Doklady Biological Sciences, Vol. 380, 2001, pp. 492–495. Translated from Doklady Akademii Nauk, Vol. 380, No. 5, 2001, pp. 714–717. Original Russian Text Copyright © 2001 by Ostroumov.

GENERAL BIOLOGY

Responses of Unio tumidus to Mixed Chemical Preparations and the Hazard of Synecological Summation of Anthropogenic Effects S. A. Ostroumov Presented by Academician V.N. Bol’shakov February 26, 2001 Received February 28, 2001

Bivalve mollusks contribute significantly to the flows of matter and energy in ecosystems [1, 2]. Bivalves were used as a test object in studies on the biological effects of metals [3], synthetic surfactants, and other pollutants and xenobiotics on living organisms [4–10]. Surfactants occur in the environment as components of surfactant-containing synthetic washing mixtures (SWMs), liquid washing mixtures (LWMs), and other mixed preparations. Until the present time, there has been no detailed work aimed at clarifying whether or not SWMs affect the rate of water filtration by freshwater bivalve mollusks. The goal of this work was to study the effects of SWMs on the water-filtration activity of the freshwater bivalve Unio tumidus Philipsson, 1788. The OMO detergent was used as an example of SWMs. The mollusks U. tumidus were collected at the partially silted large-grain sand bottom of the Moscow River upstream from the town of Zvenigorod. Collected mollusks were kept in tanks with artificial aeration. A total of 20 mollusk specimens were used in each experiment; 10 specimens were exposed to SWM (variant A) and the other 10 were control (variant B). Washing mixtures were added to experimental tanks 5 min before the experiment. The SWM concentrations shown in Tables 1–3 and indicated in the discussion of experimental data are the initial concentrations added to the tanks. In addition to mollusk-containing experimental tanks (variants A and B), control tanks of the same volume (0.5 l) containing no mollusks were used to determine the cell suspension density in the absence of water biofiltration (variants C and D). One of the control tanks (variant C) contained the same concentration of SWM as the corresponding experimental tank. The rate of water biofiltration was estimated as a decrease in the optical density of the incubation medium caused by cell elimination from water and the corresponding decrease in the cell count in water. A

Moscow State University, Vorob’evy gory, Moscow, 119899 Russia

yeast (Saccharomyces cerevisiae) cell suspension was used. The S. cerevisiae strains MSU1, MSU2, and MSU3 were from the collection of microorganisms of the Faculty of Biology, Moscow State University. Yeast cells were grown for 96 h at 26–28°C in glass flasks on a flask-shaker (180–200 rpm) under continuous intense aeration. The culture medium contained 5.0 g/l (NH4)2SO4, 1.2 g/l KH2PO4, 0.15 g/l KCl, 0.2 g/l MgSO4 · 7H2O, 0.05 g/l CaCl2, and 50 ml of yeast autolysate (initial pH, 6.0) [11]. The culture medium was poured into 0.75-l flasks (150 ml per flask) and sterilized. A sterile glucose solution was added to the flasks before inoculation (final concentration, 2 wt %). The inoculate volume was 3–4 ml per flask. The inoculate was preliminarily grown for two days at 30°ë on wort agar as a slant culture. The biomass was washed off the agar slant with sterile water and used as an inoculate. The yeast biomass was provided by N.N. Kolotilova. The optical density at 500 nm was measured using a Hitachi 200–20 spectrophotometer (Japan) at an optical path length of 10 mm. The Intelligent (Automat) OMO detergent (Unilever Polska SA, Bydgoszcz, Poland) was used as a test surfactant. Two series of experiments with different strains of S. cerevisiae were performed. The S. cerevisiae strains MSU1 and MSU2 were used in experimental series 1 and 2, respectively. The S. cerevisiae cell count was the same in the two series (7.5 × 106 per ml). The mean fresh weight of mollusks with shells in variants A and B of experimental series 1 was 19.2 and 19.0 g, respectively. The mean fresh weight of mollusks with shells in variants A and B of experimental series 2 was 19.16 and 18.99 g, respectively. The water temperature in experimental series 1 and 2 was 19 and 19.5°C, respectively. It was shown in our experiments that, in the presence of the initial SWM concentration of 100 mg/l, there was a significant decrease in the rate of water filtration by U. tumidus (Table 1). For example, during 1 h of filtration, the optical density of the incubation medium in experimental (in the presence of SWM, variant A) and control (variant B) samples decreased to 0.554 and 0.295, respectively. Therefore, the optical

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Table 1. Dynamics of optical density of the incubation medium in the studies on the effects of the SWM OMO (100 mg/l) on the filtration of S. cerevisiae MSU1 cells by mollusks Unio tumidus (experiment 1) Experiment Control Control (without Time after addition (+SWM; +mollusks) (–SWM; +mollusks) mollusks +SWM) of cells, min (Variant A) (Variant B) (Variant C) 10 20 30 40 60 70

0.670 0.667 0.644 0.653 0.554 0.566

0.660 0.585 0.525 0.442 0.295 0.242

0.713 0.715 0.676 0.666 0.598 0.581

Control (without mollusks –SWM) (Variant D)

A/B, %

0.718 0.706 0.677 0.618 0.587 0.442

101.5 114.0 122.7 147.7 187.8 233.9

Table 2. Dynamics of optical density of the incubation medium in the studies on the effects of the SWM OMO (50 mg/l) on the filtration of S. cerevisiae MSU2 cells by Unio tumidus mollusks (experiment 2) Experiment Control Control (without Time after addition (+SWM; +mollusks) (–SWM; +mollusks) mollusks +SWM) of cells, min (Variant A) (Variant B) (Variant C)

Control (without mollusks –SWM) (Variant D)

A/B, %

5

0.711

0.794

0.693

0.671

89.5

20

0.644

0.556

0.702

0.671

115.8

35

0.614

0.448

0.648

0.660

137.1

60

0.557

0.373

0.655

0.629

149.3

density of the experimental sample (in the presence of SWM) was significantly (187.8%) higher than this value in the control. The filtration-induced decrease in the optical density (0.716 units of optical density) during 70 min of incubation was (0.716–0.242 = 0.474 units of optical density) and (0.716–0.566 = 0.150 units of optical density) in the control and experimental samples, respectively. Therefore, the rate of the filtration-induced decrease in the optical density of the control samples was significantly (316%) higher than in the experimental samples. This effect can be attributed to the SWM-induced inhibition of the elimination of unicellular organisms from water by mollusks. It also follows from Table 1 that, in the absence of mollusks (i.e., in the absence of water filtration; variants C and D) the optical density of the incubation medium was significantly higher than in the control variant with mollusks and without SWM (variant B). Similar results were obtained with the cell suspension of the other strain of S. cerevisiae. It follows from Table 2 that incubation with SWM (50 mg/l) caused a decrease in the rate of water filtration. The rate of elimination of unicellular organisms from water by mollusks in the presence of SWM (variant A) was lower than in the control (variant B). The optical density of the incubation medium at the end of the experiment (after 60 min of incubation) in the experimental samples in the presence of SWM (variant A) was signifiDOKLADY BIOLOGICAL SCIENCES

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cantly (149.3%) higher than the optical density of the control samples (variant B). Similar results were obtained with the cell suspension of the third strain of S. cerevisiae. In addition, it was found in experiment 2 (S. cerevisiae strain MSU2) that, in contrast to variant A (in the presence of SWM), pellets were excreted by mollusks after 20 min of incubation of the control samples in variant B (in the absence of SWM). Although pellets were also excreted by mollusks in variant A, they appeared significantly later (after 3 h of incubation) and their amount was significantly smaller than in variant B. Therefore, the pellet excretion by mollusks was inhibited in the presence of SWM (variant A). Thus, the results of our experiments showed that the synthetic detergent OMO tested in this work caused a decrease in the rate of water filtration by freshwater mollusks. In other words, this detergent inhibits the filtration activity of mollusks and decreases the efficiency of elimination of suspended particles from water. This conclusion is consistent with experimental results obtained on marine mollusks. For example, it was found in our experiments [4–7] that SWMs and LWMs of various types were capable of inhibiting the water filtration by marine mollusks (Table 3). Therefore, certain concentrations of the mixed detergents (SWMs and LWMs) reduce the filtration activity of many species of bivalve mollusks (water filtrators). Losk-Universal,

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OSTROUMOV

Table 3. Inhibition of the filtration activity of organisms limiting the population size of phytoplankton by certain chemical substances Chemical substance SWM OMO SWM Losk-Universal SWM Tide-Lemon SWM IXI SWM Deni-Automat SWM Lanza SWM Vesna-Delikat LWM E LWM E Triton X-100

Concentration, mg/l 50–100 7–20 50 10–50 30 20 1 2 2 0.5

Organisms Unio tumidus Mytilus galloprovincialis Mytilus galloprovincialis Mytilus galloprovincialis Crassostrea gigas Crassostrea gigas Crassostrea gigas Mytilus galloprovincialis Crassostrea gigas Mytilus edulis

Effect of organic substance

References

+ + + + + + + + + –(Exposure time, 30–60 min)

This work [9] The same [6] The same " " " " "

Note: (+) effect of inhibition of elimination of unicellular organisms during filtration is observed; (–) lack of statistically significant (95%) effect of inhibition.

Deni-Automat, Vesna-Delikat, Tide-Lemon, Lanza, LWM E, and some other mixed detergents also exert similar effects. In addition to synthetic surfactants, many SWMs and LWMs also contain phosphorus compounds. The content of sodium tripolyphosphate in widely used detergents such as Bio-S, Kristall, Kashtan, and many other reaches 3–40 wt %. Phosphates stimulate phytoplankton growth. At certain concentrations of SWMs, the phosphate-induced stimulation of phytoplankton growth may exceed the potential inhibition effect of the surfactant [12–15]. Therefore, mixed detergents exert two types of hazardous effects on ecosystems: phosphate-induced stimulation of phytoplankton growth and surfactant-induced inhibition of biofiltrators. Because biofiltrators are an effective natural factor of unicellular plankton population control, the two types of the detergent-induced effects on an ecosystem facilitate the phytoplankton population growth. Therefore, these effects are summed, thereby increasing the hazard of the ecosystem collapse. Thus, the results obtained in this work are of particular interest in the context of discussion of the potential ecological danger of pollutants for integral functions of ecosystems. Synecological summation of the effects of anthropogenic factors on plankton populations and biofiltrators is of particular concern. Therefore, ecological situations should be analyzed based on the synecological approach to the problem. In other words, the interaction between populations of plankton organisms and biofiltrator-consumers should be taken into consideration in studies on the ecological effects of SWMs and LWMs on these populations. The results obtained in this work are consistent with the conclusions drawn in the preceding works [7–9].

ACKNOWLEDGMENTS I am grateful to Prof. J. Widdows, and to V.D. Fedorov, V.V. Malakhov, A.G. Dmitrieva, and other researchers at Moscow State University, as well as Yu.Yu. Dgebuadze, N.S. Zhmur, and Yu.P. Kozlov, for stimulating discussion. I am also grateful to E.A. Kuznetsov, N.N. Kolotilova, and O.S. Ostroumov for their expert assistance in some experiments. This study was supported by IBG and the Open Society Foundation (RSS project no. 1306/1999) and partly supported by the MacArthur Foundation (Program for Global Safety and Stable Development). REFERENCES 1. Alimov, A.F., Funktsional’naya ekologiya presnovodnykh dvustvorchatykh mollyuskov (Functional Ecology of Freshwater Bivalves), Leningrad: Nauka, 1981. 2. Strayer, D., Caraco, N., Cole, J., et al., BioScience, 1999, vol. 49, pp. 19–27. 3. Malakhov, V.V. and Medvedeva, L.A., Embrional’noe razvitie dvustvorchatykh mollyuskov v norme i pri vozdeistvii tyazhelykh metallov (Embryonic Development of Bivalves under Normal Conditions and under the Exposure to Heavy Metals), Moscow: Nauka, 1991. 4. Ostroumov, S.A., Donkin, P., and Staff, F., Vestn. Mosk. Gos. Univ., Ser. 16: Biol., 1997, no. 3, pp. 30–36. 5. Ostroumov, S.A., Donkin, P., and Staff, F., Dokl. Akad. Nauk, 1998, vol. 362, no. 4, pp. 574–576. 6. Ostroumov, S.A., Biologicheskie effekty poverkhnostnoaktivnykh veshchestv v svyazi s antropogennymi vozdeistviyami na biosferu (Biological Effects of Surfactants as Related to the Anthropogenic Impact on the Biosphere), Moscow: MAKS, 2000. 7. Ostroumov, S.A., Dokl. Akad. Nauk, 2000, vol. 371, no. 6, pp. 844–846. DOKLADY BIOLOGICAL SCIENCES

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RESPONSES OF UNIO TUMIDUS TO MIXED CHEMICAL PREPARATIONS 8. Ostroumov, S.A., Dokl. Akad. Nauk, 2000, vol. 372, no. 2, pp. 279–282. 9. Ostroumov, S.A., Dokl. Akad. Nauk, 2000, vol. 374, no. 3, pp. 427–429. 10. Ostroumov, S.A., Dokl. Akad. Nauk, 2000, vol. 375, no. 6, pp. 847–849. 11. Ostroumov, S.A. and Kolotilova, N.N., Toksikol. Vestn., 2000, no. 5, pp. 43–44. 12. Ostroumov, S.A. and Kolotilova, N.N., Toksikol. Vestn., 2000, no. 2, pp. 31–32.

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13. Ostroumov, S.A. and Kolotilova, N.N., Vodnye ekosistemy i organizmy-2 (Water Ecosystems and Organisms 2, Moscow: MAKS, 2000, p. 60. 14. Kolotilova, N.N. and Ostroumov, S.A., Problemy ekologii i fiziologii mikroorganizmov (Problems of Ecology and Physiology of Microorganisms), Moscow: Dialog–MGU, 2000, p. 66. 15. Ostroumov, S.A., in Avtotrofnye mikroorganizmy (Autotrophic Microorganisms), Moscow: MAKS, 2000, pp. 134–135.