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TF4005 07 Chapter 2.fm Page 27 Friday, November 11, 2005 1:42 AM 2 Organisms and Methods 2.1 Organisms: Substantiation of Choice and Aspects of Methods Used The subjects of investigation were typical representatives of the main trophic levels and large taxa from prokaryotes to eukaryotes including cyanobacteria (Synechococcus Näg.; Stratonostoc linckia (Roth) Elenk., f. muscorum (Ag.) Elenk. = Nostoc muscorum Ag., and others), marine bacteria (Hyphomonas (ex Pongratz 1957) Moore, Weiner and Gebers 1984), green algae (Scenedesmus quadricauda Bréb., Chlorella vilgaris Beijer, Bracteacoccus minor (Chodat) Petrova, and others) diatomic algae (Thalassiosira pseudonana Hasle et Heimdal), euglena (Euglena Ehr.; Euglena gracilis Klebs), mollusks (Unio tumidus Philipsson s. lato, U. pictorum (L.) s. lato, Crassiana crassa (Philipsson) s. lato, Anodonta cygnea (L.) s. lato, Mytilus edulis L., M. galloprovincialis Lamarck, Crassostrea gigas Thunberg, Limnaea stagnalis (L.), Mercenaria mercenaria), annelids (Hirudo medicinalis L.), macrophytes (Pistia stratiotes L., Elodea canadensis Michaux), seedlings of angiosperm plants (Sinapis alba L., Fagopyrum esculentum Moench, Lepidium sativum L., Oryza sativa L., Camelina sativa (L.) Crantz, Triticum aestivum L., and others). These objects were of theoretical and practical interest due to the details of their ecology, their role in the ecosystems, and the possibility of their use as biological resources. Diverse biological material made it possible to obtain broader and more substantiated conclusions on the possible role of synthetic surfactants as pollutants. Below we describe the justification of the choice of the objects (organisms) and methodological aspects of their use. The nomenclature of cyanobacteria is given according to Gollerbakh et al. (1953); of marine phytoplankton, according to Tomas (1997). The nomenclature of vascular plants of Russia and adjacent countries (territories of the former USSR) is given according to Cherepanov (1995). The nomenclature of invertebrates is according to Zatsepin and Rittikh (1975), and Zatsepin et al. (1978). 2.1.1 Prokaryotes 2.1.1.1 Cyanobacteria (Cyanophycota) This essential group of phototrophic prokaryotes uses water as the donor of electrons and, thus, produces oxygen in the light. The genus Synechococcus (class © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 28 Friday, November 11, 2005 1:42 AM 28 S.A. OSTROUMOV Chroococcophyceae, order Chroococcales) is one of the four main genera of marine cyanobacteria. This genus also includes the species that occur in freshwaters and in terrestrial habitats. Synechococcus develops in eutrophic waters and can reach large concentrations of cells in seawater. It is an important component of phytoplankton and is involved in biogeochemical fluxes of elements through marine ecosystems. It is capable of nitrogen fixation and, thus, is one of the main suppliers of nitrogen to seawater (e.g., Kondratyeva et al. 1989; South and Whittick 1987). Cyanobacteria can reach a share of 60% of all chlorophyll in marine ecosystems in the upper 50 m and about 20% and more of the total primary production (e.g., Sieburth 1979). In this study, along with the other species of cyanobacteria, we studied cyanobacteria of the combined genus Synechococcus. These unicellular cyanobacteria (less than 3 µm in size) are widespread in the open regions of the seas. They were also found in the seas of the Arctic Basin (Mishustina et al. 1994). The Synechococcus strains used were from the collection of the Woods Hole Oceanographic Institution (U.S.). The WH7805 strain (GC contents in the DNA, 59.7 mol.%) are immotile pink cells. The strain was isolated by L. Brand (cruise 48 of the “Oceanus”), sample dated June 30, 1987. The WH8103 strain (GC contents in the DNA, 58.9 mol.%) are motile yellowish cells; the strain was isolated by J. Waterbury (cruise 92 of the “Oceanus”) from the sample dated March 17, 1981. The strains were maintained in the laboratory of J. Waterbury on medium SN (see [18, 20] in the paper by Waterbury and Ostroumov 1994). The cells were cultured at a temperature of 22°C and at a permanent illumination of 20 (micro Einstein) m –2 s –1. Syntheticsurfactant solutions added to the cultures were sterilized by filtration through Acrodisc sterile filters (Gelman Sciences) with pore diameter of 0.45 µm. The absorption spectra were recorded using a Shimatzu UV 3101PC spectrophotometer to characterize the culture. Along with the native spectra, the spectra of samples with addition of sucrose to the dish (1.5 g per 3.5 ml of cell suspension) were measured, which allowed us to decrease the light scattering. We also used the strains Anabaena sp. CALU 811, Cylindrospermum sp. CALU 306, Synechococcus sp. CALU 742 from the collection of the Laboratory of Microbiology, Biological Research Institute, Leningrad University. The cultures were grown in 50-ml conical flasks on liquid medium (20 ml each) of the following composition (g/l): KNO3, 1; K2HPO4, 0.2; MgSO4, 0.2; NaHCO3, 0.2; CaCl2, 0.05; trace elements, 1 ml (solution in medium no. 6). Growth conditions: 25°C, 2000 lux. Xenobiotic NS was added to the medium at concentrations of 0.1, 0.5 and 1 mg/l. The effect of the xenobiotic on the culture growth was judged by the amount of cell biomass, which was determined by drying aliquots to a constant weight at 105°C. Strain 33 of Nostoc muscorum Ag. was isolated from calcareous soil in the Kirov Region. Strain 235 was isolated from soils contaminated with oil (Almetyevsk, Tatarstan Republic). The cultures were grown in a medium containing per 1 liter (in g): KNO3, 1.0; K2HPO4, 0.2; MgSO4 ·7H2O, 0.2; CaCl2, 0.15; NaHCO3, 0.2; and 1 ml of a solution of trace elements. The trace-element solution contained (in g/l): ZnSO4 ·7H2O, 0.22; MnSO4, 1.81; CuSO4 ·5H2O, 0.79; (NH4)2Mo7O4 ·4H2O, 1.0; FeSO4 ·7H2O, 9.3; CaCl2, 1.2; Co(NO3)2H2O, 0.08; EDTA, 10.0; H3Bo3, 1.989. Distilled water was used. The inoculate was added into each flask by 1 ml. The inoculate of Nostoc muscorum cyanobacteria was preliminarily homogenized by an © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 29 Friday, November 11, 2005 1:42 AM BIOLOGICAL EFFECTS OF SURFACTANTS 29 electromechanical homogenizer (5,000 rpm for 1 min). The growth flasks contained 50 ml of the medium each. Each variant was represented by two repeats. The cultures were incubated at an illumination of 3000 lux and at room temperature. 2.1.1.2 Marine heterotrophic bacteria Hyphomonas (ex Pongratz 1957) Moore, Weiner and Gebers 1984, 71VP Gram-negative pleiomorphic bacteria, chemoorganotrophs, require the presence of amino acids as the source of carbon (Moore and Weiner 1989). They are included in the group of budding and/or appendaged bacteria (Bergey’s Manual of Determinative Bacteriology, 1989, Vol. 3) and are related to the first organisms that inhabit hard surfaces in marine water and form a biological film. The film then becomes a basis for colonization of this biotope by other periphyton organisms. They are widespread in biological films at hard surfaces in various marine ecosystems, in biofouling on the surfaces of hydrotechnical constructions and ships. Thus, these bacteria are of practical importance. They have a specific lifecycle, which includes budding of the daughter cells from the end of the hypha. The maternal cell attaches to a hard substrate, while the budded daughter cell is transported away by the water flow and precipitates on a substrate in another place, attaches to the surface, elongates, and buds off a new generation of daughter cells (Moore and Weiner 1989). The effect of synthetic surfactants on these bacteria was barely studied; particular effects of nonionogenic and cationic surfactants were not studied in detail. The Hyphomonas bacteria were grown on S-1 medium proposed by the author. The composition of the medium is as follows: 1 l of NaCl solution (22 g/l) was added to 1 l of Marine Broth 2216 medium (0.5% peptone, 0.1% yeast extract; on sterile seawater; Difco Laboratories, Detroit). This medium is more advantageous as compared with the media used earlier as it is more economic and convenient for the experiments, where bacterial growth is recorded in the optical density measurements. Addition of TX100 was made before that of the inoculate. The inoculate (5%, v/v) was a 1-day culture grown on the same medium (S-1). The optical density (600 nm; optical path, 10 mm) after the inoculation was about 0.05. Cultivation and incubation were carried out in a thermostatted room at 25°C without mixing. The density of the culture in all experiments was measured spectrophotometrically at a wavelength of 600 nm. Additions of TX100 were sterilized by passing them through a Sterile Acrodisc bacterial filter (Gelman Sciences), 0.2 µm. The experiment included two repeats unless indicated otherwise. 2.1.1.3 Other objects The bacteria Rhodospirillum rubrum were kindly provided by the group of Prof. V.D. Samuilov (Moscow State University, Department of Cell Physiology). © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 30 Friday, November 11, 2005 1:42 AM 30 2.1.2 S.A. OSTROUMOV Eukaryotes 2.1.2.1 Diatomic algae Thalassiosira pseudonana Hasle et Heimdal (=Cyclotella nana Guillard clone 3H in Guillard & Ryther) (class Bacillariophyceae, order Biddulphiales, suborder Coscinodiscineae, family Thalassiosiraceae (9 genera)) (according to the other classification: class Centrophyceae, order Coscinodiscales, family Thalassiosiraceae, 11 genera, predominantly in marine plankton). The species of the genus Thalassiosira Cleve (about 80 modern and fossil species) are widely represented in all geographical zones, in the plankton of seas and saline reservoirs. It is a characteristic representative of the typical and mass species of marine diatoms, one of the dominating groups in marine plankton, very important as a feed resource for many species of commercial fish. The diatoms make a significant contribution to the global processes of atmospheric carbon fixation and oxygen evolution, participate in the processes of selfpurification of reservoirs, and are used in assessing the sanitary state of waters. The algae were grown on medium f/2 (Giullard and Ryther 1962) without FeCl3 and EDTA. In order to prepare the medium, water was preliminarily filtered through a polycarbonate filter (Nucleopore, 0.2 µm). The cells were counted in a Fischer hemacytometer after preliminary fixation by addition of Lugol’s solution (50 ml in 1 ml of algal culture). The initial density of the culture was 3·104 cells/ml in all variants. The culture in the stationary growth phase was used for inoculation. The illumination regime in the inoculation was as follows: light, 14 h; darkness, 10 h; intensity of illumination was 254 (micro Einstein) m –2 s –1. Temperature, 17°C. 2.1.2.2 Green algae Five classes including the Protococcophyceae, which is sometimes considered as an order. The Protococcophyceae are ecologically diverse, are present in plankton, benthos, neuston, and periphyton (the epiphyte and epizoan forms), and are common in land habitats and in soil. They are present in many types of reservoirs including fish ponds, some types of precipitation tanks, biological ponds, and filtration fields of urban water treatment facilities. Thus, they take an active part in self-purification of water by the ecosystems. Most of the Protococcophyceae are euryhaline and eurythermal organisms. The species of the general Scenedesmus and Chlorella became classical plant-cell objects and models, which were used to study many aspects of biochemistry and physiology. The Protococcophyceae are actively studied to be used for intensifying the purification of polluted waters and producing protein and vitaminized fodder. By the role they play in the natural ecosystems and biogeochemical processes of the biosphere they can be second (not always) only to the diatoms (Gollerbakh 1977; Matvienko 1977; Kondratyeva et al. 1989; South and Whittick 1987). Bracteacoccus minor (Chodat) Petrova. Strain 200 was obtained from the Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg (no. 867-1 in their collection). Strain 219 was isolated from volcanic ash collected © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 31 Friday, November 11, 2005 1:42 AM BIOLOGICAL EFFECTS OF SURFACTANTS 31 on the ash plateau free of vegetation in the vicinity of Tyatya Volcano (Kunashir Island). The algae were grown in a medium containing in 1 1 (in g): KNO3, 1.0; K2HPO4, 0.2; MgSO4 ·7H2O, 0.2; CaCl2, 0.15; NaHCO3, 0.2; and also 1 ml of trace element solution. The solution of trace elements contained (in g/l): ZnSO4 ·7H2O, 0.22; MnSO4, 1.81; CuSO4 ·5H2O, 0.079; (NH4)2Mo7O24 ·4H2O, 1.0; FeSO4 ·7H2O, 9.3; CaCl2, 1.2; Co(NO3)2H2O, 0.08; EDTA, 10.0; H3BO3, 1.989. Distilled water was used. The growth flasks contained 50 ml of medium each. Each variant was made in two repeats. The algal cultures were incubated at an illumination of 3000 lux at room temperature. Scenedesmus quadricauda Breb. The cultures were grown on Uspensky nutrient medium no. 1 in Luminostat at a temperature of 24–25°C and illumination of 2000 lux. The sources of illumination were fluorescent lamps LB-40. The cultures were grown in 250-ml Ehrlenmeyer flasks. The number of cells was determined by direct calculation in a Goryaev chamber. In the experiments with sodium dodecyl sulfate (SDS), the initial density of cells was 3.16 million/ml (experiment 1) and 2.47 million/ml (experiment 2). Pulverized podzolic soil from the Kirov Region was used in the experiments with soil cultures (the collection site was the experimental field of the Kirov Agricultural Institute). The soil had the following agrochemical characteristic: pHsal = 4.6; P2O5, 37.3 mg/100 g; K2O, 3.2 mg/100 g; humus, 1.2%. Soil (30 g each) was placed in Petri dishes, where 10 ml each of an aqueous solution of TDTMA (in distilled water) was added at concentrations of 0.1 and 0.05 mg/ml. Distilled water (10 ml each) was added to control dishes. Soil samples were incubated in the light at a room temperature and 70% humidity relative to complete water capacity (watering with distilled water by weight). Abundance of the algae in the soil cultures was determined by the generally recognized method of the direct count of cells in the soil suspension using a light microscope. The author thanks Prof. E.A. Shtina for consultations and assistance in this part of work. We also used other algae in the experiments. They were grown on standard media mentioned in respective chapters of this work and in papers we published. 2.1.2.3 Euglenas Euglenophyta (about 1000 species, half of them found in Russia and republics of the former Soviet Union) generally inhabit internal continental reservoirs. They possess all major types of nutrition: autotrophic, saprophytic, holozoic (peculiar of higher animals); are capable of mixotrophy. They are involved in self-purification of aquatic objects, the water of which contains many organic substances. The species of the Euglena genus are capable of mass development in reservoirs that can lead to water blooming. This is a favorite object for cultivation in laboratories with the objective to study the effect of various factors. They are promising for use in purification of polluted waters and for cultivating in the photoautotrophic life support systems (Safonova 1977; Kondratyeva et al. 1989). © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 32 Friday, November 11, 2005 1:42 AM 32 S.A. OSTROUMOV The culture Euglena gracilis Klebs var. Z. Pringsheim was grown photoorganotrophically in 100-ml flasks at a temperature of 26°C and illumination of 1,500–2,000 lux. The medium of the following composition was used (in g/l): NaCl, 0.1; MgSO4 ·7H2O, 0.4; KH2PO4, 0.4; CaCl2 ·6H2O, 0.05; glucose, 10.0; L-glutamic acid, 2.0; (NH4)2SO4, 1.0; vitamin B1 (0.2% solution), 0.2; vitamin B12 (0.01% solution), 0.2, solutions I and II, 1 ml each per 1 l of medium. In order to prepare solution I, 695 mg of FeSO4 ·7H2O and 930 mg of Na2EDTA was taken and dissolved in warm bidistilled water, pH was adjusted by NaOH, and then water was added up to 100 ml. Solution II was prepared by taking (in g per liter of bidistilled water): ZnSO4 ·7H2O, 10.0; MnSO4 ·4H2O, 2.2; H3BO4, 12.2; Co(NO3)2 ·6H2O, 1.0; NaMoO4 ·2H2O, 1.2; CuSO4 ·5H2O, 0.001. The strain E. gracilis obtained from the algal collection of Göttingen University (Germany), no. 1224-5/25, was used. Bidistilled water was used to prepare solutions and media. The volume of the inoculate, which was sampled in the mid-logarithmic phase of growth, was 5 ml at the onset of the cultivation. 2.1.2.4 Plant seedlings Seedlings are recommended as one of the priority objects for biotesting in the field of water quality studies (Unified methods for water quality studies. Part 3. Methods of Biological Analysis of Waters, ed. by M. Gubachek, Moscow, 1975). They are used in the arsenal of methods of the U.S. Environmental Protection Agency (U.S. EPA 1982) and other U.S. agencies (U.S. Food and Drug Administration 1987), and European agencies (European Organization for Economic Cooperation and Development 1984). The method is highly economic and efficient from the point of view of the information versus biotesting cost ratio. Plant seedlings can be used in laboratories (industrial enterprises, chemical institutions), where more susceptible organisms do not survive. Therefore, the method is free of one disadvantage of highly-sensitive test objects – they are not always capable of living under conditions of inplant laboratories, where the air can be polluted with chemicals. Seedlings serve as an alternative to animal testing, which is important from the humanitarian point of view and in the view of official recommendations by the International Union of Toxicology (IUTOX). In 1985, the IUTOX Executive Committee published an official statement that “alternative methods of testing, which do not require the use of animals, should be in common use (after their comprehensive scientific testing)” (see Telitchenko and Ostroumov 1990). High economic efficiency of this biotest is important for Russia under the current conditions of science financing. Biotesting on plant seedlings was performed by several authors – including in Russia, in the laboratories by V.B. Ivanov (Ivanov 1974, 1982, 1983, 1986, 1992), N.V. Obroucheva (Obroucheva 1992) and in the West (e.g., Wang 1987; Wang and Williams 1990; Davies 1991; Davies et al. 1991). The method was successfully used in the laboratory headed by Professor Ivanov to assess a broad class of biologically active substances (BAS), including compounds important for pharmacology. (The author is deeply grateful to V.B. Ivanov and all colleagues at the laboratory for numerous consultations and discussions of the results). In studies © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 33 Friday, November 11, 2005 1:42 AM BIOLOGICAL EFFECTS OF SURFACTANTS 33 carried out under the supervision of Professor V.N. Maksimov, the method was used to assess the toxic impacts of metals. The method was also successfully used at the Department of Soil Science, Moscow State University, to assess the toxicity of various biological preparations. This method is one of the main tools in investigating allelopathic substances. We wrote in detail about this trend of BAS studies in Chapter 3 of an earlier book (Ostroumov 1986). The method was used in the Central Botanical Garden, Ukrainian Academy of Sciences (A.M. Grozdinsky, E.A. Golovko and other specialists; the author is grateful to them for the seeds of cress). The method was also used at the Institute of Hydrobiology (Kiev) for water quality assessment (Sirenko and Kozitskaya 1988). Though this method was recommended by the U.S. Environmental Protection Agency (U.S. Environmental Protection Agency 1982), it was comparatively rarely used in the U.S. The variant of the method we used was more advanced methodologically compared to the works by Wang and by Davis and co-authors in the sense that they did not use information about the effect of chemicals on the ratio of germinated and nongerminated seeds. Introduction of the integral morphogenetic index, which unites information on the effect of a tested chemical or polluted water on both processes – germination of seeds and elongation of a seedling – was a methodological improvement. Various plant test objects were used (Ostroumov 1990, Table 1). Traditional techniques and some less traditional variants and approaches were evaluated. A list of some major effects, based on the biological activity of substances – various surfactants and some pesticides (Maksimov et al. 1988), is given in Ostroumov (1990) (Table 2 therein). The following details of some evaluated variants of methods should be noted (Goryunova and Ostroumov 1986; Nagel et al. 1988; Ostroumov and Maksimov 1988; and others). 1. The group of methods for estimating the biological activity of substances and pollution of aqueous medium by their effect on seeds (at 100% germination) and further growth of seedlings. In order to estimate the biological activity (BA) of a substance or aqueous medium at 100% germination of seeds, a Petri dish (10 cm in diameter) with the seeds of test objects put on filter paper was filled with a corresponding water solution (usually 7, 10 or 15 ml). Controls were filled with distilled water (DW) or settled tap water (STW). Incubation was carried out in the dark at room temperature or at 26–28°C. After a time interval t1 the length of the seedlings (hypocotyl + root or only root) was measured. Further measurements were made at times t2, t3, and so on. The measurement results were processed statistically using nonparametric methods (see below). Calculations of the mean rate of elongation and percentage of inhibition were used (Ivanov 1974). This group of methods was used for white mustard Sinapis alba, buckwheat Fagopyrum esculentum, cucumber Cucumis sativis, watercress Lepidium sativum and other objects. In the summary table (Ostroumov 1990, Table 1), the methods are denoted as 1, 4, 5, 7, 8. 2. The group of methods for estimating the biological activity of substances and pollution of aqueous medium by their effect on the elongation of preincubated seedlings. When the effect of a tested substance at the initial stage of germination was to be avoided, and the objective was to study its effect on the elongation of the seedlings, the experiment was carried out as follows (see Ivanov 1974). The seeds were first preincubated in DW or STW. Then seedlings (of predominantly fixed length) were © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 34 Friday, November 11, 2005 1:42 AM 34 S.A. OSTROUMOV picked out and placed in Petri dishes with tested solution at different concentrations. All dishes contained different volumes of solutions (usually 7, 10 or 15 ml) and an equal number of seedlings. Control seedlings were transferred into new Petri dishes with the water used to prepare the test solutions. The length of the seedlings at the beginning of incubation (t1) was measured. Then the incubation was carried out in the dark and the length of seedlings was measured (t2, t3, etc.). Elongation of the seedlings in the test solutions was compared with that of the control seedlings. The results were processed statistically. The method has the following restrictions: preincubation in DW or STW may slightly smooth the effect and decrease the sensitivity of the method as compared to the methods of group 1. The methods of biotesting on seedlings was evaluated in a large cycle of works by V.B. Ivanov (e.g., Ivanov 1974) and in our studies on buckwheat, rice, and other test objects. In the summary table (Ostroumov 1990, Table 1) the methods are denoted as 1b, 5b, 5c, 6. 3. The group of methods for estimating the biological activity of substances and pollution of aqueous medium by their effect on the degree of germination. Some (but not all) tested substances significantly decrease the proportion of germinating seeds. It is easiest to estimate this proportion when the germination of control seeds is 100%. If part of the controls does not germinate, the following relation is used to estimate the effect of the substance tested: E = [(M0 – Mc)/(N – Mc)]·100%, where N, Mc, and M0 are the numbers of seeds taken for testing at each concentration, of control seeds that failed to germinate, and of seeds that failed to germinate at a tested concentration of the substance, respectively. The biological meaning of this relation is that it gives an algorithm to reveal, to a certain degree, what proportion of the seeds would not germinate due to the effect of the substance tested. This approach was tested in experiments to study the effect of surfactants on F. esculentum and Allium cepa. It can be used in any experiment where not all control seeds germinate. 4. The group of methods for estimating the biological activity of substances and pollution of aqueous medium by their effect on the mean length of seedlings. Some substances can slow down elongation of seedlings without greatly decreasing germination, whereas others can inhibit both processes. Therefore, it is of interest to develop a method of biotesting and processing of the results, which would take into account and integrate the effect of substances both on the growth rate and germination of seeds. We proposed and tested the following method. Seeds were put into Petri dishes with the tested solution. Then the length of seedlings was measured and the number of seeds that did not germinate was recorded. Further processing and calculation of the mean length of the seedlings took account of the seeds that failed to germinate as seedlings with conventional zero length. The value obtained upon averaging was called the apparent average length (AAL) of the seedlings. The parameter thus calculated combined information on the effect of a substance both on the length of seedlings and germination of seeds. This approach was tested on F. esculentum and Oryza sativa. In the summary table (Ostroumov 1990, Table 1), the corresponding methods are denoted as 1a and 5a. © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 35 Friday, November 11, 2005 1:42 AM BIOLOGICAL EFFECTS OF SURFACTANTS 35 Other variants of the methods tested are based on the hypocotyl orientation disorders in seedlings affected by biologically active substances (BAS). The orientation of hypocotyls in those experiments was registered by eye. At a certain stage of development the overwhelming majority of Camelina sativa hypocotyls are oriented vertically. An example of such a study is given in the chapter on nonionogenic surfactants. Processing of the results of experiments with seedlings: after the initial results are obtained, they need to be statistically processed. The “Statgraphics” package was used. After calculating the mean length (or apparent average length) of seedlings, in some experiments it is reasonable to calculate the rate of elongation (V) and the percentage of inhibition (I) using the relations V = [x(t2) – x(t1)]/(t2 – t1), I = (1 – xexp /xcontr)·100% = [(xcontr – xexp)/xcontr]·100% where x(t1) and x(t2) are the mean lengths of seedlings at times t1 and t2; xexp is the mean length of seedlings in the variant where BAS of tested (polluted) aqueous medium are active; xcontr is the mean length of seedlings in the control. The Student’s t-test was used to assess the statistical significance of the difference between xexp and xcontr. Nonparametric criteria were also used, such as Wilcoxon’s test and Kolmogorov–Smirnov test. The use of these criteria is provided by the “Statgraphics” package. However, it can compare only samples of the same volume using Wilcoxon’s and Kolmogorov–Smirnov tests, though in practice samplings can have different volumes; this is a drawback of this package. It is eliminated in the “Statis” statistical package developed by A.P. Kulaichev at the Biological Faculty of the Moscow State University. 5. Methods of experiments on the effect on the rhizoderm cells. Seeds of buckwheat F. esculentum, white mustard S. alba or soft wheat Triticum aestivum were put in Petri dishes on filter paper. Solutions of nonionogenic surfactant Triton X-100 (Schuchardt) (7–15 ml) in distilled water were added into the dishes, and incubation was carried out in the dark. The same volumes of distilled water were added into control dishes, and the incubation was performed likewise. Experiments with F. esculentum cultivar Shatilovskaya-5 were performed in two variants. Variant 1. A total of 17–20 seeds was put in a Petri dish and 10 ml of test solution was added. Incubation was carried out at 27°C. When the concentration of surfactants increased, the number of germinated seeds decreased, which led to reduction in the total number of seedlings. The number of seedlings that failed to fix was registered in 45 h. Variant 2. Seeds were soaked not in a surfactant solution (as in variant 1) but in distilled water and then incubated. In 21 h, medium-length seedlings were transferred into new Petri dishes with Triton X-100 solutions. Ten seedlings were put in each dish. The number of seedlings that failed to fix was registered 43 h after the onset of soaking in distilled water. In experiments with S. alba VNIIMK, 15 seeds were put in a dish and 7 ml of test solution was added. Incubation was carried out at 18°C. © 2006 by Taylor & Francis Group, LLC TF4005 07 Chapter 2.fm Page 36 Friday, November 11, 2005 1:42 AM 36 S.A. OSTROUMOV In experiments with T. aestivum, 3 or 4 seeds or seedlings of winter wheat (cultivar Zarya) were put in a Petri dish, and 7–15 ml of Triton X-100 solution in distilled water or an equal volume of distilled water was added. In one of the variants of the experiment, seedlings were put on perforated disks, and the roots were completely immersed in water through the holes. Incubation was done at 27°C in the dark. 2.1.2.5 Mollusks Marine and freshwater mollusks were used. The role of mollusks is important both in the fishing industry and as a mariculture component. The total catch of marine bivalve mollusks exceeds that of all other groups of invertebrates taken together. In the monetary respect, the role of invertebrates (including mollusks) is more significant than in the weight aspect (Moiseyev 1985). Bivalve mollusks are of great importance as part of biofouling. Some bivalve mollusks became dangerous intruders (e.g., Dreissena polymorpha). Bivalve mollusks are included on the list of species of the Red Books of Russia and other republics of the former Soviet Union. (See also The IUCN Invertebrate… 1985.) The nomenclature used is according to Zatsepin and Rittikh (1975) and Zatsepin et al. (1978). 2.1.2.5.1 Freshwater mollusks The following mollusks were used in the study: Unio pictorum (L.) s. lato (common pearl clam), U. tumidus Philipsson s. lato (common cline shaped pearl clam), Crassiana crassa (Philipsson) s. lato (=U. crassus) ((thick) oval pearl clam), Anodonta cygnea (L.) s. lato (common anodonta). Family Unionidae, order Actinodontida, subclass Schizodonta (=Palaeheterodonta), class Bivalvia. The organisms were collected in the Upper Moskva River on the stone sandy silted bottom at a depth of 40–60 cm. The rate of filtration was determined by the decrease of the optical density of the incubation medium as a result of the decrease in the number of algal, cyanobacterial or Saccharomyces cerevisiae cells preliminarily added and removed by filtration. The author thanks N.N. Kolotilova and E.A. Kuznetsov for assistance. If not indicated otherwise, a typical experiment was as follows. Eight mollusks of U. pictorum were placed into beakers with 1.5 l of settled tap water (STW), i.e., tap water that had been kept at room temperature for at least 1–2 days. In variant A (control, no surfactant), 8 mollusks of 20.8 to 30.4 g in weight (mean weight, 24.3 g; wet weight with the shell) were put in a beaker. In variant B (with surfactant), 8 mollusks of 21.4 to 36.7 g in weight (mean weight, 26.1 g) were in a beaker. In both variants, a suspension of S. cerevisiae cells was preliminarily added into the water (SAF-Moment, S.I. Lesaffre, 59703 Marcq, France). The final concentration (dry weight) was 263.1 mg/l. Besides, an additional control was performed (variant C). In variant C, the beakers contained STW with a suspension of S. cerevisiae cells without mollusks. In variant C, no surfactant was added. The beakers in all three variants were incubated at a temperature of 17°C. Aliquots were taken and the optical density was measured at 500 nm (Hitachi spectrophotometer 200-20, optical path 10 mm). © 2006 by Taylor & Francis Group, LLC