Bioassay tests were set up with two organo-phosphorus pesticides Diazinon and Leptophos, which are bought under the trade names Basudin and Phosvel, respectively. Diazinon is often used as pest irradicator, for killing chironomid larvae, while Leptophos is claimed as killing both chironomid larvae and the fish Tilapia mossambica, leaving milkfish (Chanos Chanos) unharmed.
The objectives of the studies were to investigate the toxicity of these pesticides to the above mentioned fish and shrimp and also to study the uptake and loss of the pesticides in these organisms. The persistences of these pesticides and their eventual breakdown products were equally investigated. Concentrations are usually expressed in ppm (parts per million) or ppb (parts per billion = 109), which concern for ppm; mg/1 for water and mg/kg for solids and for ppb μg/1 or μg/kg respectively.
The toxicity tests were performed as single dosage toxicity tests in conformity with the application of pesticides in the ponds. This implies that during exposure no constant concentration of the pesticides in the water occurs. The gradual decrease of concentration depends on the persistence of the pesticide and the sorption to solids, like sediment, detritus and glass walls (for aquaria tests).
Sixty litre aquaria have been used, each usually filled with forty litres of brackish pond water, stocked with conditioned specimens of: milkfish fingerlings (Chanos chanos), Tilapia (Tilapia mossambica) of approximate equal size (6–7 cm) shrimp (Metapenaeus monoceros) of about 8 cm length, shrimp (Penaeus merguiensis) of 0.9 cm length, and chironomid larvae (Tendipes (chronomus) longilobus). The concentrations applied of Diazinon and Leptophos ranged between 0.05 and 2.0 ppm, calculated as initial dosage concentrations.
The results of the toxicity experiments are presented in Figure 1-A expressed in LT50 (lethal time for 50% mortality) for various concentrations of the applied pesticides. Approximately 100% mortality often occurs in a period 1½–3 times the LT50. From these results, the LD50 (lethal dosage for 50% mortality) has been estimated for 24, 48, 72 and 96 hours (Figure 1-B), as index for the toxicity effect of the pesticides. These LD50 values might be higher than when determined with chronic exposure tests, since the concentrations at only single dosage decrease after addition and within 24, 48, 72 and 96 hours.
From the surviving specimens of the toxicity tests, their behaviour was followed by replacing them in the original pesticide-free pond water. From them, and from those previously killed, samples were taken for Diazinon and Leptophos analysis (see 4.2.2).
For both the Diazinon and Leptophos aquaria test, water, specimens etc. were treated with benzene as extract organic solvent. With the Leptophos pond experiment, hexane was used instead of benzene. Clean-up procedures have been used for the hexane extracts only (with H2SO4), and proper calibrations have been carried out for Diazinon with the FPD equipment, and for Leptophos with both the FPD and ECD equipment.
The results from the aquaria tests are reported in Table 3. A budget calculation was made from these results (Table 4), while uptake and loss of Diazinon with Tilapia and loss with Chanos as function of the time is shown graphically in Figure 2.
Since with Leptophos surviving Chanos fingerlings died 5 days after the toxicity tests, and high concentrations were equally found in these specimens (cf. Table 3 -B) the aquaria results have been verified in the field by an additional pond experiment. In a new pond of 200 m2 surface, with an average depth of about 35 cm, Tilapia and Chanos were stocked. After conditioning of the fish for a week, spraying with Phosvel (30% Leptophos) was carried out at 0.5 ppm active ingredient. The mortality of fish was determined, and the concentrations of Leptophos in killed and surviving specimens was determined. The results are summarized in Table V.
Both Diazinon and Leptophos are effective killers of chironomid larvae and shrimp at initial concentrations of 0.5 ppm. However for Diazinon the efficiency relative to Chanos, is different to that claimed by Tang and Chen (1959). These authors found a safety factor of 10, defined empirically as the ratio of the threshold concentration where all stay alive at least for 96 hours, for Chanos fingerlings and the concentration where above and in 48 hours all chironomid larvae died. Our data suggest a factor of only 0.03 since in our experiments the chironomid larvae supported a 20 times higher concentration than in the experiments of Tang and Chen (0.075 ppm). This is accounted for mainly by the difference of the tests, since those of Tang and Chen were carried out in the ponds. Diazinon is strongly adsorb to the soil, which increases dosage to chironomid larvae.
Leptophos is indeed more toxic to Tilapia than to Chanos, while the toxicity of Diazinon to these species was not significantly different. With Leptophos Tilapia, at least those being used in the aquaria, were killed from 0.5 ppm concentration, which is at a relative high level of Leptophos. Some Chanos fingerlings, however, died after being replaced in pesticide-free water. By repeated aquaria tests, the same phenomenon was observed, where the presence of food (klekap algae and rice bran) made no real difference. The deceased fingerlings were very slim, and obviously had not taken much food.
This phenomenon can at present only be explained by the possibility that during the exposure to Leptophos containing water (5 days) some fingerlings might have been affected by Leptophos. Those affected also accumulated much more Leptophos, while the more probable healthy specimens could regulate the Leptophos content. Loss occurred only in the healthy specimens: for the other the metabolic processes were disturbed.
The pond experiment confirmed more or less the aquarium experiment for Leptophos. Tilapia was mainly killed, and the various sizes of Chanos survived. The residue concentrations in the dead Tilapia and the living Chanos, however, did not differ too much, which means that there is no great difference of uptake. The reason of a stronger effect on Tilapia than on Chanos is difficult to explain. Perhaps the higher lipid content in Chanos flesh (7%) relative to Tilapia flesh (4%), and the lower lipid content in the liver for Chanos (0.7%) against 15% for Tilapia liver plays a role. This would also mean that a changing content of fat, due to starvation might change the sensibility of Chanos to Leptophos: however, this is a hypothesis, although it would explain why some of the fingerlings were killed in the aquaria experiments.
This result and the relative long persistence of Leptophos and its metabolites in Chanos and sediments up to 40 days, is not in favour of Leptophos for being used in aquaculture. Damage to Chanos might occur, and if Leptophos is applied less than 40 days before harvesting, the residue concentrations are still detectable. For aquaculture such a restriction is difficult to impose on fishfarmers. On the other hand there are often solutions possible to avoid mixed fish cultures with Chanos, for example by better screening of the pond inlets and outlets. It is interesting to observe from these experiments that aquatic organisms, exposed to sublethal concentrations of pesticides are able to regulate the content of pesticides, and that such regulation is an active rapid process. This is illustrated for Diazinon in Figure 2 (for Leptophos see Table 3 -B), and is also later discussed for other pesticides in 5.2.1.
Uptake of Diazinon in living fish (Tilapia control) seems to be a rapid process taking only a few hours, while loss from healthy fish, replaced in pesticide-free water occurs at a slower rate. Expressed in terms of a so-called biological half-life for Diazinon, values between 10 and 36 hours were found.
Another interesting finding is that the concentrations in killed organisms seem to have some relationship to the concentration in the water and through this with the time of death.1 This might be explained by a disturbance of the metabolic processes which for healthy organisms regulate the level of pesticides. The supposition is made that at sublethal exposure the organisms are able to regulate the concentrations, while at lethal concentrations, in particular, when the organisms die slowly, this regulating system becomes out of hand. However, for a definite conclusion more work would be needed which could not be carried out in the course of the present study.
Another point of interest is the eventual occurrence of breakdown products from Diazinon and Leptophos in water and organisms. For Diazinon no breakdown products have been detected, which still contained organo-phosphorus.
The other eventual organic breakdown compounds escape detection in the FPD equipment. Leptophos contains, besides the organo-phosphorus group, a halogenated organic group, which allows detection by the ECD equipment. From the possible breakdown products, 4 bromo2,5 dichlorophenol and desbromo, leptophos were detected (Table 5). These compounds occurred at comparable concentrations the pesticide itself, with some retarding maximum concentrations as a function of time.
Concerning the recommendations for the application of Diazinon and Leptophos for aquaculture, only a positive recommendation can be given for Diazinon. The pesticide showed effective chironomid larvae killing, has a relative low persistence and low biological halflife and does not have breakdown products of known toxic properties. However, Diazinon also kills shrimp, thus its application, use or storage should be restricted or avoided in the neighbourhood of shrimp cultures.
For Leptophos, as already mentioned above, as a selective fish killer, it has not been fully proved that Chanos would remain unharmed, and the application of this pesticide cannot yet be recommended.
