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FISHERIES RESEARCH BOARD OF CANADA . Translation Series No. 582

WATER POLLUTION DUE TO WASTE FROM SULPHATE PULP FACTORIES

By Hilding Bergstrbm and Sten Vallin

Original title: Vattenfbrorening genom avloppsvattnet fran sulfatcellulosafabriker.

From: Meddelanden frgn Statenp undersUnings- och F5reksansta1t f5r.etvattensfisket, No-. 13, 1 19 pp., 1937. Kungl. LantbruksstYrelsen. (Mitteilungen der Anstalt fnr, Binnenfischerei , bel Drottningholm, Stockholm.). ,

Translated by Lari_Ohman Bureau for Translations, Foreign Language Division, .Department of theSecretary of State of Canada

Distributed'by: Fisheries Research Board of Canada Biological Station, St. Andrews, N.B.

1965 Swedish State Agricultural Board Report from the State Institute for the Investigation of Fresh Water Fishery. No. 13. Water contamination due-to the waste water from sulphate pulp factories. By Hilding Bergstrbm and Sten Vallin.

With 10 tables.

Page 3 HarmÈul effects due to the waste water from a sulphate factory in a water course has been subject to previous investigations by among others Ebeling, Klingstedt and Vallin, in particular with regard to fishery. The oxygen content of the water, the relative per- centage of dissolved organic substance détermined by permanganate consumption, reaction or pH-value and sulphide reaction usually do not give any appreciable indications of the contamination of the water, apart from a limited area immediately downstream of the factory drainage. In spite of this it has in certain cases been found, sometimes at a rather long distance from the factory, a noticeable poisonous effect on existing fish, in more severe cases resulting in fish death and a more or less pronounced deterioration of the taste of the fish due to stestances contained in the waste water from the factory, which effect could have been prevailing even if the fish otherwise seemed unaffected. At the mentioned investi- gations it has been shown also by comparative experiments with pure substances, that among those in the waste water existing organic compounds the resin acids are a highly effective poison for fishes. Primarily, the effect on the water course has been investigated and in certain cases the poisonous effect of the total waste water. However, in order to establish what actions should be taken for the operation of a sulphate factory in order to reduce as much as possible the damaging effect of the waste water, it is necessary to investigate the effect of different kinds of fluid in the waste water, which are generated during the manufacturing process,-- different groups of condensate, diffusion water, rinse water, "slime" water, etc. It is of course also necessary to take into account the quantitieis of the different kinds of the waste fluids in order to get a correct picture of the rele they play in the total waste water. The present investigation has been performed in accordance with the mentioned viewpoints. Chemical investigations, the determination of the amount of various waste products,etc., has been made by the Carbonization Laboratory (Kolningslaboratoriet) in Stockholm, the investigations of the Poisonous effects of the various waste fluids by the State Institute for the Investigation of Fresh Water Fishery, Drottningholm. • - At the investigation of poisonous effects the Institute's filtered P. Mâlarwater has been used as diluting water. It has a_ pH value of about 7.3 and an alkalinity corresponding to about 1.00 cubiccenti- meter 1-normal HC1 per liter. In all series of experiments where the pH-value in the various samples have exceeded 8, it has been regulated with hydrochloric acid to some value between 7.0 and 8.0, usually between 7.2 and 7.6. In order that the fish will be affected by the acidity or alkality only, a pH value below 5.0 or above • about 9.0 is required. Oneliter samples have been used at the experiments and therefore only relatively small test fishes, 5 to 6 cm long, could be considered --Spa11 roaches, summerlings of salmon and in some cases perch. The fish have, before,the experiments, been kept in the Institutes' aquariums with through flowing water. Moderate airing has been done during the experiments with alkaline fluids, diffusions water etc., where otherwise com- paratively rapid oxygen reduction could occur. On the other hand has airing not been done at most experiments with condensate products, etc. The test time has been about 5 days or somewhat more. The solutions have been renewed once a day during the experiments in order to prevent a marked reduction of the poisonous effects, in particular when concerning the samples with airing. The following number code has been used in the records to indicate the condition of the fishes during the course of the experiments. Code 0 = No effect 1 = Swlmming around somewhat restlessly 2 = Swimming aroUnd restlessly. Breathing accelerated. 3 . Swimming at intervals, in between the body stiff. 4 = Spasmodic trembling. 5 = Noticeable balance disturbances during swimming. 6 = At times resting in side - or recumbent position. = Swims (rushes) against the surface and falls back in side - or recumbent position 8 = Stays more constant at rest in side - or recumbent position at the surface or the bottom. The respiratory motions still noticeable. = Stays constahtly at rest in side - or recumbent position. Long intervals between the respiratory motions. Dead.

0 = No effect. 1 = MinUte effect. - -7-11(5tie#ab1e -'efrect. T 7 =:Strong -effect. 8 9 =1,rer'y strong effect. Page.5 Such a nuMprical scale for the gradation of the poisonous effects Makes it easier to keep record of the experiments, make the record more perspicuous and can also be used for graphically representing the course of the poisonous' effects. -3 In order to obtain a better perspicuity of the quantitative importance of the various waste fluids with regard to water impurities the concept of poison unit has been introduced. Thus a fluid contains e.g. 500 poison units per liter if it requires a dilution of at least 500 times of fresh water in order that a salmon-fry can survive in the diluted solution for 5 days. This definition of the concept poison unit is of course to some extent related to the special conditions of that water course at which the factory concerning this investigation is situated. The most important kind of fish in this water course is salmon. In particular the 2- to 3-year old salmon-fries, from the point of view fishing, are the most important resources. At this investigation as well as at previous investi- gations it has been found that the salmon-fries are much more sensitive than other fishes used, roach and perch, to the waste water from a sulphate factory. The determinatiOn of the number of poison units of the various waste fluids has to some extent been estimated approximately. If it was found during the experiments that the salmon-fry in a dilution of 1:20 died already after 2 to 3 days, but in a dilution of 1:40 was hardly or not at all affected after a minimum of 5 days, the number of poison units was estimated to 30 per liter. In some cases, even at the most diluted solution, the salmon-fry has died with'in a shorter time than 5 days. In such cases the number of poison units has been estimated somewhat higher than the mentioned most diluted solution.

The following fluids from a sulphate pulp factory are those which essentially can be assumed to have a polluting effect on the water courses and damage the fishing in these. 10 Çondensate . of steam from the pulp boilers = boiler condensate, 2. Condensate of steam from the diffusion containers = 'diffusion condensate.„ 3. Condensate of steam from the evaporation of the sulphate air = air condensate. 4. Fluid that follows the pulp from the diffusion containers = diffusion water. 5. Water that follows the slime = slime water.

The amount of these fluids and their compositions can vary for different factories. At the factory related to the present investi- gation the raw turpentine oil was separated from the condensate and is thus not included in these. The lye has been evaporated 10° to about 289 B. The condensate has been let out from the conden- sers at about 30°. The soap separated from the lye has been treated on liquid resin. The diffusion containers have usually been rinsed. As an example it can be mentioned that the following quantities of the above-mentioned waste fluids resulted from 1 ton of pulp manu- factured:

350 1 . Boiler condensate 000.0090000 OOOOOOO 90000000 liter

2. Diffusion condensate 0000000 OOOOO 00090000000 600 liter 3. Air condensate .••a00000•e•oo•osee•seo••ou 5000 liter 4. Diffusion water .••...•...... •...... 32000 liter

.2 • Slime water 00000000000 OOOOO o OOOOOOOOOOOOO 00 1400 liter

In addition comes about 5 times as much volume of water, mainly rinse water for the pulp. The total amount àf waste water per.ton pulp was therefore in this case 240.000 liter.

The results of the experiments concerning the poisonous effects of the various waste-fluids are presented in Tables 1-VIII after the text. They are summarized in the table below. Thus for the pro- cessing of 1 ton pulp per hour a waterflow of at least 9.700.00 liter per hour (the waste water from the f1uid included), assuming that all these waste fluids were disposed off without 'pretreatment and that their poisonous effects remained unchanged, • is required in order that a salmon-fry could survive at least 5 days in the mixture of river water + wuste water from the factory concerned. Thus a minimum water flow of 2.4 m per second is required for the processing of 1 ton pulp per hour under the above-mentioned conditions.

Table 1 Poison units Per ton pulp Waste fluids per liter liter poison units

. Boiler condensate 3000 350 10 50000

Diffusion condensate 500 600 300000

Ly.e condenSate 70 5000 350000

Diffusion water 250 32000 8000000

Slime water 10 1400 14000

Total 9714000 Paze 7 Boiler condensate, which after separation of raw turpentine contains, among other things, poisonous, volatile compounds such as methyl alcohol, sulphuretted hydrogen, thio-alcohols, ammonia, etc., had in the investigated case a very stong poisonous effect (Table 1) with about 3000 poison units per liter. Diffusion condensate (Table III), which mainly contains the same compounds as the boiler condensate, although much less concentrated, had a poisonous effect of about 1/6 of the condensate mentioned, or 500 poison units per liter. However, the poisonous effect of these tdo condensates can be considerably reduced by a simple evaporation process (Tables II and IV). The condensates from the lye-evanoration process (Table V) are much less poisonous than the boiler and diffusion condenSates and has only 70 poison units per liter. Since the quantities of the lye condensates is quite big, about 5000 liter per ton pulp, they should still be considered in the poisonous effect of the total waste water. The collective' poisonous effect of the various condensates, after the evaporation of the boiler and diffusion condensates corres- ponds to only a few per cent of the poisonous effect of all the waste water. In this case the evaporation from the diffusion con- densate was made difficult due to the fact that it was mixed with lye. By a properly performed evaporation process the poisonous effect of the condensates can thus be reduced to a trifle. However, the poisonous substances of thèse condensates give the water and the fish a bad taste and smell even at very low concentration, when the life of the fish is not endangered. In this way they represent a completely different hazard than the diffusion water, in which the resin acids form the poison. Thus with all certainty the substances contained in the condensates, particularly sulphur compounds, are primarily responsible for the deterioration of the taste of fish, which can occur downstream of a pulp factory. Apparently only very low concentrations of these substances are required therefore. Thus both from the point of view of fishery and protecting the water from bad taste it is important that the condensate fluids, after the separation of turpentine, also by further treatment are freed as completely as possible from these substances before they are let out into the water course. The slime water has a much weaker poisonous effect than the other waste fluids, in the present case estimated to only 10 poison units per liter or 14.000 units per ton pulp, which corresponds to only a fraction of a per cent of the total poisonous effect, and is thus of no practical importance, presuming that a sufficient and well-organized rinsing of the slime is arranged. A direct outlet of the slime with the waste water into the water course will be harmful from the point of view of fishery, since the heavy slime will settle on the bottom over an area downstream of the outlet. P.8 Mixed with the mud from the bbttom it then forms a black -6 nasty-smelling and sterile bottom layer. However, usually the slime is allowed to settle in certain tanks and the slime water from these can be allowed out into the water course without noticeable harmful effects, in spite of its dirty consistency. The diffusion water is the main contributor to the poisonous effect of the waste water. Assuming that the turpentine oil is separated from the condensates and that these are later on freed from alcohol, then the diffusion water is responsible for 95% of the total poisonous effect for the case investigated. The poisonous effect of that diffusion water which was let out with the waste water was found to be quite sei;ông--250 poison;units per liter--(Table IV), but first of al],, the large volume of this waste water--32000 liter per ton pulp--is the determining factor for the poisonous effect of the total waste water. The disposed diffusion water contains at large weaker concentrations of the same substances as the thin-lye and the black-lye. The most important substances in the diffusion water are lignine, resin acids and fatty acids. Of these substances the resin acids have previously been found to have a strong poisonous effect on fish. From a newly published investigation of "the occurrence of resin acids in black-lye and rinse water" (H. Bergstrft and K.N. Cederqvist) we take as an example that when sampling the'wood of pine to 1 ton pulp it contained:

resin acids • .... • • e 32.8 kilogram fatty acids OOOOO 29.2 kilogram neutral oils •...•.. •. 5.9 kilogram

Total 67.0(9) kilogram Thus if all the resin acids, fatty acids and neutral oils of the wood were obtained as liquid resins at the pulp boiling, 67 kg per ton pulp would have been obtained. However, only about 20-25 kg liquid resin per ton was obtained. The largest loss is represented by the soap that with the lye went to the sodambouse for evaporation and burning. Also a noticeable loss was the soap dissolved in the diffusion water and thus let out into the water course. The distribution of the resin acids of the woods per 1 ton pulp was estimated as follows: prciduced liquid resin • ...... 12.0 kg in diffusion water .., 6.4 kg remainder to soda-house . • ....• 14.4 kg

Total 32.8 kg 7 Page 9 It is undisputable that resin acids bound to alkali are a strong poison for fish. But there are also other substances in the diffusion water which are poisonous, which investigations have shown and the results are given in the table below Table II Number of poison Test units Der liter

Black-lye or thin-lye diluted 1:10 250 Black-lye without resin acids and fatty acids, diluted 1:10 50 Soap solution of liquid resin, 500 mgr/1 100 Soap solution of resin acids, 500 mgra 200 Soap solution of fatty acids, isolated from liquid resin, 500 mg/1 50 Soap solution of oleic acid, 500 mg/1 25

Thus the fatty acids bound to alkali in the liquid resin account for 10-15% of the poisonous effect of the resin acids calculated on the same amount of the acids. The diffusion water contains about the'same amount of the two kinds of acids. Black-lye which is obtained from the extracted wood, i.e., without resin acids and fatty acids, shows also a certain poisonous effect, about 1/5 of that of the normal black-lye. The diffusion water is, as already mentioned, the major contributor to the poisonous effect of the total waste water from the factory. The mentioned investigation of the occurrence of resin acids in black-lye and rinse water has shown ) however, that by relatively simple and economical arrangements the poisonous effect ol even the diffusion water can be considerably reduced. The poisonous effect of a general sample of the total waàe water Tdas also determined and showed (Table VIII) that it represents about 30 poison units per liter, which means that it has to be diluted 30 times in order that a salmon-fry can survive at least 5 days in the diluted test. The amount of resin acids has been determined colorimetrically with chloro sulphon acid (according to Cohen). Thus a general sample of the diffusion water showed a resin acid content of 250 mg per liter and a similar sample of the total waste water contained 32 mg per liter. Thus in this investigation the number of poison units for each of the various waste fluids, or in other words their total poisonous effect, has been determined before they levebeen mixed with the water of the water course. However, it must be made quite clear, that it is not possible to get a true value of the poisonous effect in the water course by a simple calculation of the degree of P 0 10 dilution based on the existing water flow and the amount of waste water. On one hand it can take several kilometers downstream of a waste water outlet at one side of .a streaming water course until the waste water is evenly mixed with the river water. As previously shown (Vallin) a local increase in the influence on fish can also occur, e.g., in backwater in coves, below falls and currents in the water course. On the other hand one can also count on 'a gradual reduction in the poisonous effect of the waste water as it mixes with the river water. During this mixing a certain precipitation of free resin acids may occur, which are relatively harmless for the fish. The determination of resin acids in the river water has hitherto concerned the total amount. For more exact investigations it would be necessary to determine how largeportion of the total amount that is free resin acids. A direct slow oxidation of the resin acids to carbon dioxide and water occurs even at normal temperature. Furthermore the resin acids are probably also gradually broken down biologically in the water course. Finally, it must be mentioned that the limiting values obtained from the experiments, for fatal poisonous effect on fish of the various waste prOducts, do not lend themselves to the conclusion that a lower concentration of these substances in the river water would be •-absolutely safe. Thus if,e.g.,the limiting value for fatal effect on salmon-fries of resin acids has been shown experimentally to be 1 or 2 mg per liter, it is quite possible that even a fraction of a mg per liter can be harmful even if a direct fish death would not occur. The fish might be affected in the way that they go away from the infected water, their feeding possibilities may be worsened and the spawning affected. Such long-lasting chronic effects of poisonous substances in low concentration are very little investigated and also difficult or even impossible to determine experimentally. However, many things indicate that they often are of great importance with regard to harmful effects on the stock of fish.

Summary The objective of the present investigation has been, that by determining the poisonous effect of different waste fluids from a pule mill, to find a basis for where and how suitable measures in the production could be undertaken in order to reduce the harmful effect of the waste water primarily with regard to fishery. Naturally the quantities of the different waste fluids must be con- sidered in this.case. In order to have some means of relative com- parison the concept of poison unit has been introduced. The number of poison units per liter fluid corresponds to the degree of dilution necessary in order that a salmon-fry shall live at least 5 days in the diluted solution. Page 11 Ëoiler and diffusion condensates have each a strong poisonous - effeOt ev6n after the separation of raw-turpentine. The lye con- densates,are considerably iess poisonous. However, the poisonous effect of the first-mentioned condensates can be considerably reduced by a simple distillation process and the summed poisonous effect of the different condensates are in this way reduced th only a few per cent of the total poisonous effect of the waste water. Certain substances in the condensates, primarily various'sulphur compounds, can, however, even in small concentrations give bad taste to the water and the fish. It is therefore important that, before the condensates are disposed of in the water course, they are freed from these compounds to as high degree as possible by an effective distillation, eventually by some further process. By a well-organized rinsing of the slime the slime-water has been found to have virtually no poisonous effect. The major contribution to the poisonous effect of the waste water from a pulp factory was in the investigated case coming from the diffusion water, primarily the resin acids, but also of fatty and oleic acids. Even the diffusion water from extracted wood, thus without resin, fatty and oleic acids, has been found to have a certain poisonous effect. A recently performed investigation by H. Bergstrft, K. N. Cederqvist och K. G. Trobeck has shown that the harmful effects of the diffusion water can be considerably reduced by comparatively simple arrangements.

Literature.

G. Ebeling: Investigations of the effect of waste water from pulp factories on fishery. Journal of Fishery, Vol. 28, No. - 4 1 1930. F. W. Klingstedt: On the effect of the waste water of the pulp factories on the stock of fish. - Journal of Pulp and Paper, Finland, 1933. S. Vallin: The pulp factories and the fishery. I. New Swedish Journal for Fishery, No. 21, 1933. I I The pulp factories and the fishery. Experimental investigations. Report from the State Institute for the Investigation of Fresh Water Fishery, No. 5, 1935. Hilding Bergstrbm and K. N. Cederqvist: The occurrence of resin acids in black-lye and rinse water. Swedish Paper, No. 5, 1937. - 10

Tables

Table 1 0 Boiler condensate. Temp. 3.70 -10.00 (Centigrades) No airing. Dilution Hour - 1:500 Salmon Rouch SRSRSR 55m/m

= dead Footnote: Number of poison units estimated to 3000 per liter.

Table II. De-spirited boiler condensate. Temp. 6.1°-8.7°. No airing.

Table III. Diffusion condensate. Temp. 3.7°-10.0°.

Table IV. De-spirited diffusion condensate. Temp. 6.0°-7.9°. No airing.

Table V. Lye condensate. (From the lye evaporation). No airing.

Table VI. Diffusion water. Temp. 3.7°-10.0°.

Table VII. Slime water. Temp. 6.0°-8.9°. No airing.

Table VIII. Sample from "the big outlet". Temp. 3.7°-10.0°. No airing. ' WE 1.1DPÂRY Alieleves FISHERIES IIESEIRCH IOARD OT cararix t. C.

FISHERIES RESEARCH BOARD OF CANADA Translation Series No. 581

On the formation of capsules around larvae of Anisakis sp. in the tissues of the shorthorn sculpin Myoxocephalus scorpius

By T. O. Prusevich

Original title: K izucheniiu formirovaniia kapsul okolo lichinok Anisakis sp. v tkaniakh kerchaka Myoxocephalus scorpius.

From: Trudy Murmanskogo Morskogo Biologicheskogo Instituta, Vol. 5, No. 9, pp. 265-273, 1964.

Translated by G. N. Kulikovsky, Bureau for translations, Foreign Language Divison, Department of Secretary of State of Canada

Fisheries Research Board of Canada Biological Station, Nanaimo, B. C.

1965 Concerning the Study of Capsule Formation near /265 the Anisakis sp. Larvae in the Tissues of Sculpin Myoxocephalus Scorpius

By T. O. Prusevich (Laboratory of Comparative and Experimental Embryology. Chief - B. P. Tokin)

(From "Trudy Murmanskogo Morskogo Biologicheskogo Instituta" /Transactions of the Murmansk Sea - Biological Institute/, USSR Academy of Sciences, No. 5 (9) p. 265 - 273)

One of the aspects of the study of interrelations between the parasite and the host, is the study of the parasite's adaptation to the organism, in which it had, in the course of its evolution, found a habitation medium (the medium of first order according to Pavlovsky, 1934, and Dogel, 1947). The physiological adaptation of the parasite progressed to suppress the immunological reactions of the host directed against it (Shulman, 1958; Shultz and Davtyan, 1955). In the light of the study of problems of the immunity of the embryos promoted by Tokin (1955), the study of the peculiarities of the immunological reactions (phagocytosis, inflammation, capsule formation) at contact spots between the larvae of parasitic worms and the host's tissues, present a consid- erable interest. The goal of the present paper is the histological study of the process of the formation of the capsules around the larvae of Anisakis sp. at artificial infection of the sculpin (Myoxocephalus scorpius) by the latter, and also a comparative study of the capsule formation around live and dead (killed through boiling) larvae, in order to clarify possible differences in the reactivity of the tissues. 2/265-266

The Methodology of the Studies

Larvae of parasitic nematodes taken from cod's liver were transplanted into sculpins with a body length ranging between 19 and 23 centimeters. The liver was placed in an aquarium in which the sea water was changed daily. After 5-6 days the larvae, which had.emerged from the capsules, were transferred into an aquarium with running water, in which they lived another 1-3 days. 2-3 hours before the operation the larvae were placed for the marking purpose, into a dense batch of carmine dissolved in sea water. One part of the larvae were first killed by boiling them for 5 minutes. The laparotomy in the sculpins was carried out without observance of any rules of aseptics. The larvae were placed on the liver. Live anisakid larvae were introduced into ten sculpins, and dead anisakid larvae were introduced in another ten sculpins. Each .sculpin 'received two larvae. The abdominal wall was sewn in layers; the muscles - by an inter- mittant suture, and the skin by a continuous one. After the operation the sculpins were kept in aquaria with running water. Four, twelve, and twenty-four hours and 30 days following the transplant- ation of the dead larvae, pieces with marked larvae were cut out of the liner of the tested fish, these pieces were fixated in the Bouin's solution and were sealed in celluloid. The sections were strained by the three following methods: according to Mallory in the picro-fuchsins; and by trioxyhematein of Hansen.

Tests in Connection with the Introduction of Live Larvae of Anisakis sp. into the Body Cavity

In the early periods of the observation, i.e. 4, 12, and 24 hours after the beginning of the operation, the body of a spirally rolled larva appeared plunged in the parenchyma to a greater or lesser depth. Within 24 hours, we observed a picture of destruction of small areas of the liver parenchyma /266 around the body of the larvae, ruptures of walls of some blood vessels (Fig. 1), small hemorrhages, ' and formation of thrombi. The larva body was covered with fibrin. During the first 24 hours of the test, the quantity of free blood and fibrin increased. When the larvae plunge to a considerable depth into the

a 3/266-268 parenchyma of the liver, the liver cells were compressed and acquired fusiform shape; the blood-carrying lacunae were subjected to distension. At the same time, in the area of more superficial attachment of the larva and at a certain distance from this area, the hyperemic blood vessels of the liver became dis- tinctly visible (Fig. 2). In one instance, 4 hours after the beginning of the test, one larva began to "bore itself" deep into the liver parenchyma. In the preparations one saw nothing but small areas of disintegrated liver cells and minor hemorrhages. In the first 4-24 hours of the test there was distinct swelling and homo- genization of the collagen layers of the serous membrane adjoining the larva, and also infiltration of the fibrin by the cell elements (Fig. 3). Within 12 hours of the beginning of the tests, an accumulation of leucocytes, of-phago- cytoses of the grains of carmine, and of the tissue detritus etc., was clearly visible near the larvae body. In some cases the larva was surrounded by a layer of various leucocytes. Towards 24 hours after the beginning of the test,.the leucocyte infiltration of the. fibrin increased, we observed, in the serous membrane of the liver, individual fibroblast elements, hystiocytes, and some- times groups of mesothelium cells. The mesothelium, together with individual leucocytes and histiocytes, phagocytize the grains of carmine. The number of dark-nuclear leucocytes, which are in direct contact with the larva, increased. 9 days after the operation, a net of fibrin fibres of various thickness was clearly visible around the parasite's body. A leucocyte ridge formed. At the border to the liver parenchyma were wandering cell elements, mainly of connective-tissue origin. 30 days after the operation, the four layers were morphologically isolated near the Anisakis sp. larvae (Fig. 4). The first layer, belonging to the coils of the parasite body, consisted of dying cell elements. It appeared to corres- pond to a leucocyte layer of earlier periods of observation. The width of the first layer varies considerably, in certain areas it was poorly expressed. The second layer (Fig. 5) consists of fibroblastic elements, one may conditionally distinguish two zones in it: the first zone is an interior zone, it adjoins the layer of dying-off cells and consists of fibroblasts with /268 small, dark, baciliform nuclei; one may assume that these are fibroblasts of hematogenous origin (Zavarzin, 1937); the second zone consists of young and 4/268-269 larger fibroblasts with oval or elongated nuclei, and with distinctly appearing cytoplasma. The presence here of mytotically dividing fibroblasts indicates that a part of these cells have connective-tissue origin (Zavarzin, 1937). Between these two zones there are transitional cell forms - smaller fibroblasts with baciliform light-coloured nuclei. The third layer consists of porously arranged diverse cell elements, among which, however, the fibroblasts predominate. One succeeds in observing mytoti- cally dividing cells, and also two-nuclei cells, which are in the stage of amytotic division. Various leucocytes, extra-vascular erythrocytes, and eosino- philic cells occur here. Blood capillaries grow into the third layer. The fourth layer is located at the border with parenchyma of liver, and consists of more numerous and wide collagen bundles, fibres, and various cc-l1 have many histiocytes and dark-nuclear leucocytes. elements. We The liver parenchyma underwent change in areas adjoining the areas bordering on the capsule, while this was in the process of being formed around the Anisakis sp. The liver cells are de-differentiated, the amount of their fat drops is sharply decreased, between the cells we see wandering cell elements of the hematogenous and of connective-tissue origin. Near large lumps of the carmine, which are arranged in 3-4 layers, the fibroblastic strands are formed. The "skeleton" of the capsule is formed of individual collagen fibrills and bundles. There are islands of homogenous basic substance, which is inten- sively tinted, according to Mallory, by the aniline blue similar to the collagen. In preparations impregnated with silver, one always observes, in the formed capsules, regularly arranged argyrophil fibres (Mikhaylova, Prazdnikov, Prusevich, in the present compendium). Zavarzin (1937) connects their formation with the fibroblasts of hematogenous and histiogenous origin. The participation of these cells in the formation of the capsule in our material permits us to assume, that here also, the development of the fibrous basic substance is connected with the formation of the argyrophil fibres. Thus, the later periods of development of the capsule near the /269 Anisakis sp. larva are characterized by the predomination of elements of the fibroblastic series, by the decrease in the amount of the hematogenous elements, and by the growth of blood vessels into the capsule. The formation of the capsule is accompanied by insignificant destructive processes in the bordering liver parenchyma. 4/269-270

Our material contained two cases of localization uncharacteristic of the Anisakis sp. larvae: the larvae did not curl up spirally on the surface of the liver, but penetrated into the organ. In one instance the intrusion of the larva into the liver began 4 hours after the operation. Histological study has shown that the larva has destroyed the glisson capsule and entered the liver parenchyma to a relatively considerable depth (Fig. 6). Along the circum- ference of the opening "drilled" by the larva is an accumulation of blood elements and of fibrin. In the liver parenchyma around the larva there are erythrocytes, leucocytes, and desintegrated liver cells. In another case, the perverted location of the anizakid larva was observed one month after the trans- plantation of parasites to the sculpin. In the preparations, we may see passages marked with carmine made by the larva in the liver, and the place of the larva's exist to the surface (Fig. 7). The larval passages are filled with erythrocytes, leucocytes, histiocytes, fibroblasts, and with the cell detritus. In the liver parenchyma the passages are separated by a single-layer /270 fibroblastic ridge, which, however, loses its continuity in certain sections. One may observe here removal of the fibroblasts from the larval passage and into the liver parenchyma. One may also observe an overgràwth of individual liver lobes by fibroblasts. Mytotitic figures are common in these fibroblasts. <4* Thus, the anisakid larva, which penetrates into the liver and moves there actively, has produced extensive stable reactive changes in the liver parenchyma, which obviously have much more negative importance to the host, than the changes which take place when Anisakis sp. larvae encapsulates in a section of the organ characteristic to its settlement, i.e. on the surface of the liver. The histological picture of the liver inf ection by the penetrating larvae of Anisakis sp. confirms, in our observations, the concept of the strengthening of the pathogenicity of the parasites in cases of the perversion of their location (Bauer, 1958). 5/270-271'

Test with Introduction of Anisakis sp. Larvae, Which Were First Killed by Boiling

At early periods of observation (14-24 hours), one observes insignificant haemorrhage in the liver, fibrin falls out on the surface of the larva. Various leucocytes accumulate around the larva (Fig. 8). In the serous membrane the collagen fibres swell, and a group of histiocytes, fibroblasts, appears. The larva's body impresses itself into areas of glandular tissue of the liver. In the liver parenchyma, in the region of attachment of Anisakis sp. larvae, a swelling of the blood vessels takes place, while in the liver cells there occurs a vacuolization of the cytoplasma. The destruction of cuticula begins to take place, individual sections of it are divided by cell accumulations consisting of numerous dark-nuclear and individual light-nuclear leucocytes. After three days, we may see a layer of phagocytes of hematogenous origin around the larva. Many phagocytes pentrate under the cuticula of the parasite (Fig. 9). Intensive phagocytosis of the carmine grains is recorded. Extravascular erythrocytes appear, probably as the result of destruction of blood vessel walls in the border zones of the liver. The hyperemia of the blood vessels of the liver is maintained. The serous membrane is eodematous at the points of attachment of the larva, and contains many wandering cell elements of hemotogenous and connective-tissue origin, the mesothelium is well pronounced. The mesothelium and adjoining connective-tissue elements grow in a wide belt around the larva fixated by the fibrin. Nine days after the beginning of the operation, large numbers of /271 leucocytes accumulate near the decaying body of the larva. The cuticula is disintegrated and its fragments are arranged chaotically. A portion of light- -nuclear and of large dark-nuclear leucocytes penetrates into skin-muscular sac of the larva's body. At the border to the serous membrane, we see a wide layer of dark-nuclear histiocytes and fibroblasts which propagates over the parasite, which is immobilized by the fibrin and is surrounded by the leucocytes. In the peripheral sections of the capsule, which is formed at the border to the serous membrane of the liver, extravascular erythrocytes occur. -4 6/271-272

Individual liver cells are dedifferentiated in the zone bordering on the decaying larva, their cytoplasma becomes basophilic, and the number of the inclusions decreases. There are many different leucocytes between the cells. One month after the beginning of the test e at the location of the former larva a fine-grained substance is visible, which is stained, according to Mallory, homogenously and very brightly into orange-yellow (Fig. 10). Around the destroyed larva, one may morphologically separate layers which are analogous to the ones that were found during the same post-operative period in the capsules around the live larvae, but which possess a number of peculiarities. The first layer consists of the remnants of destroyed phagocytes. This layer is in destroyed tissues of the parasite, while around the live larvae, it is located at the border with the parasites body. The second layer is fibroblastic. This layer could not be distinguished along its entire length. In some areas it is replaced by mesothelium, which overgrows the foreign body. The third layer consists of connective tissue with various cell elements, among which, however, the light-nuclear leucocytes and histiocytes, and also fibroblasts are very numerous. Macrophages occur containing 2-3 nuclei, and eosinophilic cells, Mytotically dividing cells are common. This layer has blood capillaries. Here we have more phagocytes, then in a corresponding layer of the capsule near live anisakid larvae, and comparatively fewer fibroblasts, and relatively fewer collagen fibrins. The fourth layer consists of thickened serous membrane; in this membrane one observes a considerable quantity of blood vessels, and also different wandering cell elements of hematogenous and connective-tissue origin. The collagen tissues and bundles of the serous membrane are porously arranged and form reticulate structure in sections at the border with the parasite which is being absorbed.

Discussion of the Obtained Data

The study according to the stages of the formation process of the capsules around the live anisakid larvae enables us to conclude that the encapsulation of live larvae of the parasite, in the case of their normal 7/272 attachment to the surface of the liver, is accompanied, in the early stages of the process, by insignificant destruction of the liver cells and of the walls of the blood vessels. The precipitation of fibrin ensures a solid attachment of the larvae, and the spilled blood becomes a source of alimentation. When introducing the killed Anisakis sp. larvae, the damage to the liver tissue occurs at later periods of the development of the inflammation, and is probably connected with the separation of the products of decomposition of the parasite. Around the larvae of live Anisakis sp. a connective-tissue capsule containing numerous capillaries is gradually formed on the surface of the liver. On the border with the larvae, a layer of distrophically changed tissues of the host is formed; these tissues serve as an original "barrier" between the parasite and the host. It may be assumed v that the product of metabolism isolated by the parasite, and which determine its pathogenity together with the toxines (Bazikolova, 1932), somewhat decrease the intensity of the phagocyte reactions, and at the same time, while damaging the border fibroblastic layers, they secure the possibility of transfer of the alimentary substances from the host's tissues to the tissues of the parasite, and also the removal of the excretion products. In the attachment on the liver of the killed Anisakis sp. larvae, various phagocytes, and also fibroblasts and mesothelium take an active part in the formation of the capsules. Without doubt, a certain role is played by the products of decomposition in the stimulation of the phagocyte reaction around the dead larvae. The processes of overgrowth by mesothelium of the remnants of the decomposing parasite are manifested within a month. The proliferative processes on the part of the fibroblast elements are less intensive, fewer of collagen fibrills are developed, than during the attachment of live larvae.

Conclusions

1. The reactive process, developed around the implanted Anisakis sp. larvae on the liver of shorthorn sculpin (Myoxocephalus scorpius), leads to the -It. formation of a connective-tissue capsule around the parasite which ensures the prolonged protection of the viability of the parasite. 8/272-273

2. The encapsulation of the dead larvae Anisakis sp. follows the type of resorption and isolation of the surrounding tissues.

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