Biological effects of surfactants - Chapter 2 potx

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.. Introduc

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

(Synecho-coccus 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 sperm plants (Sinapis alba L., Fagopyrum esculentum Moench, Lepidium sativum L.,

angio-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 theirecology, their role in the ecosystems, and the possibility of their use as biologicalresources Diverse biological material made it possible to obtain broader and moresubstantiated conclusions on the possible role of synthetic surfactants as pollutants Below we describe the justification of the choice of the objects (organisms) andmethodological aspects of their use The nomenclature of cyanobacteria is givenaccording 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) Thenomenclature of invertebrates is according to Zatsepin and Rittikh (1975), andZatsepin 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

2

Chroococcophyceae, order Chroococcales) is one of the four main genera of marinecyanobacteria 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 phytoplanktonand 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 toseawater (e.g., Kondratyeva et al 1989; South and Whittick 1987) Cyanobacteriacan reach a share of 60% of all chlorophyll in marine ecosystems in the upper 50 mand about 20% and more of the total primary production (e.g., Sieburth 1979) In thisstudy, 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, 59.7mol.%) 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 theDNA, 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 weremaintained 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 of22°C and at a permanent illumination of 20 (micro Einstein) m–2s–1 Synthetic-surfactant solutions added to the cultures were sterilized by filtration through Acro-disc sterile filters (Gelman Sciences) with pore diameter of 0.45 µm The absorptionspectra were recorded using a Shimatzu UV 3101PC spectrophotometer to charac-terize 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, whichallowed 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

Micro-biology, Biological Research Institute, Leningrad University The cultures weregrown in 50-ml conical flasks on liquid medium (20 ml each) of the followingcomposition (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 cellbiomass, 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 (ing): 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

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 cultureswere 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 ofamino 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 basisfor colonization of this biotope by other periphyton organisms They are widespread

in biological films at hard surfaces in various marine ecosystems, in biofouling onthe surfaces of hydrotechnical constructions and ships Thus, these bacteria are ofpractical importance They have a specific lifecycle, which includes budding of thedaughter cells from the end of the hypha The maternal cell attaches to a hardsubstrate, while the budded daughter cell is transported away by the water flow andprecipitates on a substrate in another place, attaches to the surface, elongates, andbuds off a new generation of daughter cells (Moore and Weiner 1989) The effect ofsynthetic surfactants on these bacteria was barely studied; particular effects of non-ionogenic 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 sterileseawater; Difco Laboratories, Detroit) This medium is more advantageous ascompared with the media used earlier as it is more economic and convenient for theexperiments, 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 incubationwere carried out in a thermostatted room at 25°C without mixing The density of theculture in all experiments was measured spectrophotometrically at a wavelength of

600 nm Additions of TX100 were sterilized by passing them through a SterileAcrodisc bacterial filter (Gelman Sciences), 0.2 µm The experiment included tworepeats 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)

2.1.2 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 discineae, family Thalassiosiraceae (9 genera)) (according to the other classification:class Centrophyceae, order Coscinodiscales, family Thalassiosiraceae, 11 genera,

Coscino-predominantly in marine plankton) The species of the genus Thalassiosira Cleve

(about 80 modern and fossil species) are widely represented in all geographicalzones, 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 inmarine plankton, very important as a feed resource for many species of commercialfish The diatoms make a significant contribution to the global processes of atmos-pheric carbon fixation and oxygen evolution, participate in the processes of self-purification 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 FeCl3and 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 Fischerhemacytometer 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 allvariants The culture in the stationary growth phase was used for inoculation Theillumination regime in the inoculation was as follows: light, 14 h; darkness, 10 h;intensity of illumination was 254 (micro Einstein) m–2s–1 Temperature, 17°C

2.1.2.2 Green algae

Five classes including the Protococcophyceae, which is sometimes considered as anorder 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 includingfish 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 manyaspects of biochemistry and physiology The Protococcophyceae are actively studied

to be used for intensifying the purification of polluted waters and producing proteinand vitaminized fodder By the role they play in the natural ecosystems and biogeo-chemical processes of the biosphere they can be second (not always) only to thediatoms (Gollerbakh 1977; Matvienko 1977; Kondratyeva et al 1989; South andWhittick 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

on the ash plateau free of vegetation in the vicinity of Tyatya Volcano (KunashirIsland) 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 traceelement 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 waterwas used

The growth flasks contained 50 ml of medium each Each variant was made intwo repeats The algal cultures were incubated at an illumination of 3000 lux at roomtemperature

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 2000lux The sources of illumination were fluorescent lamps LB-40 The cultures weregrown in 250-ml Ehrlenmeyer flasks The number of cells was determined by directcalculation 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 2.47million/ml (experiment 2)

Pulverized podzolic soil from the Kirov Region was used in the experimentswith soil cultures (the collection site was the experimental field of the Kirov Agri-cultural 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) wasplaced in Petri dishes, where 10 ml each of an aqueous solution of TDTMA (indistilled 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 (wateringwith distilled water by weight)

Abundance of the algae in the soil cultures was determined by the generallyrecognized method of the direct count of cells in the soil suspension using a lightmicroscope The author thanks Prof E.A Shtina for consultations and assistance inthis part of work

We also used other algae in the experiments They were grown on standardmedia 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 theformer Soviet Union) generally inhabit internal continental reservoirs They possessall major types of nutrition: autotrophic, saprophytic, holozoic (peculiar of higheranimals); are capable of mixotrophy They are involved in self-purification of aquaticobjects, 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 ofpolluted waters and for cultivating in the photoautotrophic life support systems(Safonova 1977; Kondratyeva et al 1989)

The culture Euglena gracilis Klebs var Z Pringsheim was grown

photoorgano-trophically in 100-ml flasks at a temperature of 26°C and illumination of1,500–2,000 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 preparesolution I, 695 mg of FeSO4·7H2O and 930 mg of Na2EDTA was taken anddissolved in warm bidistilled water, pH was adjusted by NaOH, and then water wasadded up to 100 ml Solution II was prepared by taking (in g per liter of bidistilledwater): 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 theinoculate, which was sampled in the mid-logarithmic phase of growth, was 5 ml atthe 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 areused 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), andEuropean agencies (European Organization for Economic Cooperation and Devel-opment 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 inlaboratories (industrial enterprises, chemical institutions), where more susceptibleorganisms do not survive Therefore, the method is free of one disadvantage ofhighly-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 thehumanitarian point of view and in the view of official recommendations by the Inter-national Union of Toxicology (IUTOX) In 1985, the IUTOX Executive Committeepublished an official statement that “alternative methods of testing, which do notrequire the use of animals, should be in common use (after their comprehensive sci-entific testing)” (see Telitchenko and Ostroumov 1990) High economic efficiency

of this biotest is important for Russia under the current conditions of sciencefinancing 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., Wang1987; Wang and Williams 1990; Davies 1991; Davies et al 1991) The method wassuccessfully used in the laboratory headed by Professor Ivanov to assess a broad class

of biologically active substances (BAS), including compounds important forpharmacology (The author is deeply grateful to V.B Ivanov and all colleagues at thelaboratory for numerous consultations and discussions of the results) In studies

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 theDepartment of Soil Science, Moscow State University, to assess the toxicity of vari-ous biological preparations This method is one of the main tools in investigatingallelopathic 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 BotanicalGarden, Ukrainian Academy of Sciences (A.M Grozdinsky, E.A Golovko and otherspecialists; the author is grateful to them for the seeds of cress) The method was alsoused at the Institute of Hydrobiology (Kiev) for water quality assessment (Sirenkoand Kozitskaya 1988) Though this method was recommended by the U.S Environ-mental Protection Agency (U.S Environmental Protection Agency 1982), it wascomparatively rarely used in the U.S The variant of the method we used was moreadvanced methodologically compared to the works by Wang and by Davis andco-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 integralmorphogenetic index, which unites information on the effect of a tested chemical orpolluted 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) Traditionaltechniques and some less traditional variants and approaches were evaluated A list

of some major effects, based on the biological activity of substances – varioussurfactants and some pesticides (Maksimov et al 1988), is given in Ostroumov(1990) (Table 2 therein) The following details of some evaluated variants ofmethods 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 sub-

stance or aqueous medium at 100% germination of seeds, a Petri dish (10 cm in eter) with the seeds of test objects put on filter paper was filled with a correspondingwater solution (usually 7, 10 or 15 ml) Controls were filled with distilled water (DW)

diam-or settled tap water (STW) Incubation was carried out in the dark at room

tem-perature or at 26–28°C After a time interval t1 the length of the seedlings cotyl + root or only root) was measured Further measurements were made at times

(hypo-t2, t3, and so on The measurement results were processed statistically using metric methods (see below) Calculations of the mean rate of elongation and percent-age of inhibition were used (Ivanov 1974) This group of methods was used for white

nonpara-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 seed- lings 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 firstpreincubated in DW or STW Then seedlings (of predominantly fixed length) were

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 anequal number of seedlings Control seedlings were transferred into new Petri disheswith 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 lings in the test solutions was compared with that of the control seedlings The resultswere processed statistically The method has the following restrictions: preincubation

seed-in DW or STW may slightly smooth the effect and decrease the sensitivity of themethod as compared to the methods of group 1 The methods of biotesting onseedlings 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 is100% If part of the controls does not germinate, the following relation is used toestimate 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 atested concentration of the substance, respectively The biological meaning of thisrelation is that it gives an algorithm to reveal, to a certain degree, what proportion ofthe 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 nation, whereas others can inhibit both processes Therefore, it is of interest to devel-

germi-op a method of biotesting and processing of the results, which would take intoaccount and integrate the effect of substances both on the growth rate and germi-nation of seeds We proposed and tested the following method Seeds were put intoPetri dishes with the tested solution Then the length of seedlings was measured andthe number of seeds that did not germinate was recorded Further processing andcalculation 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 uponaveraging was called the apparent average length (AAL) of the seedlings Theparameter thus calculated combined information on the effect of a substance both onthe 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

Other variants of the methods tested are based on the hypocotyl orientation orders in seedlings affected by biologically active substances (BAS) The orientation

dis-of hypocotyls in those experiments was registered by eye At a certain stage dis-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 actants Processing of the results of experiments with seedlings: after the initialresults are obtained, they need to be statistically processed The “Statgraphics”package was used After calculating the mean length (or apparent average length) of

surf-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 themean 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 ence between xexp and xcontr Nonparametric criteria were also used, such as Wil-coxon’s test and Kolmogorov–Smirnov test The use of these criteria is provided bythe “Statgraphics” package However, it can compare only samples of the samevolume using Wilcoxon’s and Kolmogorov–Smirnov tests, though in practicesamplings can have different volumes; this is a drawback of this package It is elimi-nated in the “Statis” statistical package developed by A.P Kulaichev at the Bio-logical Faculty of the Moscow State University

differ-5 Methods of experiments on the effect on the rhizoderm cells Seeds of

buck-wheat F esculentum, white mustard S alba or soft buck-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 incubationwas carried out in the dark The same volumes of distilled water were added intocontrol 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 ofsurfactants increased, the number of germinated seeds decreased, which led toreduction in the total number of seedlings The number of seedlings that failed to fixwas 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 ferred into new Petri dishes with Triton X-100 solutions Ten seedlings were put ineach dish The number of seedlings that failed to fix was registered 43 h after the onset

trans-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

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 indistilled 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 werecompletely immersed in water through the holes Incubation was done at 27°C in thedark

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 marinebivalve mollusks exceeds that of all other groups of invertebrates taken together Inthe monetary respect, the role of invertebrates (including mollusks) is moresignificant than in the weight aspect (Moiseyev 1985) Bivalve mollusks are of greatimportance 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 alsoThe IUCN Invertebrate… 1985.) The nomenclature used is according to Zatsepin andRittikh (1975) and Zatsepin et al (1978)

class Schizodonta (=Palaeheterodonta), class Bivalvia

The organisms were collected in the Upper Moskva River on the stone sandysilted bottom at a depth of 40–60 cm

The rate of filtration was determined by the decrease of the optical density ofthe incubation medium as a result of the decrease in the number of algal, cyano-

bacterial 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), 8mollusks 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 (dryweight) 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 variantswere incubated at a temperature of 17°C Aliquots were taken and the optical densitywas measured at 500 nm (Hitachi spectrophotometer 200-20, optical path 10 mm)