264 EMBREY SOME EXPERIENCES IN THE USE OF PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS. SOME EXPERIENCES IN THE USE OF COPPER SULPHATE IN THE DESTRUCTION OF ALGIE. BY G. EMBREY F.I.C. (Read at the Meeting Jane 6 1917.1 IN the year 1870 the author had frequent opportunities for observing the evil effects of the rapid growth of Algce in an artificial lake at Sutton Coldfield Warwickshire. The growth of weeds during the spring and summer was so rapid that the lake was almost useless for boating or fishing. Attempts were made to destroy the weeds by fixing scythes to the sides of a punt and dragging it through the water the weeds *being removed at intervals by means of rakes; this method was however costly and not very effective. In 1872 a railway line was constructed between Sutton Coldfield and Walsall; an embankment consisting of ferruginous sand passed over the narrow end of the lake at which the water entered.This sand wahing into the lake during a rainy period effected in one season what the cutting method had failed to do in seven: the vegetation was almost entirely destroyed and this particuh pool cleared of weeds. In 1873 the author observed the bad dour and colour of the drinking-water in Glouceater. On inquiry he learned that it was due to plants growing in the reservoirs and he suggested tde use of ferrous sulphate. One of the reservoirs was so treated and in a month the A l p were destroyed. About this time trouble arose at a tannery the skins being stained red by oxide of iron wmhed out of the service pipea.This was wrongly attributed to the ferrous sulphate used in the reservoirs and for this reaeon experiments were discontinued. Until 1908 the weeds were removed by rakes worked from punts. At interval5 the pools were emptied and the organic mud removed fifty or sixty men being frequently employed at this work. During the months of July August and September the water was badly coloured and geve out 80 offensive an dour that it waa unpleasant to use it even for a warm bath. In 1901 the United States Department of Agriculture directed the performance of experiments for removing objectionable AZp from water-cress beds by me8118 of copper sulphate. The result was satisfactory the water-cress plant being in DO way injured. In 1904 and 1905 this department issued Bulletins Nos.64 end 76, the first entitled " A Method of Destroying or Preventing the Growth of Algrar and Certain Pathogenic Baoteria in Water-Supplim;" and the eecond " Copper as an & w i d e and Disinfeatant in Water-Supplies," Both of thege were written by Memrs. G. F. Moore and I(. F. KeUermsn COPPER SULPHATE IN THE DESTRUCTION OF ALG& 265 In 1008 the author obtained permission from the Corporation of Gloucester to carry out experiments a t their Witcombe Reservoirs which were a t that time choked by a species of Chara. These plants are propagated by means of the fertilisation of spores contained in an archegonium. The bursting of the arche-gonium sets free myriads of minute greenish cells which gave the water a distinctly green colour and were also thought to be the cause of the offensive odour.The pools being periodically stocked with rainbow-trout it was desirable to use as little copper sulphate as possible. Following the instructions of Messrs. Moore and Kellerman 1 part of the salt was added to 3,000,000 parts of the water : this removed the odour but not the Chara. It was then decided to employ 1 part of the salt to 1,000,000 parts of the water; this destroyed the Chara and the fish. At this period (1908) it was generally thought that Chara was the chief cause of both colour and odour and one observer rather unwisely suggested that it was caused by a new species and named it Chara foetida. The author collected a number of the plants and found them largely covered with Spongilla ;fEuviatilis and probably other organisms.These were carefully removed and the cleansed Chara no longer gave out the peculiar odour while it was very evident in the washings. A portion of the Iatter was then used to inoculate a nutrient agar-agar medium. This on incubation developed Proteus vulgaris-an organism well known to produce a fishy odour. This seemed to account for the trouble yet I always had ljome doubts as to its sufficiency. In 1913 Dr. Houston’s valuable work “ Studies on Water-Supply,” appeared, and on p. 100 the following occurs “ At the beginning of the year 1913 part of London (Hammersmith Kensington and Hampstead) experienced a somewhat serious ‘ taste visitation,’ the Algal growth causing the objectionable taste and smell being chiefly composed of Tabellaria with some Asterionella.” This gave me a new clue and as the water from the reservoirs a t Witcombe passes through a battery of Bell filters it was easy to collect the washing and examine for minute organisms.This practice has been continued weekly since the latter end of 1913 and I am now convinced that Chara and its paresites only contribute t o a small extent in the production of the unpleasant odour. The most remarkable thing is that in Dr. Houston’s experience in the London water Tabellaria was the predominant organism and there were only a few Asterion-ella while a t Witcombe the reverse was the case Asterionella was present in abun-dance but only a few Tabellaria. A considerable number of the former were col-lected and dried; the mass was then placed in a fat extractor and the oil extracted with petroleum ether.After the removal of the solvent the residue gave out the identical odour which had caused the trouble. The odour associated with diatoms has generally been ascribed to an essential oil but this is not so in Asterionella. The substance removed by petroleum ether is certainly a fatty oil coloured with diatomin-a body closely allied to chloro-phyll. Works on microsoopy make little reference to this diatom which considering its wide distribution is surprising; even such an excellent treatise as Dallenger’s edition of Carpenter makes no reference to it. A good description of it is given i 366 EMBREY SOME EXPERIENCES IN THE USE OF ‘’ Microscopy of Drinking-Water,” by George Whipple. The figure given a t the end cf Dr.Houston’s studies is different from the photographs prepared for this paper. The frustules each conbin three or four rounded openings and before collecting into stars each moves fairly quickly through the water. It seems probable that the motion is effected by the protrusion of protoplasm through these holes; if this be S O the reason for the presence of a lubricating body becomes apparent. In what manner then does the copper sulphate destroy these organisms ? Water collected from limestone rocks contains sufficient calcium bicarbonate to bring about the following reaction The calcium will unite with sulphuric acid to form the mlphate and the copper will become basic carbonate; this will then give up carbon dioxide and yield a hydroxide which accumulates on the bed of the reservoir.This is the usual explanation but an observation made by the writer in 1911 shows that under certain conditions the hydroxide will become oxide. As the water WGS lowered in the pools both cuprous and cupric oxides were observed red and black bands being visible the red band below or immediately above the water and the black band some distance above this. This points to a probability that oxide of copper is the real poisoning a.gent and if my suggestion be correct that the fatty oil is to serve as a lubricant the oxidation of this may cause the death of the plant. EFFECTS OF THE TREATMENT. 1. Slight increase in permanent hardness. 2. Evolution of an offensive odour for two or three days. 3. Increase in the number of bacteria.The slight increase in permanent hardness is easily accounted for. The evolution of the offensive odour is probably due to the setting free and The increase in the number of bacteria appears to be due to the decomposition ovidat,ion of the fatty oil. of the AEg(e and quickly ceases as shown by the following observations: Number of Micro-Date. organisms per c.c 1913. April 12 sulphate of copper added . . 150 , April 25 . . . . . . 200 ,? May 10 . . . . 110 1914. April 18 sulphete of copl-~er ecldetl . . 140 , May 2 . . . 320 , May 16 . . 480 , May 27 . . 350 , June 17 ,. * . a . I . 260 1915. April 23 sulphate of copper added . . 180 ,) Nay 20 . . . I 150 , May 6 . * . 21 FIG. 1.-ASTERIOXELLA FORMOSA WI’TH FOUR FRUSTULES.(MAGNIFIED 600 DIAMETERS. ) FIG. 3.-hfODE IN WHIOH FRUSTULEX FORM V’S AND CHAiXS PHIOR TO FORMISG STARS. FIG. 2. - ASTERIONELLA FORXOSA PARTLY FOXMED STARS ; DIFFLUGIA IN LOWER POWION. FIG. &-MUD FROM BOTTOM OF RESERVOIR CON-SISTING ALMOST ENTIRELY OF ASTERIONELLA. To faca page 266 COPPER BULPHATE IN THE DESTRUCTION OF ALGAE 267 Nzmber of Micro-Date. organisms per C.C. 1916. April 12 sulphate of copper added . . 125 , A ril 19 . . * . a . . 170 * . 196 148 , &y 8 1917. April 28 sulphate of copper added . . 170 , May 12 . . * . 240 ) May 20 . a . . I * . , May 26 ,. 186 After the purification certain organisms increase rapidly. Three of these were very noticeable Di$lugia2 Anurea cochlearis and Anurea aculeata ; these probably wt as scavengers and use up the poisoned diatoms.PRACTICAL APPLICATION. It would seem advisable to add the sulphate of copper not later than the end of April so as to destroy desmids and diatoms and thereby prevent the formation of an organic mass into which the root-like thallus of Chara can penetrate; this view is supported by the fact that in these pools Cham is now rarely found. Experience has shown that 1 part of copper sulphate to 3,000,000 parts of water is sufficient and this amount has no injurious effect on the fish. It is generally recommended to place the copper sulphate in a canvas bag and trail this a t the stern of a boat but this has not proved so effective as allowing the crystals t o fall into the water and in this way produce greater concentration at the bottom of the reservoir where most of the diatoms may be found, It has been the practice a t Witcombe to fix a copper tank perforated with holes having a diameter of 4 mm.a t the stern end of the punt and allow the crystals to fall through the holes; in this way they quickly reach the bottom of the pools. COMPARISON WITH OTHER METHODS. Of the methods for the prevention of Algal growths the following points may be 1. Covering the reservoirs This is expensive and not always effective. 2. Constant cleansing Expensive and not effective. 3. Frequent agitation' Fairly effective but not always convenient. 4. Copper sulphate Inexpensive safe when properly controlled ; quite effective, increasing in efficiency each year.The experience a t Witcombe has shown that where similar conditions prevail, reservoirs in which a potable water is stored may be kept free from weeds colourless and odourless by a simple and inexpensive method. In conclusion I would mention that my success has been largely due to the confidence placed in me by a sympathetic and intelligent Municipal Authority and the ready help of my fellow officials-via. the Medical Officer of Health and the City Surveyor. noted 268 EMBREY SOME EXPERIENCES IN THE USE OF DIsCUssIoN. Dr. S . RIDEAL said that his attention had been drawn to this subject about twelve or thirteen years ago by some experiments made in America by Moore and Kellerman which had for their object the prevention or checking of the growth of Spirogyra in water-cress beds.Dr. Baines collaborated with him and they studied the action of copper sulphate copper chloride and metallic copper as ‘‘ algicides.” They soon found that the activity of the copper salts was proportional to the weight of copper in them-i.e. that it was an ionic reaction based on the weight of copper present copper sulphate and copper chloride being equivalent salts if the weight of copper was the same in each case It was then found that the action of copper was selective as regards different kinds of organisms; and in studying London tap-water it was found that when different quantities of copper salts were used different organisms survived so that in that way one could get in a series of bottles with different proportions of copper aImost pure cultures of the different organisms present.It was found too that a piece of metallic copper 1 centimetre square, put into 100 C.C. of water gave off sufficient of the metal to kill the B. coli strepto-cocci etc. present and this seemed to suggest that possibly the use of a copper tank or the passing of water or sewage eiluent through a sufficient length of copper pipe would result in the killing off of all typhoid coli etc. It also suggested the idea of a copper water-bottle for military use and such a bottle had actually been made. The use of copper in that way however had never been developed though he thought it would probably prove valuable if the surface of the copper could be kept clean and in a condition to do its work.A friend living in India had observed that the natives who drank with impunity polluted water such as that of the Ganges always used copper vessels for its conveyance and it might be that the use of copper for the purpose had been adopted as the result of some instinctive knowledge that the water would be thereby purified. In the East, earthenware pots were also used for water because of their cooling action and it might be worth while to study what difference there was if any between the effects of water stored in copper and earthenware respectively. Europeans in India usually used tinned copper vessels for storing water and possibly the use of ionic metals in a fine state of division might be among the means of bringing about germicidal action for it was difficult to see why the aame effects which were produced by metallic salts should not be produced by sheets of metal suspended in the reservoir or conduit.Copper had been looked upon aa a poison but M i . Embrey had shown that it produced no bad effect on fish in the reservoir in question while the Minority Report of the Departmental Committee on Food Preservatives on the question of the use of copper in preserved peas showed that it was not a t all agreed that the use of copper in food should be prohibited. Dr. Molson about the same time advocated the wearing of copper discs during cholera epidemics not as a charm, but as a scientific prophylactic ‘‘ on the ground that cholera is absolutely unknown among the workers in copper mines,” and said that such discs were used by Baron Fredericks &B far back as 1894 in Russia a t Nijni Novgorod during a plague of cholera there.The practice of using metallic copper discs against cholera wa COPPER SULPHATE IN THE DESTRUCTION OF ALGB 269 common in this country and in France much earlier than this (British Medical Journal 1893). Professor K. C. BROWNING referring to the use of copper vessels in India said that a friend who had made a considerable study of the sacred writings of the East had informed him that in more than one passage reference was made to the fact that water put into a copper vessel was less likely to cause disease. Certainly the use of copper and brass vessels waswery common among all classes in the East. As to the question of copper being poisonous the United States Food and Drugs Board after exhaustive experiments came to the conclusion he believed in 1913, that the presence of copper in food or drink should be entirely prohibited and it is now an offence against the laws of the United States for copper to be present in any food or drink.It is true that there are ;people who can ingest small quantities of copper salts for a short time without apparent harm but this is of course no argument in favour of allowing the presence of copper salts in food especially as some individuals show a marked idiosyncrasy for copper. In the case of water this objection would not hold goad if the copper is com-pletely precipitated before it reaches the consumer. Lieut. E. K. RIDEAL said that copper had been long in use as a germicide, not only in the case of water but for a variety of other purposes which did not seem to bear out the cupric and cuprous oxide theory.For instance in France very large quantities of copper sulphate were used in the form of Bordeaux mixture for the destruction of phylloxera while for sterilising the su.rface of seeds copper sulphate was a most efficient agent 1 part of copper in 700,000,000 being suecient for the sterilisation of a wheat seedling. When that strength was exceeded the seedling itself appeared to suffer. As a matter of fact,verylittlewaiunderstood about the mechanism of disinfection. Experience had shown that colloidal metals did react with micro-organisms and on the other hand it was known that micro-organisms were really sus-pensoid colloids protected by emulsoids ; the trivalent elements aluminium and iron, and also hydrogen ions would cause precipitation of such organisms owing chiefly to the electrostatic charge on the latter.But precipitation did not necessarily mean the death of the organism. Precipitation was also brought about by some divalent metals such as mercury and copper and in the case of copper the cupric ion was more effective than the cuprous firstly because of its higher electrostatic charge, and secondly because the mechanism by which the cupric ion was adsorbed on to the cell wall was different. Copper reacted with cellulose and alscr with’chitin-the cupro-ammonium process for determining cellulose being of course well known-while wool on treatment with cupric ammonium sulphate yielded amino-acid copper complex with the wool chitin.The activity of the copper ions however, was much greater towards cellulose than towards chitin which latter was the main constituent of bacteria while cellulose predominated in the higher forms of life. It was therefore reasonable to suppose other things being equal that copper would be more inimical to organisms in which cellulose predominated than to those in which chitin predominated; and such seemed to be the case concentrations of from 1 in 1,000 to 1 in 100,000 having in the American experiments been found necessary to kill bacteria while from 1 per million to 1 in 3 million would effect th 270 EMBREP SOME EXPERIENCES IN THE USE OF removal of AZp and similar higher forms of life. Now since in the organism after absorption of the copper free copper is not present it seemed to follow that the copper must act in the form of a cupramine base; and since the reaction with cellu-lose occurred in presence of ammonia one might postulate a reaction with the amino-acids in the cell wall of the organism.It would therefore appear that in tracing the germicidal action of copper on any cell two different reactions must be followed-namely first of all direct absorption due in certain cases to the electro-static charge or in other cases to chemical a%hity-e.g. Ehrlich’s parisitotropic groupings-or to the absorptive power of the cell membrane itself; and secondly, chemical reaction with the cell constituents in regard to which it appeared possible that an amino-compound was formed in the case of both copper and chlorine.Chloramines which were coming more and more into use as germicides were more active than chlorine itself since the amino-compound reacted with greater avidity with the cellulose or chitin. Chlorine too gave good results as an “algicide,” and possessed the advantage that anaerobic conditions were not so easily set up, while there would be no bad-smelling chloramines such as were produced from animal matter since the vegetable amino-compounds were relatively unstable as compared with those yielded by animal matter, Mr. W. T. BURGESS remarked that as was well known organisms like AZgce and diatoms flourished in water containing relatively large quantities of nitrates, so that it would be interesting to know the proportion of nitrates in the water of the reservoir in question a t different times of the year.Re should also like to ask whether when the Chara was present in large quantities any considerable reduction was noticed in the hardness of the water. Some varieties of Cham had the power of not exactly secreting but a t any rate causing the crystallisation of relatively large quantities of carbonate of lime on their structure. M.r. J. H. JOHNSTON said that he understood that some varieties of Alga? were capable of becoming acclimatised to small doses of copper sulphate so that to be effective the dose had to be continuously increased. He had no personal experience of this and understood that i t only occurred late in the season. Mi. A. CUTON CHAFMAN said that Mr. Embrey’s observation as to the in-crease in the number of bacteria at first followed by a decrease was paralleled by an experience which he had had in connection with certain breweries in which the amount of yeast produced had been very small the yeast being deteriorated and the outcrop scarcely more than was put in.That was found to occur in copper-lined fermenting tuns and particularly in those tuns in which the workmen had scoured the copper very thoroughly. In some experiments which he was thereby led to make on the growth of yeast in wort containing minute and gradually in-creasing quantiiies of copper he had found that the effect of the copper a t fist was t o stimulate the growth of the yeast i t being only when a certain point was reached that the toxic effect became marked the yeast ultimately being killed.The selective action of copper extended so far that he believed he was right in saying that copper waa actually a necessary constituent in certain bodies (e.g. molluscs and crusta-ceans) while everyone would remember Professor Church’s obtaining from the feathers of certain species of parrots the compound which he called turacin con COPPER SULPHATE IN THE DESTRCCTION OF ALGB 27 1 taining about 6 per cent. of copper. Predictions as to the effects of copper on any organism should always be avoided but it was undoubtedly shown by M?. Embrey’s experiments and by others on the same subject that as regards Alqce and similar lowly organisms of that description it was markedly toxic and did its work in an admirable manner. Lieut. RIDEAL mentioned that the late Henry Crookes in experiments made with discs of metal in Petri dishes each containing a specific organism had found indications of the stimulating action referred to.Round each disc there appeared first a zone showing no growth then a zone showing stimulated growth, and then a zone showing normal growth. These effects were obtained with several metals including copper zinc and nickel and with a great variety of organisms. M i . BURQESS remarked that experiments had also been made in Petri dishes with various metals used for dental purposes the result being to show that the most effective from a germicidal point of view was the ordinary copper alloy. =.EXBREY in reply said that in the case of these reservoirs it did not seem that metallic copper played any part in the destruction of the Alqce since it was hardly conceivable that reduction to the metallic state could have taken place, while the bands of cupric and cuprous oxide were perfectly distinct; but in face of what had been said it was clear that one could not safely generalise as to this.It would be remembered too that Mi Burgess some time ago had called attention to the fact that the growth of Algce in condensers could be prevented by the intro-duction of a coil of copper wire; but he had found that after a time the copper coils, although still effective in preventing the growths were completely covered with oxide. He had asked the opinion of several friends as to the smell of the oil obtained from Asterionella and they all agreed that it suggested rancidity showing that it was a fatty and not an essential oil; and it might perhaps be conjectured that it was the oxidation of the oil that killed the organism. With regard to nitrates these were never found in this water. He had not tried what effect the Chara might have had on the hardness of the water and it was now too Late for the Chara had all disappeared