The combined effect of petroleum products and humate on daphnia

Stom D.I., Dagurov A.V.
Research Institute of Biology at Irkutsk State University

Recently, more and more data are accumulating on the ability of various humic substances to reduce the negative effect of certain toxic compounds on various organisms [1, 2, 3]. Among the pollutants, both in volume and in scale, oil and oil products are especially distinguished [4, 5]. Therefore, it seemed interesting to check the possibility of weakening the toxic effect of the latter in the presence of humic substances.

Objects and research methods

The experiments used oil of the Markovskoye field of the Irkutsk region and oil products: summer diesel fuel (GOST 305-82), heating oil M-40 (GOST 10585-75), unleaded motor gasoline with an octane rating of 76 and 93 (GOST 2084-77), aviation B-95/130 gasoline (GOST 1012-72). As a source of humic substances, they took the “Humat” preparation manufactured by AgroTech Gumat LLC.
The toxicity of humates and petroleum products was evaluated in a laboratory culture of cladocera Daphnia magna (Straus) [7]. The Daphnia test is included in most national and international standards for water quality research and has a number of policy documents on the use of biological testing in practice [8, 9]. When applied, the main criterion of toxicity is the survival of crustaceans. In our experiments, an additional indicator of the toxic effect of the tested substances on Daphnia was the transition of crustaceans to the air-water interface [10, 11]. This test reaction is a significantly more sensitive indicator for the presence of emulsions of oil and oil product pollution than the death of Daphnia.
For daphnia cultivation, experiments, and control, dechlorinated tap water was used (the intake of the Angara River above Irkutsk). For experiments, juveniles of one litter at the age of 2 days were selected. In each experimental vessel with a volume of 30 ml were placed 10 crustaceans. Experience and control had at least 6 replicates with three parallels in each.
Emulsions of oil and oil products with concentrations of 0.5; 1; 1.5; 2; 2.5 ml / l was obtained by pouring the required amount of oil and oil products to 1 liter of water, followed by stirring for 60 minutes on a magnetic stirrer. 
To prepare the mother liquor of humate, a weighed portion of the corresponding finely dispersed air-dry preparation of humate 0.5 g was dissolved in 1 liter of water, then the mother liquor was centrifuged at 7 thousand rpm. for 5 minutes and then filtered through a paper filter (white tape).
To obtain saturated solutions of hydrocarbons, 2 liters of glass was poured into glass measuring cylinders almost to the top of the water, and a layer of oil products 1 cm thick was carefully layered on their surface with a pipette. The vessels were equipped with a device allowing sampling of the solution from under the hydrocarbon film. The dissolution process took place at a temperature of 20 - 220 C in an atmosphere of air for 30 days. In the experiments, saturated hydrocarbon solutions were used without dilution, with 2 times dilution with water and 2 times dilution with a humate solution.
For statistical processing, generally accepted methods were used [12, 13] using a personal computer and the Statgraf 3.0 and Excel 97 software packages. Conclusions were made when the probability of an error-free forecast was P³0.95.

Research results and discussion.

At the first stage of the study, concentrations of humate solutions were selected that did not have a visible effect on daphnia during a 24-hour exposure. They turned out to be solutions of humate prepared from the initial sample of 0.5 g / l. 
As can be seen from table 1 in the studied concentration range (from 0.5 to 2.5 ml / l), oil emulsions and tested oil products caused the transfer of crustaceans, although to different degrees to the surface layer. From the same table, as well as from table 2, it follows that humate preparations in the tested concentrations in most cases prevented, or at least significantly reduced the ability of emulsions of oil, oil products and their individual hydrocarbons to initiate the emergence of Daphnia. In this regard, the effect of humate solutions on oil and diesel fuel is especially effective: in the absence of humate, 100% of crustaceans appeared on the surface, and in its presence 100% Daphnia floated in the thickness of the emulsion (Table 1). It was previously shown that, of the wide variety of substances tested, only surface-active agents prevented the transfer of crustaceans to the surface film caused by emulsions of petroleum products. Humate not only prevented Cladocera from floating into the surface film, but although in a smaller number of experiments and not so significantly, it still weakened the toxic effect of emulsions of oil and oil products.
In some cases, for example, in experiments with emulsions of diesel fuel at concentrations of 0.5 and 1 ml / l, the difference in mortality of crustaceans in the presence and absence of a humate solution was 50%. In the emulsion of diesel fuel with a concentration of 2 ml / l in the absence of humates, the complete death of all crustaceans occurred after 5 hours, and with the addition of humate after 24 hours. The concentration of 2.5 ml / l caused a complete suppression of the activity of crustaceans after 4 hours, and when humates were added, the crustaceans remained active for 5 hours.

Humate was less effective in reducing the toxic effects of aviation gasoline. Antagonism was even weaker in mixtures of fuel oil and humate. But the least pronounced was the weakening by humate of the effect on oil daphnia emulsions.
At the same time, it can be seen from the materials presented in Table 1 that solutions of the humate preparation in this concentration prevented the transfer of daphnia to the surface layer caused by the action of the emulsion, or at least reduced the number of crustaceans leaving practically all oil products leaving the thickness of the aqueous emulsion. 
Since, unlike emulsions of oil products in their solutions, crustaceans did not leave the water column on its surface, the question arose: would humate preparations weaken not only the toxic effect of emulsions, but also solutions of oil and oil products?

Analysis of the materials of table 3 shows that humates weakened toxicity for crustaceans not only emulsions, but also oil solutions. For example, with a daily exposure of diluted twice with saturated diesel fuel solutions with water, the number of daphnia survivors was 33.3% less than with their corresponding dilution with humate solutions. 
A weakening or even complete suppression of the behavioral reaction of Daphnia ascent to the surface and a decrease in toxicity were also observed in experiments with individual aromatic and aliphatic compounds that are part of petroleum products (Table 2 and Table 4).
In mixtures of oil emulsions with humates, over the entire range of studied concentrations, weakening or even complete prevention of the behavioral reaction of the emergence of crustaceans into the surface layer was recorded. Thus, the addition of humates to emulsions of oil and oil products, reduced the toxic effect of the latter for daphnia. 
The authors are grateful to Dr. I.V. Perminova and Professor H. Steinberg for valuable discussions and consultations. This work was financially supported by the NTP “State support of the regional scientific and technical policy of the Higher School and the development of its potential”, project No. 477 and the Russian Federal Property Fund (grant No. 99-04-49612).
Table 1-Effect of emulsions of petroleum products in the presence and absence of humates on the survival and ascent to the surface of daphnia during 24-hour exposure (in% of control - dechlorinated water).

Note: humate solutions with an initial concentration of 0.5 g / l do not have a visible effect on daphnia.

Table 2 - Effect of oil hydrocarbon emulsions in the presence and in the absence of humates on the survival and ascent to the surface of daphnia during 24-hour exposure (in% of control - dechlorinated water).

Note: humate solutions with an initial concentration of 0.5 g / l do not have a visible effect on daphnia.

Table 3 - The effect of mixtures of equal volumes of saturated solutions of oil, oil products and humate on the survival of daphnia, in% of the control - water.

Table 4 - The effect of emulsions of aromatic hydrocarbons of oil in the presence and absence of humates on the survival and ascent to the surface of daphnia during a 24-hour exposure (in% of control - dechlorinated water).