Pesticide soil contamination mainly affects earthworm male reproductive parameters
Eduardo Bustos-Obregn, Roger Iziga Goicochea
Biology of Reproduction Unit. Program of Morphology, ICBM Faculty of Medicine, University of Chile, Santiago, Chile
Asian J Androl 2002 Sep; 4: 195-199
Keywords: earthworm; reproduction; soil contamination; organophosphoric agropesticide
Aim: To explore the effect of exposure to commercial Parathion®(Pc) on the reproductive parameters (sperm and cocoon production and genotoxicity on male germ cells), the survival, the body weight and the gross anatomical changes in Eisenia foetida. Methods: Three doses of Pc (1478, 739 and 444 mg/kg of soil) and three time intervals of exposure (5, 15 and 30 days) were used. Results: All treated animals were affected. An acute genotoxic effect, revealed by DNA fragmentation (comet assay), was seen by 5 days. Alterations in reproductive parameters were conspicuous in regard to the number of sperm, cocoons and worms born, and the histological observation of the gonads and seminal receptacles. In addition, the body weight and survival rate were decreased. Neuromuscular function was also affected. Conclusion: Earthworms are suitable bioindicators of chemical contamination of the soil, their advantage being their easy and economical handling.
Earthworms have been used as biomarkers for assessing chemical environmental pollution . In the agricultural areas of many countries worldwide there is an increasing concern about soil contamination due to the widespread use of agropesticides . Among them, organophosphoric compounds have been indicated to cause reproductive damage. Commercial Parathion®(Pc), a highly toxic organophosphorate, is used in Latin America, and its application endangers the environment and the public health [3, 4].
Due to the low cost, easy handling and ethical issues related to mammalian assays, many other animal species and even bacteria have been used for these studies [5-10].
Because of their living habits, earthworms are exposed to chemicals present in their terrestrial environment [11-13]. Therefore, it was believed to be of interest to analyze the effect of an organophosphoric agropesti-cide Pc, on the reproductive and general parameters of the earthworm Eisenia foetida as indicators of soil chemical pollution.
2 Materials and methods
2.1 Animals and LC50
Sexually mature earthworms (Eisenia foetida) kept under standard conditions were used . The LC50 for Pc (0,0-diethyl-0-p-nitro phenyl monophoshate 80 %, Anasac, Chile, imported from Cheminova Co, Denmark) was determined by rearing groups of 30 worms in inert plastic receptacles with 3 kg of soil of 30 % humidity, to which aqueous solutions of Pc at 1600, 2000, 2200 and 2400 mg/kg of soil were added. The final humidity in the soil was 40 % (pH 6.5; 20 % organic matter and particle size under 2 mm). Controls were added distilled water. Groups of 30 worms were treated in duplicated assays (n= 60, per dose). There is a wide range for the time of determining the LC50 in earthworms, ranging from 7 days to 6 weeks [11, 14, 15]. Nine days was the shortest time in which an acute and important lethal effect was appreciated at high concentrations of Pc. Therefore the percentage of dead and alive worms was determined at this time to assay for the LC50, which was calculated to be 2217 mg Pc/kg of soil.
The subsequent experiments were run using 0, 2/3; 1/3 and 1/5 the LC50 (equivalent to 0, 1478, 739 and 444 mg Pc/kg soil, respectively). Groups of 40 worms were treated in duplicated assays (n=80, per dose). A test duration of 3 weeks seems to be sufficient to assess effects on reproductive parameters and growth, however, tests periods may vary from 7 to 30 days [11, 14-17]. Observations were done by 5, 15 and 30 days to evaluate the evolution of sub lethal effects at short, middle and long time post-exposure to Pc, including assessment of mean weight of the worms, gross anatomical and behavioral changes, and analysis of seminal vesicles, testes, seminal receptacles and ducts. These organs were dissected out, macerated in 1 ml of PBS (pH 7.4) and used to count the sperm. The sperm number corrected by the worm weight (in mg) is taken to represent the sperm production by each worm.
The anterior segments of earthworm, between prostomium and clitellum which contains testis, seminal vesicles and receptacles, fixed in Bouin's solution were processed for routine light microscopy. Sections (5 mm) stained with H.E. were digitalized using a video system and the Osiris program for image analysis.
Capsules (cocoons) were recovered from control and experimental worms at the time intervals scheduled and were reared in Petri dishes with humid, non-contaminated soil for 5 weeks to observe the number of worms that emerge from the cocoons.
An assay of genotoxicity of Pc in the male germ cells was done using the comet assay [18-20] at 5 and 15 days after exposure of the worms to Pc.
2.3 Statistical Analysis
Data analysis was carried out with Statistical Software Instat. Sperm count, histometric data of seminal receptacles, number of surviving animals, and weight changes were evaluated using analysis of variance and Dunnett's multiple range test. Cocoons production, genotoxicity on male germ cells, and gross anatomical changes were evaluated using chi-square test.
Table 1 refers to the percentage of survival of the worms under the doses and at the intervals studied. It can be seen that at the low Pc dose, survival is compromised only at long time after exposure (30 days), whereas at the high dose, marked effect is found at 15 days and no survivors by 30 days.
Table 1. Percentage of survival of E. foetida exposed to three concentrations of Pc at 5, 15 and 30 days after exposure.
Table 2 shows the total number of sperms per worm and in Table 3, values are corrected for body weight. It can be seen that in Table 2 there is no much changes in the sperm number after Pc. When corrected for body weight, low and mid doses show significantly higher figures at 15 or 30 days.
Table 2. Total number of E. foetida spermatozoa exposed to three concentrations of Pc at 5, 15 and 30 days after exposure, mean SD, n = number of worms. cP<0.01, compared with controls ( Dunnet test).
Table 3. Total number of E. foetida spermatozoa per mg of body weight exposed to three concentrations of Pc at 5, 15 and 30 days after exposure, mean SD, n = number of worms. bP < 0.05, cP < 0.01, compared with controls (Dunnet test).
In agreement with these results, the seminal receptacle diameter, their lumina and luminal area are larger in worms treated with mid- and high Pc doses. There seems to be no change in the hatchability of capsules obtained from treated worms, since the number of worms was not different from controls.
Table 4 shows the histometric data obtained for the seminal receptacles. Table 5 shows the number of cocoons and of worms born from them after 5 weeks of rearing.
Table 4. Seminal receptacle diameter (SR), luminal diameter (L-SR) and luminal area in E. foetida exposed to three concentrations of Pc. mean SD, cP<0.01, compared with controls ( Dunnet test).
Table 5. Total number of capsules and worms born in E. foetida exposed to three concentrations of Pc. bP<0.05, compared with controls (Dunnet test).
Data on body weight of the worms (controls taken as 100 %) are given in Figure 1 for the 3 doses of Pc used at the different time intervals analyzed. A marked weight loss is seen by 15 days. Figure 2 and 3 show the percentage of germ cells with damaged DNA at 5 or 15 days of exposure to the 3 doses of Pc, as revealed by the comet assay. The damage observed by 5 days with the three Pc doses is evident only with the highest dose.
1. Changes of body weight in E. foetida exposed to three concentrations
of Pc at 5, 15 and 30 days after exposure. compared with controls, control
= 100 %.
Concerning gross anatomical changes in all worms only coiling was observed with the two higher doses of Pc by 5 and 15 days after exposure and also at 30 days with the lowest dose. Locomotion and feeding of control worms was normal under routine inspection and much less active at all periods for the Pc treated animals. Edema was seldom seen, unrelated to the Pc dose at 15 or 30 days. Constriction or evisceration that has been reported for other chemicals in the literature  was not observed.
Mortality has been the most frequently used parameter to evaluate the chemical toxicity in earthworms [11, 12, 14]. It is postulated, however, that survival is less sensitive from an ecotoxicological point of view . The lethal effect of a chemical is not a necessary consequence in intoxication and sub lethal effects may be produced. Therefore, growth and reproduction, addressed in the present work, have been recommended as useful sub lethal criteria [22,23].
Weight loss in our observations is a valuable indicator of physiological stress, related to the degree of intoxication and time of exposure [1, 24]. A similar trendhas been reported for organochlorine pesticides intoxication [1, 11, 25] and for the effects of fungicides and herbicides in Lumbricus terrestris and Eisenia foetida [26-28].
Coiling, another symptom seen in 100 % of the Pc treated worms, is the consequence of alteration in muscular function elicited by organophosphoric pesticides, which may explain the difficulties for locomotion of the intoxicated worms and their relative inability to feed themselves. These facts are related with weight loss. Reproduction is also interfered, since worms find their partner less easily and copulation is abnormal in terms of mating posture. There is probably also an alteration in the neuromuscular system involved in sperm transfer. Ejection of sperm seems also to be hindered and therefore a large number of spermatozoa is found in intoxicated worms in spite of a clear effect on sperm production under Pc treatment.
Numerous reproductive parameters have been studied in earthworms exposed to various xenobiotics, cocoon production, hatching, viability of the worms produced [11, 14, 15, 29, 30] and sexual maturation . All these are very important issues, but require a very long time of observation. Sperm count seems to be a very sensitive marker [1,16], though in our observations this parameter was probably interfered by the weight loss suffered by the treated worms.
Although the number of capsules produced by Pc treated worms was clearly decreased, the viability of worms hatched from them was not noticeably affected. This may imply that the quality of the ova is not damaged by Pc. Also the sexual characteristics of the worms were not affected.
Genotoxic evaluation, such as that performed using the comet assay, may prove to be a sensitive and useful tool for detecting effect of different chemical pollutants in a variety of organisms, from plants to mammals . It clearly shows that Pc is genotoxic for male germ cells of E. foetida in our experimental conditions, which may be related to increased apoptotic rates of these cells, as has been demonstrated in mice .
In conclusion, reproductive parameters of earthworms exposed to agropesticides seem to be useful bioindicators of soil pollution and promising tools to predict genotoxic damage of the germ cell elements.
Cikutovic M. In: Pathologies in earthworm: sub lethal biomarkers of xenobiotic
toxicity. Dissertation. University of North Texas. 1991.
to: Dr. Eduardo Bustos-Obregn, P.O. Box 70061, Santiago 7, Chile.