the use of biological tests for the examination of...

THE USE OF BIOLOGICAL TESTSFOR THE EXAMINATION OF PESTICIDES

ALFRED KAMIIESKI

Medical Academy, Chair of Medical Biology, 90-64 7 Lód, Pkc 9-Maja 1,Poland

ABSTRACTThe increasing input of chemicals to the environment points to the need tocontrol the input of pesticides. This requires a detailed analysis, not only ofpesticide efficiency, but also of ecological response of the environment to apesticide, before it is introduced for common application. Such an analysisshould cover: (1) spectrum of species sensibility, (2) results of qualitativeand quantitative bioassay analyses, (3) community response to the pesticidecontamination, (4) neutralization, toleration and adaptation abilities ofecosystems, (5) share of a given pesticide in total toxity caused by the chemicalcontamination, (6) ecological predictions of environment contamination withpesticides. All these features of a pesticide can be expressed in numbers orby graphs with numerical axes. The paper presents a project, based on personalexperiments, of an application of multi-assay bioindicatory analysis for theecological evaluation of pesticides already used or being introduced for use.The experimental animals were: Asellus, Lumbriculus, Cloeon, Daphnia,Planorbarius, Lebistes. They were used as bioindicators of phospho-organicinsecticide applied in 0.01—0.001 p.p.m. concentrations. The basis for bio-indication of poisons in environments, and their characteristics in relation tothe above six points were accomplished with the help of bioassay and the appli-

cation of models of aquatic and terrestrial ecosystems.

I. GENERAL METHODOLOGICAL PRENCIPLES

The use of living organisms for studies of pesticides has a rather longhistory"2' . The main objective of these tests, which have been conductedon many species and with different techniques, is to estimate:

(1) Practical value of pesticides,(2) Mechanism of action,(3) Damage to human health,(4) Methods of detection and quantitative evaluation.

The increasing contamination of the environment with chemicals and athreat to the normal functioning of biocoenoses made it necessary to extendthe above-mentioned range of investigations so as to provide informationon the ecological consequences of the use of pesticides in the quantities neededfor plant protection. The rate of treatment with pesticides is limited by healthfactors and by the need to protect the environment against excessive contami-nation.

255

ALFRED KAMINSKI

Ecological screening should involve determination of the major environ-mental effects of a pesticide and in particular:(1) The biotaxonomic spectrum of toxic activity (BSTA), i.e. threshold con-

centrations contaminating the bionts being representative of pollutedbiocoenoses.

(2) Response of ecosystem structure and function to pollution,(3) Persistence of pesticides in the environment,(4) Effect of pollution with pesticides on the cumulative toxicity of all the

coexisting chemical pollutions in the environment.In all, the obtained information should extend the scientific criteria of

pesticide evaluation and facilitate their qualitative optimization and rationalapplication.

Long-term field and laboratory experiments are needed in ecologicalstudies. The other disciplines involved here, such as biology, medicine,physicochemistry and agricultural technology, provide results which arefrequently difficult to coordinate and interpret objectively.

Because of the complexity of the environmental (ecological) toxicology ofapplied poisons it is difficult to carry out such investigations in one labora-tory.

Rapid evaluation of the properties of a pesticide (particularly one whichis being introduced on to the market) requires the use of environmental—toxicological techniques which should be as simple as possible.

The objective of this report is to present such a simplified system basedexclusively on biological test analysis (TA).

TA is a research process in which a test biont (TB) or an ecological testsystem(ETS), for instance, a laboratory model of the ecosystem, performs therole of a double indicator in relation to the pesticides, which permits theworker:(1) to detect and quantitatively estimate biologically active substances,(2) to obtain a complete toxic—dynamic characteristic (TDC).

The primary reason for the use of TA for both purposes was to preserveas far as possible the structure of the polluted material (water, soil, air, tissues)as a result of elimination of complex, physical and chemical treatments.

The second idea was to introduce as a subject of analysis the quantitativedetermination of the actual toxicity of pesticides under existing environ-mental conditions. This actual toxicity is the net value of all additive, syner-gistic and antagonistic interactions in environments polluted with pesticides.

The third idea was to adjust the technique so that it could be used in con-junction with any chemicophysical analysis (CPA) and not in competitionwith it. This adjustment consists of an appropriate stimulation of the sensi-tivity and selectivity of TA. For this purpose it was necessary to broadenthe TA method by the introduction of new and particularly sensitive testbionts (STB) and to develop new analytical criteria for the identification ofpesticides. It was also necessary to create an artificial (control) analyticalenvironment common to all the TB, and to unify the techniques of investiga-tions in different natural environments.

The fourth idea was to minimize and speed up the process of analysis sothat it could keep pace with the environmental—toxic processes stimulated by

256

THE USE OF BIOLOGICAL TESTS FOR THE EXAMINATION OF PESTICIDES

pesticides, without inhibiting them artificially, whether they were instan-taneous or long-term.

The fifth idea was a maximum utilization of one TA conducted simul-taneously for many TB species to obtain the greatest possible quantity ofdata characterizing the toxic—dynamic properties of a pesticide.

Differences between the TA and CPA methodsQualitative identification of a pesticide with the TA method consists of the

assessment of a set of symptons of the test response (TR) on TB (or ETS) thatwill be discussed below. As is well known, the quantitative estimation of atoxicant consists of the assessment of a threshold concentration (C50),which produces a TR in 50 per cent of the TB population.

Thus, TA indicates the effect of biologically active substances, and on thebasis of replicability of this direct effect a conclusion may be drawn as to thenature and concentration of the active poison. It therefore provides directinformation to the toxicologist or to the specialist applying pesticides inpractice.

CPA, by comparison, is based on measurements of chemical reactions andphysical processes not related to the actual biological activity of the analysedsubstance. From the point of view of environmental (ecological) toxicologyCPA provides information which may have little direct bearing on the naturalenvironment under study. This is particularly so if factors not detected by CPAact to inhibit or stimulate the effects of a pesticide. Such a disjunction mayalso occur if the natural structure of a substance is broken down during itspreparation for CPA.

Thus, TA measures the results of direct effects under conditions closelyapproximating to the natural environment. It also measures the biologicaleffects of the activity of pesticides under replicable artificial conditions createdby the investigator. But these artificial conditions must always be within thetolerance limit of TB. which can always be measured precisely in controlsystems. Therefore, the purity of the TB reagent is analogous to the purity ofa chemical reagent or to the efficiency of a measuring instrument. This purityis determined by the limit of tolerance of the analysis to side factors (factorswhich are present but not analysed) which should not disturb the physiologi-cal homeostasis (condition) of TB. The role of the energy potential of homeo-stasis (EPH) for the practical application of TA will be analysed in the sectionconcerned with methods. But it should be noted here that a TB particularlysensitive to pesticides, i.e. STB, allows the TA to be carried out in such ashort time that many side factors, including other poisons, will not be ableto disturb either EPH or the specificity of TR to a definite pesticide.

With TA one can determine not only particular pesticides but also thecumulative toxicity of the environment (water, soil, air, biomass) contaminatedwith chemicals. And in that case TA cannot be replaced by any other method.

Quantitative biochemical analysis (BCA) of organophosphoric pesticides,based for instance on the acyl choline matrix4'5'6 and which is in some wayintermediate between CPA and TA, is characterized by a rather high sensi-tivity but it is not specific for particular toxicants and it is sensitive to the sidefactors. Matrix (receptor of a pesticide) in this method is 'naked'. not beingprotected by the cover provided by the TB biomass in each TA. Due to the

257

PAC—42/1-2/K

ALFRED KAMINSKI

homeostatic nature of TB and the readily visible response of such an organismto pesticides, TA gains a valuable selectivity and loses none of the advantagesof BCA.

Searching for new TB and, particularly, STB consists of the selection ofsmall and common carriers of a reactive and specific receptor of the toxicants,which are surrounded by the most effectively functioning EPH cover. Thiscover should eliminate side factors, efficiently transmit the subject of analysisto the receptor, and produce a multisymptomatic TR, which is the basisof the functioning of TA. Methodologically more similar to TA are the testsof pesticides conducted on cellular organelles, on chloroplasts for instance7,involving biochemical measurements of the effect of the degree of inhibitionof Hill's reaction but not taking into account the external symptoms of theTR, which could provide additional criteria of pesticide identification,besides the determination of a threshold concentration.

Similarly, the measurements of the inhibition by pesticides of enzymeactivity in TB8 represent an interesting junction of the methodologicalprinciples of TA and BCA, which, however, do not specify toxic substancesin the analytical sense. The above-mentioned techniques are of great im-portance for the idea of TA functioning although they have a rather theoreticaland auxiliary character.

Certainly, the present short report cannot provide a more complete outlineof the theoretical bases of TA nor ecosystemic test analysis of pesticides (ETA).

However, to characterize the 'ideology' of this technique, it should bestated that even the most detailed CPA cannot provide reliable evidence forthe lack of toxic contamination of the environment if it is used separately,without any verification by TA, while TA used separately can also provideevidence of toxicity in relation to unident/Ied poisons present in the field (in asample) independently of whether the appropriate CPA methods exist or not.Therefore, fully developed TA can provide a methodological basis for thefunctioning of a system of permanent bioindication (SPB) summing andevaluating the effects of chemical impact on the natural environment. SPBcan act through STB permanently present in the environment as naturalreactors (in situ). They play the role of micro-, macro- and megabioindicatorsof toxicity or effectiveness and ineffectiveness of treatments with chemicals.

The first step to create the STB at particular levels of organization oforganic matter is a simple TA and ecological test analysis (ETA) leading tothe selection of STB and to modelling of ecological processes of environ-mental contamination in the laboratory. Parallel and successfully developedCPA techniques for pesticides represent an indispensable research instru-ment for scientific interpretation of the processes occurring in environmentspolluted with chemicals. Combination of these techniques can provide themost useful results.

II. TECHNIQUE OF TA REALIZATIONIt is impossible to present all technical details of TA in this short report

and only the main methodological principles will be illustrated by theanalysis of selected examples of pesticides and other toxic substances suffi-

258


ciently representative for the present pollution of the natural environmentwith chemicals.

(1) Subjectof TA

(a) Standard toxicants (ST)ST such as gamma-BHC (gamma-hexachlorocyclohexane), DDVP (2,2-

dichlorovinyl ester of dimethylphosphoric acid), Chlorofenvinphos (dichloro-1-1/2,4 dichiorophenylvinyl ester of diethyiphosphoric acid), PMPF (pina-coline ester of methyifluorphosphonic acid), which is an inhibitor of cholineesterase killing vertebrates, and mercuric chloride (HgCl2) were testedin the form of impregnates on pure starch. The method of complete TBwas used according to the technique described below, in aquatic andstandard terrestrial environments both being strictly replicable (ST formula—Figure 1).

0% /O.CH.C.(CH3)3 H3C—O

H3CF H3C—OO —CHC— Cl2PMPF DDVP

HC 0 052 Cl HCI/\ II Cl HH5C2—O O—C=CH H Cl

-ClCljH

Cl y-BHC

Chiorofenvinfos(ChLVF)

Figure 1. Toxicants used as examples in TA

(b) Dilution of toxicantsAll insecticide ST were first diluted in acetone at a concentration of

lg/100 ml; the highly toxic PMPF was similarly diluted in propylene glycol,and mercuric chloride in redistilled water. These concentrated solutions wereused to prepare aqueous working fluids, the concentrations of which areindicated in the tables and figures. The series of concentrations of the workingfluids, used to impregnate the starch matrix, forms a decreasing geometricprogression with the ratio two.

(c) Introduction of toxicants to ecological test systems (ETS)Aquatic ETS of a volume of 4L were contaminated with 11 of natural

water (from a culture of TB) containing an appropriate concentration of STindicated in the tables. Terrestrial ETS were contaminated by the impregna-tion of soil with aqueous-acetone solutions of ST at the concentration ofacetone tolerated by all TB.

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ALFRED KAMINSKI

(3) Experimental environments (EE)

(a) Aquatic (AEE)The mineral composition of the diluent common for the tested toxic

substances (EE) was determined for all aquatic TB. It contained the followingcompounds, excluding water of crystallization: NaCl 1.00, KC1 0.250, MgCl20.120, CaCl2 0.250, NaHPO4 0.00620, NaHCO3 0.125, and redistilled waterto 1000.0.

This composition is the equivalent of Tyrode's fluid diluted eight times.For Spirostomum the described AEE was additionally diluted eight timeswith redistilled water prepared in a quartz apparatus without metallic con-nectors as this animal is sensitive to traces of metals above 0.01 p.p.m.

The quality and proportions of the concentration of particular componentsof EE correspond to the electrolytes of vertebrate blood. So, it is possibleto use TB for the analysis of body fluids and tissues contaminated with insecti-cides.

Environmental—toxicological analyses are generally conducted in filtratednatural water coming from the TB cultures. Only when the effect of the naturalwater on the results of analysis should be determined, or a verdict evaluatingthe quality of a pesticide, comparable with that of other authors is needed,should EE be used together with an identical (international) standard of thetested substance.

(b) Terrestrial (TEE)They represent systems in many respects similar to a fluid environment,

despite their dryness. This is pure potato starch. It readily disperses toxi-cants through the impregnation and evaporation of solvent. Evaporationis either complete (acetone, ethanol, ether, benzene, cyclohexanone, water) orpartial, as in the case of water. Due to such partial evaporation a very valuablemodel approximating moist soil with any proportions of minerals or othercomponents of a natural biotop can be obtained.

Similarity between the terrestrial and aquatic EE results from the precisedispersal of a toxic substance due to impregnation, capillarity, mixing andmovements of the particles of impregnant in the presence of mobile TB,which are adapted to a loose and dry environment as they are flour-dwellingorganisms. It has been found that the replicability of the results of TA inimpregnants and aqueous solution is identical'4. Powder preparations canalso be dispersed in the starch in dry form by grinding weighed portions.

(c) Samples of natural environments (NE)The use of TA for the ecological studies of pesticides requires the determina-

tion of their residues (residual toxicity) in various materials and environ-ments (soil, sediments, tissues, air). Generally, each material containing atoxic substance can be tested by TA through the direct contact with TB.Asellus and Lumbriculus live in and feed on plant tissues. Great concentra-tion of plant material in a sample does not disturb the physiological conditionof these TB. Animal tissues, blood and soil are also tested through directcontact or in homogeneous extracts diluted according to the technical princi-ples of TA described below. Duration of the analysis of NE is limited only bya danger of the decay of test material.

266

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ALFRED KAMINSKI

(4) Test response (TR)Unlike the majority of the accepted methods of TA by which LC50 is

determined, i.e. the concentrations of toxic substances producing lethalresponse (LR) of TB, in our experiments the symptoms of TR are analysed invivo. TR is a replicable set of symptoms produced in response to the effect of atoxicant on TB. Lethal response has a supplementary importance as aspecifying criterion. TR always occurs much earlier than LR and at concen-trations (C50) many times lower (Table 2).

Motor paralysis is a common and readily visible TR to insecticides inAsellus, Daphnia, Tenebrio and Lebistes. Additional symptoms are such ashypercinesia, which precedes TR, and the sequence of paralysing particularorgans.

Organophosphoric inhibitors of choline esterase (IChE) primarily paralysebreathing (movements of the gill cover) and then the heart, which is readilyvisible through the integument of small TB. In crustaceans a prolonged agi-tation of branchial limbs is observed. It is different in the presence of IChE(acceleration) than in the presence oiy-BHC (discordance). Mercury paralysesrespiratory movements (akinesis) in Asellus.

The results of TA presented in the tables and figures concern TR inArthropoda, fish and Spirostomum; their motor activity and static reflexesare paralysed. Special attention should be paid to TR in Lumbriculus. Thisorganism is characterized by permanent mobility, and it rapidly escapes inresponse to a touch stimulus. This response can be reversed in the presence ofIChE at very low concentrations—the irritated TB falls in a tonic contraction(spiral kink) instead of escaping. This response is a result of the inhibition ofacylcholine esterases, which are very active in this TB13, and it disappears with-in 3—16 days after the separation of TB from the poison. Reversibility of TRis specific for particular IChE and represents a criterion specifying them.High concentrations of IChE result in an irreversible response leading to LR.

The differences between C50 and LC50 are also specific for particulartoxicants. Detailed symptomatology of TR is the subject of a separate publi-cation (in press) and exceeds the framework of this report. The differencesbetween C50 and LC50 also occur in relation to other TB.

(5) Quantitative TAThe methodological basis of TA is the assessment of the maximum dilution

of a toxicant (R50) which can produce TR in 50 per cent of the TB populationwithin a definite time of absorption, t. In analyses of standard insecticides,the concentration of the prepared solution is known and the assessment ofR50 provides direct information about C50. When the material polluted witha toxicant of unknown concentration is analysed, only R50 can be determinedand this value can be used for the calculation of C50 by comparing it with thestandard.

The absolute concentration of a toxicant in the material can be calculatedaccording to the simple formula

C =

where C is the concentration of poison in the sample (p.p.m.), Ra50 is the

268


threshold dilution of the analysed solution, R50 is the threshold dilution ofthe standard solution, and f is the concentration of the standard toxicsubstance in the standard solution (p.p.m.).

To find the value of R50, an analytical sequence of poison dilutions mustbe prepared according to an optimum quotient of the geometric series (q).Generally, q = 2 is used to find room in the limited number of containers forthe whole set of analyses ranging from 100 to 0 per cent of respondingTB at all time-moments when TR is recorded, i.e. for numerous t. For specialpurposes and to increase the accuracy of TA, q ranging from 1.5 to 1.05 isused, e.g. for a precise estimation of pesticide quality, identification of poisonor toxicological verdicts.

(6) Calculation of R50 and statistics

R50 can be calculated according to any interpolation probit statistic'5.In our investigations Wiechnowski's method'6' 17 was used. This methodinvolves calculation not interpolation and has an element of self-control. Itneeds a regular course of TB response in the analytical sequence, i.e. a responseproportional to the dosage. This analysis may be modified by appropriateselection of the q value and by high quality of TB. The confidence intervalpresented in the tables is determined by two standard deviations. Afterfive replications of the analysis, the standard deviation does not exceed8—15 per cent, except for Daphnia magna, which is in the interval of 12—17 percent.

(7) Parameter of time in TAThe time-period (t) needed to produce TR in TB in contact with a toxicant

along the dilution gradient depends on its concentration and rate of absorp-tion from the environment. TR was measured along the decreasing gradientof concentrations at time-intervals t selected according to a geometric pro-gression. High frequency of records at the beginning of the gradient is neces-sary because of a rapid increase in TR at high concentrations of the toxicsubstance.

Lower frequency of records at the end of the gradient (low concentrations)coincides with a slow increase in TR or with the lack of increase when a com-pensation point is reached between the resorption rate and TB toleranceto the threshold concentration of a toxic substance. In the presence of y-BHCthe response of Tenebrio disappears within the range of 1—2 dilutions becauseof the metabolic adaptation of TB (internal detoxification).

The time-moment when the increase in TB response along the dilutiongradient is stopped represents an important parameter of the qualitativespecification of a toxicant. It is designated by the symbol tEA and expressedin minutes. It characterizes the critical time-moment of effective absorptionand gives the idea of lower or higher cumulation of the toxicant in TB, andalso of the chemical persistence of this substance in the environment.

The duration of TA depends on the purpose of the analysis. It is determinednot only by tEA but also by the need for collecting additional information,such as lethal response (LR), which follows TR after the period characteristicfor each toxicant. To estimate the biological activity of new insecticides by the

269

PAC—42/1-2/L

ALFRED KAMINSKI

Figure 11. A multitest TA of DDVP in fresh (natural) water, the progress of TR in dilution series,C50/t values for Tenebrio from TA in dry medium, for Mammalia after parenteral application

in mg/kg = ppm.

2 4 8 16 32 6.4t=minutes of TB contact

r=minutes of TB contact

Figure 12. Multitest TA of Chlorofenvinfos, the data analogous to those of Figure 11

270

THE USE OF BIOLOGICAL TESTS OR THE EXAMINATION OF PESTICIDES

0.0005

0.0010

0.0020

0.0040

0.0080

0.016C

0.0310

0.062

0.12 __________________

0.2E

0.50

E

0

2 4 8 16 32 64 128 256 512 1024 2048 minutes of TB contactwith toxicant

Figure 13. Multitest TA of gamma-BHC, the data analogous to those ofFigure 11

Figure 14. Multitest TA of PMPF, the data analogous to those of Figure 11

271

TA in natural water /V-BHCC=2 ppm.

/./I

./ cC'41 '/ I.

/I

TB C50/24h

Ase/lus 0.00063

Ten ebrio 0.26Leb/stes 0.32

Lumbriculus 1.0Spirostomum 1.4flrinhniq 1 6

ALFRED KAMINSKI

TA method, standards are not necessarily needed, but only detailed records ofsymptoms and the range of TR.

The course of TR for different toxicants and TB is given in figures. Pointsin the coordinate system indicate the value of C50 recorded at particulartimes of response (C50/t). When the materials of unknown concentration areanalysed, the value of R50/t is used (Figures 11—15).

EciC

0(-)

Figure 15. The influence of medium humidity (20%) on TR progress in dilution series of BHC(starch impregnation)

(8) Tests of interrupted observation (TIO) and continuous observation (TCO)Records of response at the time intervals fixed in advance (t) provide the

values of C50/t, R50/t and tEA. A set of many t values increases the basis forstatistical analysis of the toxicant and for comparison with the standard. Inaddition, the values of C50/t or R50/t provide a basis for graphic representa-tion of the results. This method can be defined as a test of interruptedobservation (TIO).

The close relation between the onset of TR in TB and toxicant concentrationmakes it possible to use continuous observation and to note the time of TRin particular TB at each concentration of the toxicant. In this way an average

value is obtained for a group of TB composed of 10—30 individuals, depend-ing on the level of the desired accuracy of the analysis. In that case, a sampleof the material under study may be used without dilution or it may be dilutedto half so that TR could be produced in all TB within 3—60 minutes. Therelation between and insecticide concentration is shown in Figure 11 and

272

y - BHC in dry and humid medium

0.

0.

TB Tenebrio molitor

t 60 120 2I0 L 80 960 1920 minutes of TB contact


Table 3. TA of DDVP steam in dynamic micro-air chamber—washer system (compare Figure 3)TB I = Tenebrio molitor; TB II = Daphnia magna

Steam source: 0.25 g of DDVP on 10 cm2 of impregnated Whatman filter-paper

DurationTest response (TR) of TB (t values, mm)

Detected concentrationof DDVP (p.p.m.)

of steam

flow-0L/hIn miniaturedynamic airchamber on

Tenebrio

In Schottwasher I on

Daphnia

In Schott washerII on Daphnia

Washer I Washer II(100 ml) (100 ml)

1 without TR 11.5 without TR 0.013 02 without TR 7.10 without TR 0.041 05

10partial TRtotal TR

2.221.43

without TRwithout TR

0.40 00.82 0

15 total TR 1.17 without TR 0.92 0300 total TR > 1.00 35.2 2.00 0.0070

Table 3. Due to the test of continuous observation. TA can be conductedduring a short time and in a small volume of the sample.

(9) Reversibility of TRTB removed from the analytical series of a toxicant (pesticide) and put

into a pure environment (AEE, TEE) are used for further investigations todetermine the degree of reversibility of TR. The highest degree of reversibilityof the response to insecticides was found in Lumbriculus (criterion of thespecification of toxic substances). Tenebrio is restored after heavy doses ofpyrethrins after a 24-h period of contact with BHC it is restored at a concen-tration of three times that of C50/24 h. The reversibility index (RvI) may beimportant for the practical evaluation of insecticides. It is calculated for aparticular t according to the formula

RvI = C5

Table 4. Reversibility of TR in fresh water after one 24-h TB contact with toxicant dilution series

Toxicant TB species

RvI values throughouTR

t restitution time .Maximum

RT = 24h 48 h 72 h 96 h RT, h RvI

DDVP Daphnia 1 0.72 + + + +ChLVF Lebistes 0.83 0.76 0.76 0.76 24 0.83ChLVF Tenebrio 0.88 0.76 0.53 0.53 24 0.88DDVP Lebistes 1 1 1 1 24 1

DDVP Tenebrio 1.1 1.4 1.4 1.4 48 1.4

7-BHC Tenebrio 2.1 3.2 3.2 3.2 48 3.2y-BHC Lumbriculus 1.6 3.5 5.3 7.6 254 10ChLVF Lumbriculus 2.0 3.7 17 32 336 103DDVP Lumbriculus 7.4 13 54 123 96 394

Abbreviations: RT = duration of restitution; RvI = (C50/RT)/(C50/24h); + denotes lethalreaction.

273

ALFRED KAMINSKI

where Cr50is the concentration in the analytical series to which TR was shiftedfor a TB absorbing a toxicant during time t, and C50/tis the threshold con-centration for TR in TB, determined during time t of absorption (Table 4).

(10) Qualitative analysis and evaluation using TADeveloped TA is primarily used to characterize toxicodynamic properties

of pesticides. Conducted on numerous TB, including the determination of theeffects on their reproduction, it provides a part of the ecological screening.This part gives a taxonomic spectrum of toxic activity (BSTA) which isspecific for each poison. On the other hand, the quantitative criteria, sympto-matology of TR, BSTA, tEA and RvI are the basis for the identification ofinsecticides by TA and simultaneous quantitative analysis.

When the material is contaminated with a mixture of toxicants, objectswith a low sensitivity to insecticides are used (Spirostomum) to detect non-pesticide toxicants, such as heavy metals and detergents, because in that casethey perform the role of STB. In further analyses particular insecticide STBare included in the TA. Preparatory elimination (without reactors)—thermal,distillation and sorptive—has an auxiliary role, the details of which cannotbe discussed here. *

Ifa sample contaminated with a toxicant of unknown quality and quantityis the subject of TA, only the detailed symptomatology of the response of theTB system and the ease of determining the value of R50 for a particularTB can be found. This is a multitest analysis of the cumulative toxicity(MACI) of industrial sewage, river, soil, atmospheric fallout etc.,independent of the amount and chemical quality of poisons contributing tothe total toxicity. MACT should be a component of the chemical indicationof environmental pollution and the creation of a sanitary—ecological securitymargin, besides that of CPA.

The studied TB are frequently many times more sensitive to the testedtoxicants than the human organism. So, this security margin is quite correctfrom the point of view of protecting nature. MACT provides a basic criterionfor the estimation of the effects of soil and water pollution with pesticides.

The CPA method of qualitative analysis of each sample of material con-taminated with pesticides provides the same number of criteria to specifypesticides as TA does. But when the concentration of toxicants is low and thesize of samples is small, the latter is preferred. When a mixture of toxicants isstudied, a system of the MACT indices on any number of TB can always befound.

(11) The use of TA for the detection of toxicological interactions (TI)

Interactions among different toxicants (synergism, addition, antagonism)are the subject of many investigations'8. Commonly used methods of TIdetermination are rather complex. In our experiments the TI screening usedwas based on the toxicological parameters of the ratio of the concentrationof particular components of the mixture. A constant 0.5 C5 /24 h of one toxi-cant is used in the presence of the whole gradient of concentrations of anothertoxicant and vice versa. Synergism occurs when the effect of the mixtureon TB is intensified. In that case the threshold TR is shifted towards lowerconcentrations. When antagonistic interaction takes place, e.g. in the presence

274


of an antidote, the threshold (limit) is shifted towards the beginning of thegradient. The ratio of weights for both the components of the mixture rangesfrom a fraction to a multiple. Due to the double versions of weight ratios,an optimum for maximum interaction can be found. In the third version theoriginal mixture of known composition is diluted in such a way that a constantratio between the weights of both components is preserved. This ratio may beobtained either on the basis of absolute weights (in which case it is not toxi-cologically defined) or on the basis of the weights proportionally related tothe value of C50 for each of the components, which provides a better situationfor the proper interpretation of the results.

Ill. ECOLOGICAL TEST ANALYSIS (ETA)(1) Objectives

This analysis, like TA, has two objectives: first, an assessment of the responseof the biont, i.e. in this case a whole natural model ecosystem (ETS) or a partof it, to contamination with a toxicant (pesticides), and secondly, the fate ofthe toxicant in contact with the components of the ETS.

These two problems are equally important from the point of view of thepractical application of pesticides. The complex objective of ETA may bedivided into smaller units by the use of laboratory ETS. Methodologicallythe following phenomena can be distinguished here:

(1) contamination and its effects on the structure and functioning of ETS,(2) detoxification by biomass, natural absorptive systems and by chemical

processes in ETS,(3) threshold of tolerance to contamination with toxicant,(4) restoration after a single contamination,(5) the above-mentioned phenomena under the dynamic conditions of a

continuous flow of the toxicant through ETS.

(2) Aquatic ETS—staticDuring the long-term experiments a static biological unit was devised,

which formed an element of the multi-component battery of ETS (Figure 16).This unit (4 litres) consists of all ecological trophic levels of the natural watersof alpha-mesotrophic type: sand—a layer of 4 cm, aquatic plants such asValissneria spiralis, Myriophyllum pinnatum, and aquatic TB such as Asellus,Lumbriculus, Planorbarius, Spirostomum and Daphnia. At controlled photo-period and uniform nutrition (dried leaves of Populus nigra and Populuscanadensis) this ETS can be maintained in a state of functional balance (homeo-stasis) over many months, independent of the season. The biomass of plants,animals, microplankton, microbenthos and saprobionts are able to maintaina proper nutrient cycle. The chemical properties of the water are normal. Dueto the mineralization processes a sedimentary material similar to young peatis deposited without detectable decay processes. The state of homeostasisis identified on the basis of the behaviour of animal TB sensitive to oxygendeficiency and the changes in the chemical properties of the water.

The battery of ETS plays the same role as TB in TA. TR resulting from pol-lution with toxicants can be observed in all bionts (plants and animals) and

275

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I -


Detoxication of water in static ETS urve Toxicant 05-residues of initial charge num- and C1 hber ppm.

100PMPF

90 Hg Cl2 /•7

80 ® HgCi70

— DDVP —® (2)

W4(Y-BHC

ChLVF© (2) 257

Ch LVF© (20) 38L

v-BHC(20)

Figure 17. The decrease of active toxicant residues in aquatic ETS with L5 values

ments with toxicants are replicated and the receptors are saturated, thedetoxification rate rapidly decreases. The effect of detoxification is bestcharacterized by i.e. the time to half-degradation. Also the maximumvalues of concentration for a complete detoxification by ETS in 24 h (Table 5)provides good information. They are the highest for PMPF and the lowestfor Chlorofenvinphos and y-BCH.

(4) Aquatic ETS—dynamic

The battery of ETS united in a flow system of communicating vessels(Figure 18) is subjected to permanent pollution. Its role for the analyticalecology of contamination corresponds to the role of static systems. In addition,its main objective is to carry out a complete detoxification by means of adefinite number of linked units. The efficiency of the battery, e.g. composedof five units, depends on the input of a toxicant. The required value is themaximum rate of inflow (in ml/h) ensuring detoxification along the wholebattery. The criterion of detoxification is the complete survival of STB in thelast unit and the results of quantitative analyses in the remaining units (Table6).

To start the experiment, a solution of the toxicant of known concentrationsis poured into the battery. Immediately before the start, unit I is filled withundiluted toxicant. The inflow of toxicant from the source pushes the toxicantalready present in unit I into unit II and all successive units. One of the mainpurposes of the use of the battery is to determine the tolerance to permanentcontamination. In that case samples from particular units should be takenevery day and the content of toxicant determined on STB. Also the response

277

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(6) Inversion of the ETA function

Readily metabolized IChE, such as PMPF, HgC12, DDVP and Dipterexare used for the detection of living matter in different materials of a complexstructure, such as soil or sediments. Comparison of the effect of detoxifica-tion in natural and denatured materials characterizes the content and activityof a biotic factor. The value of such a reagent increases with an increase bothin its influence upon the environment and the rate at which it is metabolizedby the biomass. At low concentrations of 0.1—1 p.p.m. its toxicity rapidlydisappears due to the metabolic activity of small biomass (small plants andanimals). The degree of disappearance is proportional to the amount ofactive biomass. The reactive system is composed of PMPF—Asellus, or DDVP—Daphnia, or HgCl2—Daphnia and Spirostomum.

The concentration of DDVP drops from 1 p.p.m. to 0.25 p.p.m. after30 mm of incubation with natural humus soil, and to 0.120 p.p.m. when it isincubated with denatured soiL So, the index of metabolic detoxificationactivity (MDA) is 4.8, the absorption of dead soil being taken as 1. The analysiswith an accuracy of 0.01 p.p.m. of detoxification effects after the incubation ofa reagent with soil takes only a few minutes (compare Table 7).

Table 7. Biodegradation and absorption of toxicants by organic detritus from an eutrophic lake.

T = 24 h, temperature 19°C, permanent aeration

Toxicant M (g) CIM (ppm.) C1 (ppm.)

Final concentration oftoxicant (Cf) in p.p.m.

Index ofmetabolic

sterilized livingdetritus Cf detritus Cf

detoxification(MDI)

DDVP 20 5 1 0.20 0.021 9.5HgCI2PMPF

1

201000

5101

0.21 0.0540.0085 0.0025

4.03.4

ChLVF 20 5 1 0.46 0.32 1.47-BHC 20 5 1 0.21 0.19 1.1

Abbreviations: M is fresh weight of detritus in 100 ml of water; CM is charge of toxicant per 1 g of detritus; Ci is initial con-centration of toxicant in the water; Cf is final residue of active toxicant in the water, and MDI = Cf /Cf,.

Quantitative analysis of metabolic activity in relation to the above-men-tioned toxicants is based on an analytical series of decreasing amounts ofsoil, sediment and tissue incubated with a toxicant at a constant concentra-tion. Complete detoxification at the beginnirTg of the series is the maximumlimit, detoxification by half is the medium limit and a lack of detoxificationis the minimum limit. These limits are modified by the value of C1/orginalconcentration of the toxicant (reagent).

This information would be useless here if there was no need for the predic-tion of pesticide doses for crop protection. It is also needed to determine theeffect of additional treatments with chemicals on the detoxification activityof different soils, and to characterize the biotic potential of the environmenton the basis of the index of detoxification capacity (MDA), besides enzymaticcharacteristics.

282


SUMMARYThe TA and ETA techniques presented here are in the first stage of develop-

ment. The basic purpose is to determine the environmental—toxicologicalcharacteristics of all toxicants polluting the natural environment, withparticular emphasis on pesticides.

The enlarged TA and ETA should increase the range of information onpesticides and other toxicants in the following directions:(1) Detectability in different materials and concentrations difficult to study

with other methods,(2) Effects of pesticides on the structure and functioning of natural aquatic

and terrestrial ecosystems,(3) Effect of the natural environment on the biological activity and persis-

tence of pesticides,(4) Stimulation of progress in the production of new pesticides less damaging

to biocenoses (with optimum BSTA),(5) Development of a bioindicatory security margin which can regulate

the quality and intensity of treatments with pesticides,(6) Current ecological estimation of the cumulative impact of chemicals

on the natural environment,(7) Ecological evaluation of new pesticides,(8) Utilization of readily metabolized toxicants and STB as reagents

characterizing the biomass of environments without disturbing theirnatural structures,

(9) Planning ecosystems more resistant to the necessary treatments withchemicals,

(10) Adaptation of the most sensitive natural ecosystems for performing therole of bioindicators both in the laboratory and in the field.

Increasing pollution of the environment produces a situation in which STBare protected in laboratories and their natural habitats are deprived of them.This situation can be improved by the application of new methods for thechemical control of pests (repellants, attractants, hormones, chemosterilants)and by lowering the total impact of chemicals. In that case STB will returnto their natural habitats as indicators of their purity and the role of STB willbe performed only by selectively sensitive pests. At the moment they shouldgradually pass to the higher levels of specific sensitivity under the influenceof new pesticides. It should be expected that TA and ETA will be still moreeffectively used for monitoring these processes.

INDEX OF ABBREVIATIONS

AEE aquatic experimental environmentBCA biochemical analysisBSTA taxonomic spectrum of toxic activityC concentrationCPA chemicophysical analysisEE experimental environmentEPH energy potential of homeostasisETA ecological test analysis

283

ALFRED KAMINSKI

ETS ecological test systemIChE inhibitor of choline esteraseLC50 lethal concentrationLR lethal responseMACT multitest analysis of cumulative toxicityMDA metabolic detoxification activityNE natural environmentR dilutionRvI reversibility indexSPB system of permanent bioindicationST standard toxicantSTB sensitive test biontt timeTA test analysisTB test biontTDC toxic—dynamic characteristictEA critical time-moment of effective absorptionTEE terrestrial experimental environmentTI toxicological interaction

REFERENCES1 J• R. Busvine, A Critical Review of the Techniques for Testing Insecticides, Londdn (1957).2 R. L. Metcalf, Organic Insecticides, New York-London (1955).

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13 W. Bratkowska, Biul. WAM, 16, 49 (1973).14 A. Kamiñski, Angew. Parasitol. 2, 86, 110 (1961).

D. J. Finney, Statistical Methods in Bioassay, Griffin: London (1952).16 A. Kamiñski, B. Zawadzki and T. Szymañska, Roczn. Nauk Roln. 95-A-4, 589 (1969).17 A. Kamiñski and T. Kisieliñski, Roczn. WIHE 2(5), 211 (1961).18 Seiroku Sakai, Insect Toxicological Studies on the Joint Toxic Action of Insecticides. Yashima

Chemical Industries: Tokyo (1960) (with 573 references).19 A. Kamiñski, Verch. Internat. Verein. Limnol. 16, 969 (1966).20 R. E. Warner, Bull. World Health Org. 36, 181 (1967).

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