cell," according to the researcher. When the body needs energy, ATP delivers phosphates and becomes ADP, but it can continue to release energy until it becomes AMP. "When ATP decreases and other species increase, this is an indicator of the energy state of the cells," says Menéndez Helman. When an organism is exposed to an environmental stressor, it has a higher energy expenditure. Consequently, measuring the balance between adenylates (ATP, AMP, and ADP) can inform whether an organism was under stress.
To evaluate the effects of the herbicide, the fish were placed, for fifteen days, in containers with water that contained 1 ppm (one part per thousand, which is equivalent to one milligram per liter) and 10 ppm of glyphosate, which are concentrations within the range of those reported in water bodies of the Pampean plain.
After exposure to glyphosate, the researcher dissected the fish, removing the liver, brain and muscle. These tissues were then processed in order to obtain the molecules and measure them using a specific technique called HPLC (high efficiency liquid chromatography). According to the researcher, "the extraction of substances is a very delicate process, because they are not very stable, and they degrade easily".
When measuring in the three tissues, both in the fish exposed to glyphosate and in those that functioned as controls, the researchers found significant differences. "In particular, in the liver and in the muscle we find that ATP levels decrease compared to total adenylates, and this shows, in some way, that when exposed to the herbicide the body is undergoing stress", confirms Menéndez Helman. These results were published in Ecotoxicology and Environmental Safety.
Toxic to neurons
In other experiments, Menéndez Helman wanted to find out if glyphosate had an effect on the nervous system of fish. To do this, he analyzed the activity of the enzyme acetylcholinesterase, whose physiological role is to inactivate a neurotransmitter, acetylcholine, at the end of synaptic transmission. The inhibition of this enzyme causes an abnormal accumulation of acetylcholine, with the consequent overstimulation of the synapse.
Precisely, certain insecticides inhibit acetylcholinesterase in some organisms. This effect occurs because organophosphate pesticides, of the phosphate subgroup, bind to this enzyme irreversibly.
"Glyphosate is an organophosphate pesticide, but it does not belong to that subgroup, so it was not expected to have any effect on the enzyme," says Menéndez Helman. In fact, some studies carried out with the glyphosate formulation showed effects, so the researcher wanted to find out what was happening with the active principle, and tried with little mothers of water.
"We did acute exposure tests, for 96 hours, at concentrations from 1 to 35 ppm, and we found that glyphosate, under these conditions, inhibited the enzyme," says Menéndez-Helman. The work was published in the Bulletin of Environmental Contamination and Toxicology.
Acetylcholinesterase inhibitor poisonings can lead to cardiorespiratory collapse and lead to death. In fish, when exposed to these contaminants, problems in balance and effects on locomotion patterns have been observed.
It should be noted, incidentally, that glyphosate along with some pesticides is already part of the list of "possible" or "probable" carcinogens. Indeed, in March of this year, this herbicide, like the insecticides diazinon and malathion, were classified by the International Agency for Research on Cancer (IARC), which depends on the World Health Organization, as “probably carcinogenic to humans ”.