Toxic effects of Tween-80 and its mixtures with oil on oligochaetes

This study investigates the toxicity of the nonionic surfactant Tween-80, both its pure form and mixtures with oil, against the Baikal oligochaetes Mesenchytraeus bungei Michaelsen, 1901. It was experimentally shown that mixtures of the surfactant under study and oil have a toxic effect on oligochaetes at concentrations, at which the action of individual substances is harmless. In addition, this negative effect intensifies with an increase in the concentration of both the surfactant and oil in the mixtures.


Introduction
The remediation of oil-contaminated media is increasingly performed using various reagents. Among them, polysorbate 80 (Tween-80, C 64 H 124 O 26 ) is attracting practical attention [1][2][3][4][5] due to its low cost and low toxicity. The use of polysorbate 80 for removing oil and other hydrophobic pollutants determines the possibility of its presence, along with oil products, in the as-decontaminated soils. All these components can enter water bodies with thawing and rain water. Despite this potential danger, the biological effects caused by the combined presence of various pollutants have been poorly investigated. In this regard, the aim of this work was to study the toxicity of the nonionic surfactant Tween-80, both its pure form and mixtures with oil, against the Baikal oligochaetes Mesenchytraeus bungei.
Prior to experiments, the collected oligochaetes underwent adaptation for 2 days under laboratory conditions using an aeration system equipped with a micro-compressor. Their incubation was carried out in glass or plastic cages filled with non-sterile Baikal water, at a temperature of 8-10°C, illumination 250-300 lux. River sand was placed at the bottom, with the layer thickness of 0.8-1.5 cm.  This non-ionic surfactant is an emulsifier and solubilizer of hydrophobic products, widely used in cosmetics [2] and the food industry, as well as for remediation of oil-contaminated soils. In our study, the effect of this surfactant was evaluated at concentrations of 1; 10; 20; 30; 40 and 50 mM.
2) Oil from the Markovskoye field of the Irkutsk region. Estimation of the survival rate of oligochaetes. In order to study effects of the Tween-80 and oil mixtures on the survival of Baikal oligochaetes, 20 ml of both the oil emulsion and surfactant solution under study were almost simultaneously poured into a Petri dish. Baikal water was added to the solutions. The experiment was started immediately after mixing the components.
10 individuals of oligochaetes with a length of about 1.8-2.0 cm were placed into each dish. The experimental dishes were maintained for 4 days under the same conditions at which the oligochaetes were cultivated. The criterion for death was complete immobilization.
Statistical data processing. Statistical data processing was performed using the Microsoft Office software package. The experiments were carried out in 5 independent experiments, 3 replicates in each experiment. The conclusions were made under the P ≥ 0.95 probability of error-free prediction.

Results
During the experiment (4 days), Tween 80 solutions did not lead to the death of Baikal oligochaetes across the entire concentration range (1; 10; 20; 30; 40 and 50 mM). However, Tween 80 mixed with oil or diesel fuel (at their initial concentrations having no visible effect on oligochaetes) caused a toxic effect. This negative effect became more pronounced upon an increase in the Tween-80 concentration.

Discussion
It was shown that mixtures of oil and the nonionic surfactant Tween-80 have a toxic effect on oligochaetes at concentrations, at which the action of individual substances is harmless. An increase in the concentrations of Tween-80 and oil in the mixtures under study intensified the toxic effect.
Presently, it is premature to talk about the mechanism underlying the observed phenomenon. Among possible explanations is the suggestion that the increased toxicity is associated with an increase in the bioavailability of oil hydrocarbons under the action of surfactants, due to an increase in the solubility (solubilization) of hydrocarbons in the presence of Tween-80 [6][7][8][9][10][11]. However, the observed effects could also have been caused by weakening of the barrier properties of biomembranes under the action of Tween-80, which facilitated the entrance of oil.

Conclusion
In this study, such concentrations of oil emulsions and Tween-80 solutions that caused no visible toxic effect on the Baikal oligochaetes Mesenchytraeus bungei Michaelsen, 1901 for 4 days were selected. At the same time, the toxicity of oil and Tween-80 mixtures at concentrations, at which the action of individual substances was harmless to oligochaetes, was observed. Notably, the observed negative effect intensified with an increase in the concentration of both the detergent and oil in the mixtures.