Anthraquinones As Inhibitors of Sulfide Production from Sulfate-Reducing Bacteria

Home , Carminic acid, Trihydroxyanthraquinone

Europaisches Patentamt 19 European Patent Office

Office europeen des brevets © Publication number: 0 525 073 B1

12 EUROPEAN PATENT SPECIFICATION

@ International publication number : WO 91/15954 31.10.91 Gazette 91/25

@ ANTHRAQUINONES AS INHIBITORS OF SULFIDE PRODUCTION FROM SULFATE-REDUCING BACTERIA.

© Priority: 18.04.90 US 510763 © Proprietor : E.I. DU PONT DE NEMOURS AND 07.02.91 US 652406 COMPANY 1007 Market Street Wilmington Delaware 19898 (US) @ Date of publication of application 03.02.93 Bulletin 93/05 © Inventor : ANDERSON, Albert Gordon 2410 St. Francis Street (45) Publication of the grant of the patent : Wilmington, DE 19808 (US) 25.05.94 Bulletin 94/21 Inventor : COOLING, Frederick Beiswanger, III 521 Cabot Drive @ Designated Contracting States : Hockessin, DE 19707-0731 (US) AT BE CH DE ES FR GB GR IT LI NL Inventor : ODOM, James Martin 28 Hilltop Road Avondale, PA 19311 (US) © References cited : Inventor : WEIMER, Paul James EP-A- 0 094 721 14 Oxwood Circle CH-A- 505 900 Madison, Wl 53717 (US) CH-A- 614 466 GB-A- 2 155 453 US-A- 4 532 117 © Representative : Jones, Alan John et al CHEMICAL ABSTRACTS, vol. 43, no. 7, 10 April CARPMAELS & RANSFORD 1949, Columbus, Ohio, US; M. ANCHEL: 43 Bloomsbury Square "Identification of the antibiotic substance London, WC1A 2RA (GB) from cassia reticulata as 4,5-dihydroxyanthra- CO quinone-2-carboxylic acid", abstract no. 3062f CO h- o If) CM If) Note : Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been UJ filed until the opposition fee has been paid (Art. 99(1) European patent convention).

Jouve, 18, rue Saint-Denis, 75001 PARIS EP 0 525 073 B1

Description

FIELD OF THE INVENTION

5 The first aspect of the invention is the novel discovery that many anthraquinones, or the corresponding anthraquinol or tetrahydroanthraquinone derivatives thereof, inhibit sulfide production from sulfate-reducing bacteria and are thus potentially useful chemicals for treating industrial situations where biological sulfide generation is a problem. Examples include the prevention of souring of oil wells with hydrogen sulfide orthe con- tamination of surface operations such as pipelines, and storage tanks with hydrogen sulfide. The second as- 10 pect is the relative specificity of the anthraquinones, anthraquinols ortetrahydroanthraquinones for the process of respiratory sulfate reduction. This aspect extends the useful application of these inhibitors to situations where only sulfate-reducers should be inhibited, but not other bacterial types, as for example, in sewage treatment where bacterial digestion is desirable but sulfide generation is not, or in the utilization of biomass for the generation of fuel gases where hydrogen sulfide is a deleterious contaminant, or in the agricultural sphere 15 where sulfide generation and consequent soil alkalinity may be a problem for rice cultivation. Hereinafter, the term "anthraquinone(s)" as used in this application is defined to include anthraquinone compounds, the corresponding anthraquinol derivatives, the corresponding tetrahydroanthraquinone compounds, the corresponding octahydroanthraquinone compounds, and the reduced derivatives thereof. It is proposed that the anthraquinones are a novel treatment for the problem of hydrogen sulfide pollution 20 by virtue of their relatively benign environmental impact, which is due to their relatively specific toxicity for respiratory sulfide generation. As a consequence, these compounds may be useful in treating situations that are incompatible with the use of broad-spectrum biocides.

BACKGROUND OF THE INVENTION 25 A recent review by Widdel (1986) in Anaerobic Bacteria in Habitats Other Than Man, pp 157-184, Eds. Barnes and Mead, Blackwell Scientific Publications, includes as sulfate-reducing bacteria the genera: Desul- fovibrio, Desulfotomaculum, Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema, Desulfosarcina and Thermodesulfobacterium. Significant inter- and even intra-generic differences exist in terms of morphologies, 30 ecological niches, and metabolic capabilities. However, all respiratory sulfate-reducing bacteria are strict anae- robes which are poisoned by oxygen. The preponderance of isolates are eubacterial and classified as Desul- fovibrio although extremely thermophilic archaebacterial sulfate-reducers have been isolated from undersea volcanoes (Stetter et al., 1987 Science 236:822-825). Thus, the term sulfate-reducer encompasses a broad spectrum of organisms across both eubacterial and archaebacterial branches of bacterial phylogeny. 35 The metabolic capabilities of the genus Desulfovibrio reflect the range of available niches. Utilization of sulfate as an electron sink and respiratory substrate is the characteristic and predominant mode of energy generation. However, nitrate, elemental sulfur, f umarate and other sulfur oxy-acids, when present, can also serve in a similar capacity for a few species. These bacteria are specialists adapted to use the metabolic end-products of primary degradative bacteria for electron and carbon sources as described by Odom et al., 1981 in 40 Trends in the Biology of Fermentations for Fuels and Chemicals, Eds. Hollaender, Rabson, Rogers, Pietro, Valentine, and Wolfe, Plenum Publishing and Odom etal., 1984 Ann. Rev. Microbiol. 38:551-592. Organic acids such as lactate, formate, and pyruvate, alcohols such as ethanol, and molecular hydrogen are the preferred electron sources for sulfate reduction. Acetate, which is the metabolic end-product of the Desulfovibrio, can be utilized by Desulfobacter as an electron donor with carbon dioxide as the sole end product (Stetter et al., 45 1 987 Science 236:822-825). Thus, the sulfate-reducers can effect total mineralization of organic matter from the level of alcohols and acids to carbon dioxide. The sulfate-reducers play a special role in methane formation as it occurs in sewage treatment and freshwater bogs. In these situations, where sulfate concentrations are very low, the sulfate-reducer enters into a symbiotic relationship with methane-producing bacteria (methanogen) wherein the sulfate-reducers actually so produce hydrogen from organic acids and alcohols only if the hydrogen is continuously consumed by the methanogen. This important process, termed interspecies hydrogen transfer, is a vital link in the food chain from complex polymers to methane (Odom et al., 1981 in Trends in the Biology of Fermentations for Fuels and Chemicals, Eds. Hollaender, Rabson, Rogers, Pietro, Valentine, and Wolfe, Plenum Publishing). The most in- tensively studied aspect has been hydrogen metabolism due to its inherent relationship with methane (fuel) 55 generation from biomass. Similarly, the implication of microbial hydrogen uptake in the phenomenon of anaerobic corrosion of steel according to the theory of cathodic depolarization has stimulated research into the the hydrogen metabolism of sulfate-reducers as well as other microbial types (Von Wolzogen Kuhretal., 1934 Water 18:147-165, Odom etal., 1984 Ann. Rev. Microbiol. 38:551-592, Pankhania etal., 1986 J. Gen. Microbiol. 2 EP 0 525 073 B1

132:3357-3365, and Ringas et al., 1987 Corrosion Engineering 44 #6:386-396). There are currently approximately 130 different industrial biocide products registered with the U.S. En- vironmental Protection Agency which are produced by over a 100 different companies in the U.S. alone. The 5 leading biocides are halogenated compounds, which make up 35% of total sales, while quarternary detergents and phenolics represent 22% of total sales. Organometallic compounds, inorganic compounds, aldehydes, ani- lides, and organosulfur compounds make up the rest of the total. None of the known commercial chemicals are specif ic for sulfate-reducing bacteria, but many have demonstrated effectiveness against sulfate-reducers. Below are discussed some of the more commonly used inhibitors against sulfate-reducing bacteria. 10 Organo-sulfur or sulfur-nitrogen compounds contain some of the more effective industrial biocides against sulfate-reducers. The isothiazoline containing compounds (produced by Rohm & Haas, Inc. under the name "Kathon®") are effective against, but not specific for, sulfate-reducers at 12 ppm (Oil and Gas Journal March 8, 1982, p253). The thiocyanate containing compounds are effective in the range of 5-30 ppm (i.e. "Cytox®"). 2,2-Dibromo-3-nitrilopropionamide (DBNPA) is produced by Nalco, Inc. and is claimed to be particularly ef- 15 fective against sulfate-reducing bacteria at 3-12 ppm. This compound is also a good surfactant and corrosion inhibitor. Recently, Nalco, Inc. has introduced an oilfield biocide claimed to be effective against sulfate-reducers. The product is essentially metronidazole, which is a pharmaceutical originally used to treat Trichosomal infections. The compound is not specific for sulfate-reducers but is claimed to be generally effective against anaerobic bacteria. 20 Acids and aldehydes are also effective inhibitors of sulfate-reducers. Glutaraldehyde is widely used in water flooding situations at concentrations in the range of 100 to 2000 ppm. The sole U.S. producer is Union Car- bide, Inc. with its own registered "Ucarcide®" formulation. Ucarcide® is claimed to protect oil fields from aerobic and anaerobic microorganisms including sulfate-reducing bacteria in water flooding situations, injection water, drilling and packer fluid, formaldehyde, which is substantially cheaper, is also used in similar concen- 25 trations. These two compounds together comprise the major biocides used in the oil field. Acrolein, an extremely toxic compound, is also effective against sulfate-reducers and is reccommended for use at concentrations in the range of 1 to 15 ppm. Quarternary amines are a diverse group of compounds containing a quarternary nitrogen atom with long chain alkyl or aromatic substituents. In the oil field these compounds may act as both corrosion inhibitors and 30 bacteriostatic agents. The compounds are used in this application in the 5-100 ppm range. At low concentrations these compounds may be bacteriostatic rather than bacteriocidal. The quarternary amines are generally less hazardous than many oil field biocides, but they must often be used in conjunction with more toxic biocides to enhance their effectiveness. Halogenated compounds, such as chlorhexidine (a biguanide), (Hibitane® from ICI, Inc.) is a widely used, 35 commercial, antimicrobial compound, and is known to be effective against a number of sulfate-reducing bacteria at concentrations of 1-10 ppm (Davies et al., 1954 Brit. J. Pharmacol. 9:192). This compound or its derivatives are generally effective against most bacterial types and are, therefore, nonspecific. Biguanides such as chlorhexidine appear to function by disruption of the cell membrane which causes release of cytoplasmic contents. There is no available data on any industrial use of this chemical to treat sulfate-reducing bacteria. 40 Inorganic compounds, such as liquid chlorine, hypochlorites, chlorine dioxide and chloroisocyanurates are strong oxidizing agents, are often found as the active constituents in bleach, and have shown effectiveness in oil field situations against sulfate-reducing bacteria. Classical inhibitors of sulfate reduction such as molybdate, selenate, and fluorophosphate anions are analogues of sulfate and have been shown to interfere with the primary enzymatic step in the activation of sulfate, 45 i.e., the adenosine 5'-triphosphate (ATP) sulfurylase reaction. Here an unstable phospho-analogue anhydride is formed in place of the phospho-sulfate bond. The consequence of this is that the bacterium eventually de- pletes its energy reserve of ATP via reaction with these analogues and death ensues (Taylor et al., 1 979 Cur- rent Microbiol. 3:101-103 and Wilson et al., 1958 J. Biol. Chem. 233:975-981). Sulfate analogues have been used to inhibit sulfate-reduct ion atconcentrationsfrom 5-20 mM (i.e., 1000-4000 ppm molybdate). These levels so are impractical from an applications standpoint but the compounds have found use as research tools (Post- gate, 1952 J. Gen. Microbiol. 6:128-142 and Saleh etal., 1964 J. Appl. Bact. 27#2:281-293). Furthermore, the sulfate analogues inhibit sulfate assimilation as it occurs in all bacteria and plants, as well as sulfate respiration, and, thus, are not truly specific for sulfate-reducing bacteria. Antibacterial activity associated with anthraquinones was first discovered in plant extracts of the genus 55 Cassia. (Patel etal., 1957 Indian J. Pharmacol. 19:70-73). Subsequent investigations revealed that the active component of leaf extracts of Cassia sp. was Rhein or4,5-dihydroxyanthraquinone-2-carboxylic acid (Anchel, 1949 J. Biol. Chem. 177:169-177). Subsequently, it has become apparent that many anthraquinones have antibacterial properties, however, it is equally clear that these compounds do not inhibit all bacterial types. In one study by F. Kavanaugh it would appear that gram positive organisms such as Bacillus sp. or Staphylococ- 3 EP 0 525 073 B1

cus are sensitive to anthraquinones while gram negative species such as E. coli or Pseudomonas sp. are rather insensitive (Kavanaugh 1947 J. Bacteriol. 54:761-767). However, even among the gram positive bacteria, antibacterial effects are sporadic and unpredictable. For example, another study showed that 1,4,6,8-tetrahy- 5 droxyanthraquinone inhibited 4 species of Bacillus, one strain of Nocardia, one strain (out of four tested) of Streptomyces and one of the Gram negative Proteus. The compound did not affect any species of E. coli, Pseu- domonas, Salmonella or Sarcina (Anke et al., 1980 Arch. Microbiol. 126:223-230 and Anke et al., 1980 Arch Microbiol., 126:231-236). A study by Bakola-Christianopoulou et al. 1986 Eur. J. Med. Chem.- Chim. Ther. 21#5:385-390, where the metal chelates of the anthraquinones were studied, showed that 1 ,8-dihydroxy-an- 10 thraquinone was inactive against B. aureus, B. subtilis, B. Stearothermophilus and S. aureus. In the same study 1,2-dihydroxyanthraquinone and 1-amino-4-hydroxyanthraquinone were either inactive against these strains or required concentrations in excess of 100 to 1000 ppm for inhibition. These workers also concluded that the metal chelates were more active than the free uncomplexed compounds and that the compounds showed the most activity against the gram positive Bacillus sp. These results are typical of the published stud- 15 ies on the antibacterial activity of anthraquinones. Swiss Patent No. 614,466 of Mycogel Laboratories Inc., Brooklyn, New York, entitled Agent for Inhibiting the Growth of Bacteria in Culture Media and Use of the Agent describes the use of compounds derived from paraquinone or their hemiquinone or glycoside derivatives as agents for use in culture media for cultivating fungi and yeasts. The rationale disclosed was that these compounds inhibit bacterial growth but not the growth 20 of eukaryotic cells such as molds and yeasts. Preferred anthraquinone derivatives claimed include those substituted with methyl, hydroxymethyl, carboxyl, aldehyde and carboxyethyl groups. Haran et al., 1981 Isr. J. Med. Sci. 17#6:485-496, demonstrated that certain diaminoanthraquinone derivatives exhibited toxicity against gram positive cocci and that gram negative bacteria were rather insensitive. The mode of action of anthraquinones on bacterial metabolism is not clear and may be multitudinous. It 25 is clear that the inhibitory effect is only observed with bacteria and not with plant, fungal or mammalian tissue, hence, the compounds are relatively non toxic to higher life forms. It is known that many anthraquinones interfere with bacterial DNA metabolism, presumably at the site of DNA directed RNA polymerase (Anke et al., 1980 Arch Microbiol., 126:231-236).). Anthraquinone-containing compounds have also been shown to inhibit mitochondrial ADP transport (Boos et al., 1981 FEBS Lett. 127:40-44). It is also known that reduced anthra- 30 quinones may react chemically with oxygen to produce the highly toxic superoxide radical and this is generally very toxic to bacteria (Shcherbanovskii et al., 1975 Rastit. Resur. 11#3:445-454). The consensus from the existing literature is that anthraquinones are not generally antimicrobial. The organisms that are sensitive to anthraquinones have been Gram positive bacteria, in particular Bacillus sp. The anthraquinones do exhibit an array of unrelated and unpredicted biological effects as briefly listed above. The 35 inhibition of sulfide production by anthraquinones is unreported in the literature and appears to be another example of an unpredicted and unrelated biological effect, particularly considering that the sulfate-reducers are Gram negative organisms. It is clear that sulfide pollution is a growing industrial and environmental concern for which there exists no truly effective or adequate treatment that is environmentally sound. The chemical treatments that are avail- 40 able have a number of shortcomings. Many of these chemicals are highly reactive with short effective lives as antimicrobials and therefore high concentrations are required. Secondly, due to their inherent toxicity, these compounds may pose a health hazard to the personnel using them. Thus a need exists for better means of controlling sulfide pollution. One key and important difference between existing chemicals and the compounds of the instant invention 45 is the relatively unreactive nature of a number of anthraquinones as a group. In fact, 1 ,8-dihydroxyanthraquinone (one of our most potent compounds) has been sold commercially as a laxative (see Physicians' Desk Reference, page 574 for Modane®, and page 575 for Modane Plus®), Many anthraquinones, including derivatives of 1 ,8-dihydroxyanthraquinone, are naturally-occurring in a number of plants such as rhubarb (Rheum officinale). Use of plant extracts include senna in Senakot® Tablet and Senokot-S® (see Physician's Desk so Reference, pages 1596-1597), and casenthranol in Dialose Plus® (see Physicians' Desk Reference, page 1979). The specific inhibitory activity of the compounds of the present invention and their lack of toxicity to many other organisms opens up entirely new possiblities for use in various waste treatment situations where con- ventional biocides cannot be used. For example, the odor associated with sulfide pollution in sewage treatment 55 is both a health hazard and an esthetic problem for many communities. More significantly, from the economic standpoint, concrete conduits are damaged from the aerobic oxidation of sulfide to sulfuric acid. This is a particular problem for cities such as Miami or Los Angeles where the distances involved in sewage transit at ambient temperatures mean long residence times, high metabolic activity and tremendous destruction of concrete structures. The current "state of the art" method to treat this problem is to precipitate the sulfide from 4 EP 0 525 073 B1

solution using massive amounts of ferrous chloride. This alleviates the odor problem but does not remove the sulfide as a substrate for acid producing bacteria (Jameel 1988 Journal WPCF 61#2:230 and Dezham et al., Journal WPCF 60#4:514). The instant invention is particularly advantageous for this kind of application. 5 SUMMARY OF THE INVENTION

The present invention relates to a process for inhibiting sulfide production by sulfate-reducing bacteria comprising contacting certain anthraquinone compounds with the medium containing the sulfate-reducing 10 bacteria.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic of an automated analyzer used to screen anthraquinones for inhibition of sulfide 15 production. Figure 2 is a trace from an automated analyzer used to screen anthraquinones for inhibition of sulfide production.

DETAILED DESCRIPTION OF THE INVENTION 20 One object of this invention comprises a process to inhibit sulfide production by sulfate-reducing bacteria comprising contacting certain anthraquinones with the medium which contain the sulfate-reducing bacteria. The term "anthraquinone(s)" as used herein is as previously defined under Field of the Invention. Many anthraquinones are commercially available. The preparation of anthraquinol derivatives from an- 25 thraquinones is known to those skilled in the art. For example, 1 ,8-dihydroxy-9-anthranol can be prepared by reduction of 1,8-dihydroxyanthraquinone using hydrogen and a nickel catalyst, K. Zahn and H. Koch, Chemi- sche Berichte, 17B, 172 (1938) or by use of phosphorous and hydriodic acid. 1,2,10-Anthracenetriol can be prepared by reduction of 1,2-dihydroxyanthraquinone using ammonium hydroxide and zinc, H. Roemer, Chemi- sche Berichte, 14, 1259 (1881), C. Grabe and C. Thode, Liebig's Annalen derChemie, 349, 207 (1906). These 30 and similar reductions are summarized in Das Anthracen und die Anthrachinone, H. Houben, Georg Thieme pub., 1929, Leipzig, pp. 193-195. Reduction methods for preparation of hydroanthraquinones are found in Das Anthracen und die Anthrachinone, H. Houben, Georg Thieme, pub., 1929, Leipzig, pp 183-186. For example, the preparation of tetrahydroanthraquinones, and various reduced derivatives are described in U.S. Patents 1,967,862 issued July 24, 1934 and 4,642,393 issued February 10, 1987; Diels and Alder, Ber., 62, 2337 35 (1929); Carothers etal., J.Amer. Chem. Soc, 54, 4071 (1932); and Euleretal., Ber., 53B, 822 (1920). The activity of various anthraquinones in the inhibition of sulfide production by sulfate-reducing bacteria can be predicted based upon analysis of the substituents present. A total of 147 different active anthraquinones were named according to the rules found in the 7th Collective Index of Chemical Abstracts. Following this convention, a multiple linear regression analysis of the activity data resulted in the creation of formula (I) 40 Activity = 28.1 +S(coeff.) (I) wherein activity refers to the percent reduction of H2S production, 28.1 is a constant, and 2 (coeff.) refers to the sum of the coefficient values. Coefficient values are given in the coefficients table, Table C, for each individual coefficient through Rs present in the anthraquinones represented by structure (A), or the corresponding derivatives thereof. 45

(A) 50

As an example, the activity of the anthraquinone substituted as shown below is predicted to be 26.4 from 55 the formula. The observed value is 26.0.

5 EP 0 525 073 B1

O Br

CH3 O

Anthraquinones having substituents with positive values in the coefficient table increase activity while those that have substituents with negative values decrease activity. Anthraquinones having substituents whose coefficients yield positive activity in formula (I) are active and those with an activity above at least 21 in the formula are most active and preferred. Those having substituents whose coefficients yield a negative activity in formula (I) are not active. The compounds may possess some substituents having negative coefficients and still retain overall activity. These negative substituents can serve other purposes, such as increas- ing solubility. It should be noted that the standard deviation of regression for the formula is 20.7; therefore, the values calculated from the formula are + 20.7.

6 EP 0 525 073 B1

TABLE C Substituent Substituent Name r.nef fieient Name Coefficient 1 CI 5.3 28 05 -5.6 2 C2 7.0 29 06 -7.8 3 C3 -9.2 30 07 -1.5 4 C4 -16.3 31 08 4.7 5 C6 -6.9 32 0R1 -23.1 6 C7 -42.6 33 0R2 -7.4 7 Carb2 35.0 34 0R3 3.1 8 Carb3 -53.0 35 0R4 2 9.1 9 Nlamine -0.2 36 0R5 31.0 10 Nlamide -19.4 37 0R6 78.4 11 Nlaryl -5.4 3B SI -4.8 12 N2amine -7.0 39 S2 -3.7 13 N2amide -7.7 40 S3 4.8 14 N2aryl 1.8 41 S5 -5.2 15 N4amine -19.6 42 S6 -11.0 16 N4amide -24.0 43 S7 -15.4 17 N4aryl -14.0 44 Cil ■ 25.0 18 N5amine 4.4 45 C12 25.8 19 N5amide 6.0 46 C13 -1.9 20 N5aryl 8.4 47 C14 -12.6 21 N6amine -1.0 48 C15 18.9 22 N6aryl -20.5 49 C16 -4.9 23 N6amine -4.6 50 C18 -12.7 24 01 12.1 51 Brl -8.4 25 02 6.0 52 Br2 7.5 26 03 -13.0 53 Br4 -14.5 27 04 -9.6 54 Br5 3.1

Number of data points = 147 Standard deviation of regression = 20.7 The abbreviations for the substituents are explained as follows: "C" refers to any alkyl or aryl group bound to the anthraquinone ring by a covalent carbon-carbon bond with the exception of carboxylic acid groups. "Carb" refers to any carboxylic acid group, C02H, bound to the anthraquinone ring. "N-amine" refers to any amino group, NR1R2, where R^ and R2 may be H or alkyl; "N-amine" also refers to N02, diazonium, or NO groups since under the reducing conditions present, these are expected to be reduced to amine groups. "N-amide" refers to EP 0 525 073 B1

RlO N-C-R2 5

R3O 10 N-S-R4 0

groups, where where R2, R3, R4 may be H, alkyl groups oraryl rings. 15 "N-aryl" refers to aryl amine groups, such as phenylamino, or a heterocyclic aromatic ring attached to the anthraquinone ring by carbon-nitrogen-carbon bonds. "O" refers to the hydroxyl group. "OR" refers to alkyl or aryl ethers. "S" refers to S03H group. 20 "CI" and "Br" refer to the chloro and bromo groups. The number with each substituent refers to the position on the anthraquinone ring to which the substituent is attached. Anthraquinones or anthraquinone precursors or derivatives suitable for the inhibition of sulfide production by sulfate-producing bacteria in the process of the present invention include the following: 25 N-[1-(9,10-dihydro-9,10-dioxo)anthracenyl]-N'-(1-methylethyl)imidodicarbonimidic diamide hydrochloride CAReg. No.: None Source: E. I. du Pont de Nemours and Company 1- Aminoanthraquinone (97%) CAReg. No.: 82-45-1 30 Source: Aldrich Chemical Co., Inc. 940 West Saint Paul Avenue Milwaukee, Wl 53233 2- Aminoanthraquinone CAReg. No.: 117-79-3 Source: Aldrich 1-Amino-4-Hydroxyanthraquinone 35 CAReg. No.: 116-85-8 Source: Kodak Laboratory Chemicals Building 70 Eastman Kodak Co. 343 State Street Rochester, NY 14650 1 ,2-Diaminoanthraquinone CAReg. No.: 1758-68-5 40 Source: Aldrich 2,6-Dihydroxyanthraquinone; Anthraflavic acid CAReg. No.: 84-60-6 Source: Aldrich Anthraquinone-2-carboxylic acid (98%) 45 CAReg. No.: 117-78-2 Source: Aldrich 1,5-Dihydroxyanthraquinone; Anthrarufin (92%) CAReg. No.: 117-12-4 Source: Pfaltzand Bauer, Inc. Division of Aceto Chemical Co., Inc. 172 East Aurora Street Waterbury, 50 CT 06708 1 ,2-Dihydroxyanthraquinone; Alizarin CAReg. No.: 72-48-0 Source: Aldrich 2,2'-[(9,10-Dihydro-9,10-dioxo-1,5-anthracenediyl)diimino]bis[5-methylbenzenesulfonic acid], di Na salt; Ali- 55 zarine violet 3R CAReg. No.: 6408-63-5 Source: Aldrich 1 ,2,5,8-Tetrahydroxyanthraquinone; Quinalizarin CAReg. No.: 81-61-8 8 EP 0 525 073 B1

Source: Aldrich 4- Amino-9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracenesulfonicacid, monosodium salt; Nuclear fast red CAReg. No.: 6409-77-4 5 Source: Aldrich 1 ,8-Dihydroxyanthraquinone; Danthron CAReg. No.: 117-10-2 Source: Aldrich 2,2'-[(9,10-Dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-methylbenzenesulfonic acid],di Na salt; Acid 10 green 25 CAReg. No.: 4403-90-1 Source: Aldrich 1 -Amino-2,4-bromoanthraquinone CAReg. No.: 81-49-2 15 Source: Pfaltz & Bauer 5- Chloro-1-anthraquinonylamine CAReg. No.: 117-11-3 Source: Pfaltz & Bauer 2-Ethylanthraquinone (97+%) 20 CAReg. No.: 84-51-5 Source: Aldrich 1-Hydroxyanthraquinone (97%) CAReg. No.: 129-43-1 Source: Aldrich 25 2-(Hydroxymethyl)anthraquinone (97%) CAReg. No.: 17241-59-7 Source: Aldrich 1-Amino-4-methoxyanthraquinone CAReg. No.: 116-83-6 30 Source: Aldrich 1-Amino-6,7-dichloroanthraquinone CAReg. No.: 5355-88-4 Source: Aldrich Benz[a]anthracene-7,12-dione (97%) 35 CAReg. No.: 2498-66-0 Source: Aldrich 1,8-Dihydroxy-3-methylanthraquinone; Chrysophanic acid CAReg. No.: 481-74-3 Source: Aldrich 40 1 0-[(3-Amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7,8,9, 1 0-tetrahydro-6,8, 1 1 -trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione hydrochloride;- Adriamycin hydrochloride CAReg. No.: 23214-92-8 Source: Aldrich 9,1 0-Dihydro-4,5-dihydroxy-9,1 0-dioxo-2-anthracenecarboxylic acid; Rhein 45 CAReg. No.: 478-43-3 Source: Aldrich (8S-cis)-8-Acetyl-10[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride; Daunomycin hydrochloride CAReg. No.: 20830-81-3 50 Source: Sigma Chemical Co., Inc. P.O. Box 14508 Saint Louis, MO 63178 1 ,2,4-Trihydroxyanthraquinone; Purpurin CAReg. No.: 81-54-9 Source: Aldrich 1-Aminoanthraquinone diazonium salt 55 CAReg. No.: 16048-40-1 Source: K&K Laboratories Division of ICN Biomedicals, Inc. 4911 Commerce Parkway Cleveland, OH 44128 2,2'-Dimethyl-[1,1'-bianthracene]-9,9',10,10'-tetrone; 2,2'-Dimethyl-1,1'-bianthraquinone CAReg. No.: 81-26-5 9 EP 0 525 073 B1

Source: Aldrich 3-(D-apio-beta-D-Furanosyloxy)-1,8-dihydroxy-6-methyl-9,10-anthracenedione; Frangulin B CAReg. No.: 14101-04-3 5 Source: Pfaltz & Bauer 2-Chloroanthraquinone 99% CAReg. No.: 131-09-9 Source: Aldrich 1 .5- Dichloroanthraquinone (96%) w CAReg. No.: 82-46-2 Source: Aldrich 1,4,5,8-Tetrachloroanthraquinone CAReg. No.: 81-58-3 Source: K & K 15 1-Chloroanthraquinone CAReg. No.: 82-44-0 Source: Aldrich 1 ,8-Dichloroanthraquinone (97%) CAReg. No.: 82-43-9 20 Source: Aldrich 2-Bromo-3-methylathraquinone CAReg. No.: 84-44-6 Source: Pfaltz & Bauer 2- (2,2,2-Trimethylpropionamido)anthraquinone 25 CAReg. No.: None Source: Aldrich 2.6- Bis[2-(dimethylamino)ethoxy]-9,10-anthracenedione; Tilorone analog R11,043DA CAReg. No.: 66686-31-5 Source: Sigma 30 2-Methyl-1-nitroanthraquinone CAReg. No.: 129-15-7 Source: Lancaster Synthesis Ltd. P.O. Box 1000 Windham, NH 03087 1-Amino-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid; 1-Aminoanthraquinone-2-sulfonic acid CAReg. No.: 83-62-5 35 Source: Aldrich 9,10-Dihydro-5-nitro-9,10-dioxo-1-anthracenesulfonic acid; 5-Nitroanthraquinone-1-sulfonic acid CAReg. No.: 82-50-8 Source: Aldrich 3- Chloro-9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid; 2-Chloroanthraquinone-3-carboxylic acid 40 CAReg. No.: 84-32-2 Source: Pfaltz & Bauer Anthraquinone CAReg. No.: 84-65-1 Source: Aldrich 45 1 ,8-Dihydroxy-3-(hydroxymethyl)-anthraquinone; Aloe-emodin CAReg. No.: 481-72-1 Source: Apin Chemicals, Ltd. Unit 1 Milton Trading Estate Near Abingdon Oxon OX14 4RS United Kingdom 7,16-Dichloro-6,15-dihydro-5,9,14,18-anthrazinetetrone; 7,16-Dichloroindanthrone 50 CAReg. No.: 130-20-1 Source: K & K 1,2,3,4,5,8-Hexahydroxyanthraquinone; Alizarin cyanin CAReg. No.: None Source: K & K 55 2,4,5,7-Tetrabromo-1,8-dihydroxy-9,10-anthracenedione; 2,4,5,7-Tetrabromochrysazin CAReg. No.: 17139-66-1 Source: K & K 1 ,2,7-Trihydroxyanthraquinone; Anthrapurpurin CAReg. No.: 602-65-3 10 EP 0 525 073 B1

Source: Pfaltz & Bauer 1 ,4,5-Trihydroxy-2-methyl-9,10-anthracenedione; Islandicin CAReg. No.: 476-56-2 5 Source: Apin 1 .4.5.7- Tetrahydroxy-2-methyl-9,10-anthracenedione; Catenarin CAReg. No.: 476-46-0 Source: Apin 1,8-Dihydroxy-3-methoxy-6-methyl-9,10-anthracenedione; Physcion w CAReg. No.: 521-61-9 Source: Apin 1 .4.5.8- Tetrahydroxy-2-methyl-9,10-anthracenedione; Cynodontin CAReg. No.: 476-43-7 Source: Apin 15 1 ,5,8-Trihydroxy-3-methyl-9,10-anthracenedione; Helminthosporin CAReg. No.: 518-80-9 Source: Apin 1- Hydroxy-2-[(6-O-p-D-xylopyranosyl-p-D-glucopyranosyl)oxy]-9,10-anthracenedione; Ruberythric acid CAReg. No.: 152-84-1 20 Source: Apin 2- Phenoxy quinizarin-3,4'-disulfonic acid, di K salt CAReg. No.: None Source: K & K (+,-)-1-Acetoxy-8-hydroxy-1,4,4a,9a-tetrahydroanthraquinone 25 CAReg. No.: 73794-49-7 Source: Aldrich 1-Amino-4-[[4-[(dimethylamino)methyl]phenyl]amino]-9,10-anthracenedione; Basic Blue 47 CAReg. No.: 12217-43-5 Source: Aldrich 30 1,5-Bis(2-carboxyanilino)-9,10-anthracenedione; Acridylic acid CAReg. No.: 81-78-7 Source: Sandoz Chemicals 4000 Monroe Road Charlotte, NC 28205 1 ,8-Dihydroxy-9-anthranol; 1 ,8-Dihydroxyanthranol CAReg. No.: 480-22-8 35 Source: Aldrich 1 ,2,1 0-Anthracenetriol; Anthrarobin CAReg. No.: 577-33-3 Source: Aldrich 1-Amino-4-bromo-2-methylanthranquinone (99%) 40 CAReg. No.: 81-50-5 Source: Aldrich 1,4-Diaminoanthraquinone (97%) CAReg. No.: 128-95-0 Source: Aldrich 45 2,6-Diaminoanthraquinone CAReg. No.: 131-14-6 Source: Aldrich 1-Amino-4[4-[[4-chloro-6[[2, 3 or4-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid; Reactive blue 2; Procion blue HB(S); Cibacron blue 3G-A; Basi- 50 len blue E-3G CAReg. No.: 12236-82-7 Source: Aldrich Anthraquinone-1,5-disulfonic acid, di Na salt hydrate (95%) CAReg. No.: 853-35-0 55 Source: Aldrich Anthraquinone-2,6-disulfonic acid, di Na salt CAReg. No.: 84-50-4 Source: Aldrich Anthraquinone-2-sulfonic acid, sodium salt monohydrate 11 EP 0 525 073 B1

CAReg. No.: 131-08-8 Source: Aldrich 1,2-Bis[(4-sulfophenyl)amino]-4-hydroxyanthraquinone; Alizarin blue black B 5 CAReg. No.: 1324-21-6 Source: Aldrich 3-Aminomethylalizarin-N,N-Diacetic acid CAReg. No.: 3952-78-1 Source: Pfaltz & Bauer 10 1-Amino-4-[[3-(ethenylsulfonyl)phenyl]-9,10-dihydro-9,10-dioxo]-2-anthracene sulfonic acid, monosodium salt; Acid blue 215 CAReg. No.: 14541-90-3 Source: Aldrich 1 -(Methylamino)anthraquinone (98%) 15 CAReg. No.: 82-38-2 Source: Aldrich 2,2'-[(9,10-Dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracendiyl)diimino]bis[5-methylbenzenesulfonic acid], di Na salt; Acid green 41 CAReg. No.: 4430-16-4 20 Source: Aldrich 2,2'-[(9,10-Dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-butylbenzenesulfonic acid]; Acid green 27 CAReg. No.: 6408-57-7 Source: Aldrich 1,1'-lminobis[4-amino]9,10-anthracenedione, sulfonated; Acid black 48 25 CAReg. No.: 1328-24-1 Source: Aldrich 1-Amino-9,10-dihydro-9,10-dioxo-4-(phenylamino)-2-anthracenesulfonic acid, Na salt; Acid blue 25 CAReg. No.: 6408-78-2 Source: Aldrich 30 4-[[4-(Acetylamino)phenyl]amino]-1-amino-9,10-dihydro- 9,10-dioxo-2-anthracenesulfonic acid, Na salt; Acid blue 40 CAReg. No.: 6424-85-7 Source: Aldrich 1-Amino-9,10-dihydro-9,10-dioxo-4-[[3[[2-(sulfooxy)ethyl]-sulfonyl]phenyl]amino]-2-anthracenesulfonic acid, 35 disodium salt; Remazol Brilliant blue R; CAReg. No.: 2580-78-1 Source: Aldrich 1-Amino-4[[3-[4,6-dichloro-1,3,5-triazin-2-yl)amino]-4-sulfophenyl]amino]-9,10 dihydro-9,10-dioxo-2-anthracenesulfonic acid; reactive blue 4 40 CAReg. No.: 13324-20-4 Source: Aldrich 1-(9,10-Dihydro-9,10-dioxo-1-anthracenyl-1,2-hydrazinedisulfonic acid, di Na salt; (1-Anthraquinonyl)-1,2-hydrazine disulfonic acid, di Na salt CAReg. No.: 54345-83-4 45 Source: K & K 9,10-Dihydro-5,6-dihydroxy-9,10-dioxo-1-anthracenesulfonic acid; Alizarin-5-sulfonic acid CAReg. No.: 6373-42-8 Source: Tokyo Kasei Kogyo Co., Ltd. c/o CTC Organics 792 Windsor Street P.O. Box 6933 Atlanta, GA30315 so N-(4-Chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide; 1-Benzamido-4-chloroanthraquinone CAReg. No.: 81-45-8 Source: Aldrich 1-Amino-4-bromo-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid, Na salt; 1-Amino-4-bromoanthraquinone-2-sulfonic acid, Na salt 55 CAReg. No.: 6258-06-6 Source: Aldrich 1-Amino-9,10-dihydro-4[[(4-methylphenyl)sulfonyl]amino-9,10-dioxo-2-anthracenesulfonic acid, Na salt; 1- Amino-4-(p-toluenesulfonamido)anthraquinone-2-sulfonic acid, Na salt CAReg. No.: 64981-00-6 12 EP 0 525 073 B1

Source: Aldrich 9,1 0-Dihydro-9,1 0-dioxo-2,3-anthracenedicarboxylic acid CAReg. No.: 27485-15-0 5 Source: Aldrich 1,1'-lminobis(4-nitro-9,10-anthracenedione) CAReg. No.: 128-88-1 Source: Pfaltz & Bauer 1- Amino-4-chloro-2-methylanthraquinone w CAReg. No.: 3225-97-6 Source: Aldrich 2.3- dimethyl-1,4-dihydroxyanthraquinone; 2,3-Dimethylquinizarin CAReg. No.: 25060-18-8 Source: Aldrich 15 6-Methyl-1,3,8-trihydroxyanthraquinone; Emodin (99%) CAReg. No.: 518-82-1 Source: Aldrich 1.4- Bis(methylamino)-anthraquinone CAReg. No.: 2475-44-7 20 Source: Aldrich N-(4-Amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthracenyl)-4-methylbenzenesulfonamide; N-(4-Amino- 3-methoxyanthraquinone-1-yl)-p-toluenesulfonamide; 1-Amino-2-methoxy-4-(p-tolylsufonamido)anthraquinone CA.Reg. No.: 81-68-5 25 Source: Aldrich [1,1'-Bianthracene]-9,9'10,10'-tetrone;1,1'-Bianthraquinone CAReg. No.: 914-20-5 Source: Columbia Organic Chemical Co., Inc. P.O. Box 1045 Camden, SC 29020 6.7- Dichloro-1 ,4-dihydroxyanthraquinone (97%) 30 CAReg. No.: 1225-15-6 Source: Aldrich 2- [[9,10-Dihydro-4-(methylamino)-9,10-dioxo-1-anthracenyl]amino]-5-methyl-benzenesulfonic acid, monosodium salt; Alizarine astral B-CF CAReg. No.: 6408-51-1 35 Source: Tokyo Kasei Kogyo Co., Ltd. 2.8- Diphenyl-anthra[2,1-d:6,5-d']bisthiazole-6,12-dione; Indanthrene yellow GCN CAReg. No.: 129-09-9 Source: Tokyo Kasei Kogyo Co., Ltd. 2-Methoxy-3-methyl-9,10-anthracenedione 40 CAReg. No.: 17241-42-8 Source: Aldrich 1,4-Bis[(1-methylethyl)amino]-9,10-anthracenedione; 1,4-Di(isopropylamino)anthraquinone CAReg. No.: 14233-37-5 Source: K & K 45 1,4-Bis[(2,4,6-triethylphenyl)amino]-9,10-anthracenedione; 1,4-Bis(2,4,6-triethylanilino)anthraquinone CAReg. No.: 116-74-5 Source: K & K 1- (2-Hydroxyethyl)amino-4-methylaminoanthraquinone; Disperse blue 3 CAReg. No.: 2475-46-9 so Source: Aldrich 1,4-Bis[(4-methylphenyl)amino]9,10-anthracenedione; Solvent green 3 CAReg. No.: 128-80-3 Source: Aldrich 2- Amino-3-hydroxyanthraquinone 55 CAReg. No.: 117-77-1 Source: Tokyo Kasei Kogyo Co., Ltd. 1-(Bromothio)anthraquinone CAReg. No.: None Source: Aldrich 13 EP 0 525 073 B1

1 ,8-Bis(phenylmethoxy)-9,1 0-anthracenedione; 1 ,8-Dibenzyloxyanthraquinone CAReg. No.: 69595-66-0 Source: Aldrich 5 1-Amino-2-(2-aminoethylthio)-4-hydroxyanthraquinone CAReg. No.: None Source: Aldrich 1,4-Bis(pentylamino)-9,1 0-anthracenedione; Oil blue N CAReg. No.: 2646-15-3 10 Source: Aldrich 1- Amino-2-bromo-4-hydroxyanthraquinone CAReg. No.: 116-82-5 Source: Aldrich 2- Propionamidoanthraquinone 15 CAReg. No.: None Source: Aldrich 1,4-Diamino-2,3-bis(2-phenoxyethoxy)anthraquinone CAReg. No.: 41313-11-5 Source: Aldrich 20 N-(5-Chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide; 1-Benzamido-5-chloroanthraquinone CAReg. No.: 117-05-5 Source: Aldrich Anthraquinone-1-arsonic Acid CAReg. No.: None 25 Source: K & K N,N'-[lminobis(9,10-dihydro-9,10-dioxo-4,1-anthracenediyl)]-bisbenzamide; 4,4'-Dibenzamido-1,1'-dianthrimide CAReg. No.: 128-79-0 Source: Aldrich 30 1 ,4,5,8-Tetraaminoanthraquinone; Disperse blue 1 CAReg. No.: 2475-45-8 Source: Aldrich 2-Methylanthraquinone CAReg. No.: 84-54-8 35 Source: Aldrich 9,10-Dihydro-9,10-dioxo-2,7-anthracenedisulfonic acid, di Na salt; Anthraquinone-2,7-disulfonic acid, di Na salt CAReg. No.: 853-67-8 Source: K & K 40 1 ,2,3-trihydroxyanthraquinone; Anthragallol CAReg. No.: 602-64-2 Source: K & K Carmine (Aluminum lake) CAReg. No.: 1390-65-4 45 Source: Fisher Scientific Co., Inc. 711 Forbes Avenue Pittsburg, PA 15219 9,1 0-Dihydro-1 ,4-dihydroxy-9,1 0-dioxo-2-anthracenesulfonic acid CAReg. No.: 145-48-2 Source: Pfaltz & Bauer 2-Amino-3-chloroanthraquinone 50 CAReg. No.: 84-46-8 Source: Kodak 1-Anthraquinonesulfonic acid, Na salt CAReg. No.: 128-56-3 Source: Kodak 55 2-tert-butylanthraquinone (98%) CAReg. No.: 84-47-9 Source: Aldrich 1 ,4-Dihydroxyanthraquinone CAReg. No.: 81-64-1 14 EP 0 525 073 B1

Source: Lancaster 1,5-Diamino-4,8-dihydroxyanthraquinone CAReg. No.: 145-49-3 5 Source: Lancaster 1-Hydroxy-4-[(4-methylphenyl)amino]-9,1 0-anthracenedione; 1-Hydroxy-4-(p-toluidino)-anthraquinone CAReg. No.: 81-48-1 Source: Sigma 1 ,4-Dimethylanthraquinone (95%) w CAReg. No.: 1519-36-4 Source: Pfaltz & Bauer 1,1'-lminobis-9,1 0-anthracenedione; Dianthrimide CAReg. No.: 82-22-4 Source: Pfaltz & Bauer 15 2-(Cyclopropylcarboxamido)anthraquinone CAReg. No.: None Source: Aldrich 1- Amino-2-methylanthraquinone; Disperse orange 11 CAReg. No.: 82-28-0 20 Source: Sigma 2- [(9,10-Dihydro-4-hydroxy-9,10-dioxo-1-anthracenyl)amino]-5-methyl-benzenesulfonicacid, Na salt; Solway purple R CAReg. No.: 4430-18-6 Source: K & K 25 2,2'-[(9,10-Dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-methyl]benzenesulfonic acid; alizarine viridine CAReg. No.: 4430-16-4 Source: K & K 1,4-Bis(ethylamino)-9,1 0-anthracenedione; Sudan blue 30 CAReg. No.: 6994-46-3 Source: K & K 1,4-Diamino-5-nitroanthraquinone CAReg. No.: 82-33-7 Source: K & K 35 N-Benzyl-9,10-dihydro-9,10-dioxo-2-anthracenesulfonamide CAReg. No.: None Source: Aldrich 2-Bromoanthraquinone CAReg. No.: 572-83-8 40 Source: E. I. du Pont de Nemours and Company 1-Fluoroanthraquinone CAReg. No.: 569-06-2 Source: E. I. du Pont de Nemours and Company 1- Cyanoanthraquinone 45 CAReg. No.: 38366-32-4 Source: E. I. du Pont de Nemours and Company 2- Trifluoromethylanthraquinone CAReg. No.: 362-21-0 Source: E. I. du Pont de Nemours and Company so All the above-named compounds except N-[1-(9,10 dihydro-9,10 dioxo)anthracenyl]-N'-(1-methylethyl)imidodicarbonimidicdiamide hydrochloride are commercially available. N-[1 -(9,10 dihydro-9,10 dioxo)anthracenyl]-N'-(1-methylethyl)imido dicarbonimidic diamide hydrochloride, however, is a novel compound and was synthesized as follows: Athree liter round bottomed flask was charged with 1 8.2 g (3.09 mole) of isopropyl amine. After cooling the flask to -70°C in a dry ice and acetone bath, 304.5 g of concentrated (37%) hydro- 55 chloric acid (3.09 mole) was added. The mixture was warmed to room temperature and water was removed with a rotary evaporator at aspirator pressure. To the dry residue was added 275.3 g (3.09 mole) of sodium dicyanamide and 760 ml of 1-butanol. The mixture was heated under reflux forthree hours and then the butanol was removed with a rotary evaporator at aspirator pressure. To the residue in the flask was added 1 800 ml of 1 ,4-dioxane. A slightly sticky precipitate was formed which was filtered and recrystallized from four liters of 15 EP 0 525 073 B1

1 ,4-dioxane to yield 379 g of N-cyano-N'-isopropylguanidine as a co-precipitate with some NaCI, 1 ,4-dioxane, and isopropylamine. Calc. for C5H10N4(NaCI)0.1(C4H8O2)0.1(C3H9N)0.15 5 Calc: C 46.94; H 8.1 8; N 38.83; CI 2.37 Found: C 46.85; H 8.27; N 38.42; CI 2.44 To 5.02 g of 1-aminoanthraquinone (0.0225 mole) was added 2.22 g (0.0225 mole) of concentrated (37%) hydrochloric acid. The mixture was dried at 95°C on a rotary evaporator to give a free-flowing powder. Then, 20 ml of 1-butanol and 4.26 g of N-cyano-N'-isopropylguanidine as prepared above were added to the flask 10 and the mixture was refluxed for one hour. The mixture was then cooled to room temperature (about 22°C). The reaction was judged to be complete due to the absence of a cyano group infrared stretching frequency at 2180 cm-1. The product was precipitated by adding 100 ml of diethyl ether and air dried to give 5.86 g of N- [1-(9,10 dihydro-9,10 dioxo)anthracenyl]-N'-(1-methylethyl)imido dicarbonimidic diamide hydrochloride having characteristic infrared absorption at 1640, 1601, 1540, and 1285 cm-1. 15 The preferred anthraquinones to inhibit sulfide production by sulfate-producing bacteria are: 1 ,8-dihydroxyanthraquinone, 1-chloroanthraquinone, 2-chloroanthraquinone, and 3-chloro-2-anthraquinone carboxylic acid. If sulfate-reducing bacteria are growing by sulfate respiration (which is the growth mode from which they derive the most energy or growth) then inhibition of sulfide production will result in cessation of bacterial growth. Cessation of bacterial growtn under these conditions may not necessarily result in cell death and does 20 not preclude the bacteria utilizing other energy yielding pathways which do not involve sulfate as an electron acceptor as previously discussed. A number of different sulfate-reducing bacteria as well as several other organisms are suitable for use in the present invention and were used in the experiments of this invention. They were all grown in BTZ-3, BTZ- 3 supplemented with other chemicals, or BTZ-4 medium. These media will be described hereinafter. The or- 25 ganisms used in the experiments were obtained from the sources shown below but they are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852. For use in the experiments below, Desulfovibrio desulf uricans G1 00A was isolated at E. I. du Pontde Nemours and Company, Wilmington, DE; D. desulf uricans API was obtained from the American Petroleum Institute; D. salexigens, D. vulgaris, D. desulfuricans 27774 and D. desulfuricans Norway were obtained from the laboratory of Dr. H.D. Peck Jr. Uni- 30 versity of Georgia, Athens GA 30601 and were grown in BTZ-3 medium supplemented with sterile sodium lactate (60 mM) and sodium sulfate (30 mM). The D. salexigens and D. desulfuricans Norway strains were grown in BTZ-3 medium supplemented additionally with sodium chloride to a final concentration of 0.4 M. BTZ-3 medium was used to grow Escherichia coli and T. denitrificans, however, the medium was then supplemented differently for each organism to contain 5 mM glucose for E. co[i and 25 mM sodium thiosulfate and 10 mM 35 sodium nitrate for T. denitrificans. Growth conditions for all the above organisms were strictly anaerobic with a nitrogen atmosphere. Where reducing agents were added to the medium a reducing agent of the following composition was used: Sodium sulfide 9 hydrate 50 mM Cysteine hydrochloride 50mM 40 in 0.1 N sodium hydroxide There may be a problem in delivering a known amount of an anthraquinone to an aqueous system because these compounds are not very soluble in water. Therefore solvent systems to deliver concentrated amounts of inhibitor accurately may be required. Suitable solvents for use herein comprise water-miscible solvents. Ex- amples of such solvents include, but are not limited to, water, detergent emulsions in water, ethanol, acetone, 45 methanol, dimethylsulfoxide, dimethylformamide, or tetrahydrofuran. Due to their aqueous insolubility, one cannot be certain that the final concentration of the compound correlates, in a linear fashion, with the amount added in the organic solvent. The water solubility of, for example, the 1,8 dihydroxyanthraquinone is 2-3 ppm, however it is demonstrated that additions of the compound in excess of this amount have additional inhibitory effect, therefore it is often necessary and beneficial to add the inhibitor in excess of its theoretical solubility. so Typically, in our experiments, the anthraquinone to be tested was dissolved to a concentration of 300 ppm in ethanol:water (80:20, 20 mM pH7 sodium phosphate buffer). This was used to deliver the compound to a growing bacterial suspension in BTZ-3 medium. While this is the preferred solvent, other solvents such as acetone, methanol-water (80:20), dimethylformamide, and tetrahydrofuran have proven to be equally effica- cious. The pH of the ethanol-water system can be from 5 to 12 with no apparent pH effect. There is no reason 55 to suspect that the compound must be delivered in the ethanol-water buffer or acetone or any particular organic solvent. In fact depending upon the particular system or anthraquinone, the solid compound could be added to it directly. Experiments have established that the compounds are effective at a final concentration of at least 0.1 ppm. When an inhibitory anthraquinone is added to a growing culture of Desulfovibrio sp. growth and sulfide 16 EP 0 525 073 B1

formation cease immediately at anthraquinone concentrations of 3 ppm. This cessation typically lasts for several days after which growth resumes. This effect has been demonstrated with several different species of Desulfovibrio in pure culture. It has also been found that the redox potential of the culture affects the potency 5 of the anthraquinone. Compounds are most effective when added to a culture in early exponential growth where some sulfide has already been produced thus rendering the medium more reduced than uninoculated medium. However compounds still show some inhibition when added to oxidized medium which is subsequently inoculated. It has been shown that use of a reducing agent can enhance the strength of the inhibition even when the inhibitor is added concurrently with the inoculum. A preferred reducing agent is sodium sulfide at 10 concentrations of between about 2 and about 4 mM in the medium. This aspect would suggest that the compounds are most effective under anaerobic, reduced conditions as would be found in, for example a sewage treatment anaerobic digestor, or in another respect the compounds may be thought of as being activated by the end-product of the sulfate-reducer which is sulfide. This invention also describes a process for the automated screening of inhibitors of sulfide production by 15 bacteria. While any sulfide-producing bacterium can be used, an isolate of Desulfvibrio desulfuricans (strain G100 A) from an oilfield was used for the experiments described. The isolation and physiological character- istics of this strain have been described previously by Weimer et al., 1988 Appl. Environ. Microbiol. 54:386- 396. The process comprises: a) maintaining a culture of sulfate-reducing bacteria in exponential growth phase by chemostat culture of the organsims; b) contacting in an automated analyzer a solution of the compound to 20 be tested with an aliquot of said culture of step a); c) adding to the compound and culture a substrate to initiate sulfide production; d) incubating the culture, compound and substrate mixture to permit sulfide production; and e) measuring the amount of sulfide produced. The system is shown schemat icily in Fig 1 . The candidate inhibitors are delivered from an automated sampler (1) into the bacterial culture stream (2) (The culture was freshly removed from the chemostat into a 158 25 ml serum vial and sparged with argon or nitrogen for about 1 hour to reduce the background sulfide level to below 0.2 mM). Immediately, small bubbles of nitrogen gas (3) are introduced into the bacterial culture/inhibitor stream to produce discrete liquid segments. After traversing this and a ten-turn mixing coil (4) (time required is about 1.6 min), substrate solution (5) (10 mM Na lactate, 5 mM Na2S04, and from 0.1% to 0.2% final concentration using a 30% stock solution of Brij 35® (v/v) (trademark of Atlas Chemical Industries, Inc. and avail- 30 able from Fisher Scientific Co., Fairlawn, NJ 07410)) is introduced into each liquid segment. The segments traverse the downstream tubing (6) in the system for about 18 minutes, at which point a small amount of 0.03N sulfuric acid (7) is added to reduce the pH to about 4.5. At this pH, sulfate reduction ceases and greater than 99% of the sulfide anions (S= and HS") are converted to H2S without lysing the bacterial cells. The H2S diffuses through a gas-permeable silicone membrane (8) and is picked up by a very dilute solution of Brij 35® surfactant 35 (9), which aids in maintaining the integrity of the individual liquid segments. The sulfide is next reacted with N,N-dimethyl-p-phenylenediamine sulfate ordihydrochloride (DPD,10) and ferric chloride (11) to produce methylene blue whose concentration is determined by the spectrophotometric flow cell (12) at the end of the reaction loop. The letters A through D on Figure 1 indicate flow rates for polyvinylchloride pump tubing of the indicated 40 collar color as follows: A = orange/green 0.10 ml/min.; B = orange/white 0.23 ml/min.; C = black/black 0.32 ml/min.; D = red/red 0.8 ml/min. Such tubing is available from Technicon Instruments Corp., Tarrytown, N.Y., as Flow Rated Pump Tube, from Precision Technology, Inc., 375 Oaktree Rd., Palisades, N.Y., 10964 as Ac- culab® Flow Rated Pump Tubes, and from Elkay Products via Fisher Scientific Co., Fairlawn, N.J., 0741 0 as Accu Rated® pump tubing. Culture, inhibitor and substrate solutions are typically added at equivalent volumes. 45 Typical reagent concentrations used are as follows: substrate = 1 .12 g sodium lactate + 0.71 g Na2S04/L; DPD = N,N-dimethyl-p-phenylenediamine sulfate ordihydrochloride at 1.19 g/L in 1.14 N HCI; ferric chloride = 3.1 g/L in 1 .14 N HCI; surfactant = 1 .0 ml/L of 30% Brij 35®. The letter T represents the number of turns of glass mixing coils. All parts used were standard Technicon® AutoAnalyzer II® parts available from Technicon® In- struments Corporation, 511 Benedict Avenue, Tarrytown, NY 10591. Similar equipment which provides equiv- 50 alent results can also be used. In order to maintain anaerobic conditions for the bacteria and to prevent oxidation of the sulfide generated during the incubation, the entire apparatus is contained inside an anaerobic glovebag (Coy Manufacturing Co., Ann Arbor, Michigan). However, sparging of the culture vessel is to be avoided, as it reduces the background level of sulfide in the culture and thus causes baseline drift. 55 The net amount of sulfide produced is determined by subtracting the background amount of sulfide produced by combining fresh culture filtrate with lactate/sulfate and with water (instead of the inhibitor solution), from the amount of sulfide produced by combining culture, lactate/sulfate, and water. The culture filtrate is obtained by filtering the sparged chemostat sample described above through a 0.2 urn filter; this filtration is performed in the glovebag to prevent oxidation of the sulfide. In order to provide a baseline measure of sulfide 17 EP 0 525 073 B1

production by uninhibited cultures, a vial of N2-sparged water is inserted between each candidate inhibitor vial in the autosampler rack. Sulfide production resulting from this combination of water, culture, and lactate/sulfate represents the amount of sulfide produced by the uninhibited culture. Atypical chart trace from an AutoAna- 5 lyzer® run is shown in Fig. 2. A indicates the 0.45 mM sodium sulfide standard plus lactate/sulfate plus water. B is the culture filtrate plus lactate/sulfate plus water. C is the cell suspension plus lactate/sulfate plus water. The numbers 1 through 12 indicate different test compounds plus cell suspension with substrate. The points indicated by a dot above each downward-tending peak represent the level of sulfide in uninhibited cultures. Note that the inclusion of a water vial between each candidate inhibitor provides a convenient marker for de- 10 termining the amount of inhibition obtained for each compound. The extent of inhibition of sulfate is calculated as follows: net mM sulfide in of inhibitor „,% ...... inhibition = 1j — - — presence x 100.nn net mM sulfide in, absence of inhibitor..... Because the automated assay is based upon the measurement of sulfide production, test compounds 15 which react with sulfide (whether by oxidation, complexation, or precipitation) will underestimate the net amount of sulfide formed and thus overestimate the extent of inhibition. The detection of such reactivity can usually be accomplished by the assay method if the cell suspension used has a moderate background level of sulfide; reactive compounds will often yield a sulfide peak below that obtained from the filtrate itself (Fig. 2, peak 9), particularly if the candidate inhibitor is tested at relatively high concentration. However, it is es- 20 sential that all compounds which appear to cause a net decrease in sulfide formation be retested for their reactivity with sulfide in the absence of active cells. This is most easily accomplished by using an automated analyzerto combine test compound, lactate/sulfate, and (instead of a bacterial suspension) a standard solution (e.g., 0.5 mM) of sodium sulfide. Reaction with sulfide is detected as a decrease in methylene blue formation that is detected by the spectrophotometer. 25 Under normal operating conditions, the instrument's recording chart is set such that full scale was equivalent to either 0.5 or 1 .0 mM sulfide. Asteady chart trace permits the detection of changes in sulfide production of 5 to 10 nM (0.01 X full scale absorbance). The relative sensitivity could be enhanced even further by in- creasing the total amount of sulfide formed (e.g., by lengthening the reaction path). The screen has been used to test a wide variety of compounds for their ability to inhibit bacterial sulfide 30 production. It has also been used to evaluate promising inhibitors with respect to concentration effects and to retention or loss of inhibitory activity following exposure to environmental stresses (e.g., extremes of temperature or salinity). The automated nature of the method permits unattended operation and maximizes sample throughput (particularly if a 144-sample automated sampler tray is used) while retaining the high degree of reproducibility afforded by the use of chemostat-grown cells. The method has been effective in rapidly iden- 35 tifying compounds which inhibit sulfide production by sulfate-reducing bacteria. The enhanced reproducibility of the assay method resulting from the use of chemostat-grown bacteria is in agreement with the report of Lagarde et al., 1976 Corrosion, Traitments, Protection, Finition 15: 275-280, who screened inhibitors by de- termining changes in bacterial cell density and sulfide production following direct addition of test compounds to chemostats containing sulfate-reducing bacteria. While their method provided reproducible data, it is not 40 practical for screening large numbers of compounds, due to the limited mumber of chemostats that can be maintained simultaneously, and to the long time required to re-establish steady-state biological and chemical conditions following addition of inhibitor. The following Examples showing the results of the screening process (i.e., inhibition of sulfide production) and other aspects of the inhibition of sulfide production by anthraquinones illustrate the present invention but 45 are not intended to limit it in any way.

EXAMPLE 1

Screening Anthraquinones for Inhibition of Sulfide Production by Desulfovibrio desulfuricans G100A: 50 Aculture of Desulfovibrio desulfuricans GIOOAwas maintained in exponential growth phase by chemostat culture of the organism. The organism was maintained in a single stage chemostat (working volume = 1 liter) continuously fed BTZ-4 medium which is BTZ-3 medium modified to contain 40 mM sodium lactate as growth limiting nutrient, and a reduced (to 0.05 mM) level of ferrous iron. The medium designated BTZ-3 consisted 55 of the following mineral base:

18 EP 0 525 073 B1

Ammonium sulfate c o . J g„ Potassium dihydrogen phosphate o.68 g (or Dipotassium hydrogen phosphate 0087 g) Magnesium sulfate heptahydrate 0 2 g Calcium chloride dihydrate 10 0 1 g

Mineral solution 10X, 1.0 ml. Iron sulfate heptahydrate 0.0042g Deionized water to a final volume of 1 liter

20 Composition of 10X mineral solution:

Nitrilotriacetic acid 12.8 g Cupric chloride 2H2O 0.254 g Manganese chloride 4H2O 1.0 g Cobalt chloride 6H2O 3.115 g Zinc chloride 1#0 g Boric acid 0.1 g Sodium molybdate 2H20 o.l g Nickel chloride 6H2O 1.84 g Deionized water to 1000 ml pH to 7.0

40 The chemostat was operated at ambient temperature (19 to 24 °C), pH 7.9 to 8.2, and a dilution rate of 0.035 per hour. Bacterial concentrations (as determined by direct counting in a Petroff-Hauser counting cham- ber and a phase contrast microscope) consistently fell between 7 and 9 x 108 cells per ml. The automated screen in this example used a Technicon® AutoAnalyzer® II as both an incubator to expose chemostat-grown bacteria to different inhibitors in the presence of bacterial growth substrates (lactate and sulfate), and as an 45 analyzer of the amount of H2S formed as a consequence of the incubation. Thus compounds were each tested with bacteria of the same strain at about the same bacterial cell density (i.e., 7-9 x 108 cells/ml) and the same stage of growth (exponential). Compounds were prepared as 300 ppm solutions in ethanol:aqueous:phosphate buffer (80:20, 20 mM Phosphate pH 7). After solubilizing the compound in this solution, the solution was diluted 10 fold and an aliquot placed in the AutoAnalyzer®. Introduction of the aliquot of compound into the bacterial 50 suspension resulted in another 3 fold dilution and thus compounds were actually exposed to the cells at 10 ppm (theoretical) concentration with 2.7% ethanol and 0.7 mM phosphate present. Controls with solvent only (minus compound to be tested) were run for each compound tested. An additional control used was chlorhexidine, a potent non-specific bacteriocide which also inhibits sulfide production. Inhibition by this compound was an indicator of the overall reproducibility of the assay and results were expressed both as total percent 55 inhibition of sulfide production and inhibition relative to chlorhexidine inhibtion. In the AutoAnalyzer® compounds were mixed with the bacterial suspension and incubated for approximately 1.6 minutes at which time the substrates for sulfide production were added (10 mM sodium lactate and 5 mM sodium sulfate). This mixture was then incubated for an additional 18 minutes to allow the formation of sulfide. The reaction was stopped by volatilizing the formed sulfide with a small amount of sulfuric acid and 19 EP 0 525 073 B1 measuring the formed sulfide as described in detail above. The resulting data are summarized in Table I. In Table I, the notation "<10 ppm" indicates that the compound was not totally soluble upon visual inspection of the sampling vials at a concentration of 30 ppm in 8% EtOH prior to the 3x dilution by the analyzer. The notation "«10 ppm" indicates that there was very little solubility upon visual inspection of the sampling vials at a concentration of 30 ppm in 8% EtOH prior to the 3x dilution by the analyzer.

Table I

Compound: N- [1- (9, 10 -da hydro- 9, 10-dioxo) anthracenyl] -N ' - ( 1 - methylethyl) imidodicarbonimidic diaau.de hydrochloride % Inhib. 83.1% (10um-3% EtOH,)

Compound: 1-Aminoanthraquinone (97%) % Inhib. 28.4% 6 lOppm in 2.7% EtOH

Compound: 2-Aminoanthraquinone % Inhib. 30.4% 6 lOppm in 2.7% EtOH

Compound : 1 -Amino-4 -hydroxyanthraguinone % Inhib. 54.3% @ <10ppm in 2.7% EtOH

Compound: 1, 2-Diaminoanthraquinone % Inhib. 26.9% @ lOppm in 2.7% EtOH

Compound: 2, 6-Dihydroxyanthraguinone; Anthraflavic acid % Inhib. 26.2% 0 lOppm in 2.7% EtOH

Compound: Anthraquinone-2-carboxylic acid (98%) % Inhib. 22.6% 6 <10ppm

Compound: 1, 5-Dihydroxyanthraquinone; Anthrarufin (92%) % Inhib. 31.0% 6 lOppm in 2.7% EtOH

Compound: 1,2-Dihydroxyanthraquinone; Alizarin % Inhib. 68.9% 6 lOppm, in 2.7% EtOH

'- compound: 2, 2 [ (9, 10-Dihydro-9, 10-dioxo-l, 5-anthracenediyl) diimino]bis[5-methylbenzenesulfonic acid], di Na salt; Alizarin violet 3R % Inhib. 30.9% 8 lOppm

!0 EP 0 525 073 B1

Compound: 1,2, 5, 8-Tetrahydroxyanthraquinone; Quinalizarin % Inhib. 80% 6 lOppm in 2.7% EtOH

Compound : 4-Amino-9, 10-dihydro-l, 3-dihydroxy-9, 10-dioxo-2- anthracenesulfonic acid, monosodium salt; Nuclear fast red % Inhib. 20.4% S lOppm

Compound: 1, 8-Dihydroxyanthraquinone; Danthron % Inhib. 82.2% 6 10 ppm in 2.7% EtOH

Compound: 2,2'-[ (9, 1 O-Dihydro-9, 10-dioxo-l, 4 - anthracenediyl) diimino]bis [5-methylbenzenesulf onic acid],di Na salt; Acid green 25 % Inhib. 38.6% 6 lOppm

Compound: l-Amino-2, 4-dibromoanthraquinone % Inhib. 44.6% 6 lOppm in 2.7% EtOH

Compound: 5-Chioro-l-aminoanthraquinone % Inhib. 57.1% 6 lOppm in 2.7% EtOH

Compound: 2-Ethylanthraquinone (97+%) % Inhib. 57.1% @ approx. lOppm in 2.7% EtOH

Compound: 1-Hydroxyanthraquinone (97%) % Inhib. 59.3% 6 lOppm in 2.7% EtOH

Compound: 2- (Hydroxymethyl) anthraquinone (97%) % Inhib. 87% 6 <10ppm in 2.7% EtOH

Compound: l-Amino-4-methoxyanthraquinone % Inhib. 57.1% 0 lOppm ir. 2.7% EtOH

21 EP 0 525 073 B1

Compound: 1 -Amino- 6, 7-dichloroanthraquinone % Inhib. 22.9% 6 <10ppm in 2.7% EtOH

Compound: Benz [a] anthracene-7,12-dione (97%) % Inhib. 52.9% 6 <10ppm in 2.7% EtOH

Compound: 1, 8-Dihydroxy-3-methylanthraquinone; Chrysophi % Inhib. 27.9% 6 <10ppm in 2.7% EtOH

Compound: 10- [ (3-Amino-2, 3, 6-trideoxy-alpha-L-lyxo- hexopyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8,11- trihydroxy-8- (hydroxyacetyl) -l-methoxy-5, 12- naphthacenedione hydrochloride; Adriamycin hydrochloride % Inhib. 55.7% 6 <10ppm in 2.7% EtOH

Compound: 9, 10-Dihydro-4, 5-dihydroxy-9, 10-dioxo-2- anthracenecarboxylic acid; Rhein I Inhib. 4B.6% 6 <10ppm in 2.7% EtOH impound: (8S-cis) -8-Acetyl-10 [ (3-amino-2, 3, 6-trideoxy- alpha-L-lyxo-hexopyranosyl)oxy] -7, 8, 9, 10-

tet rahydro- 6,8, 11 -trihydroxy-1 -methoxy-5 , 12- naphthacenedione hydrochloride; Daunomycin hydrochloride t Inhib. 62.9% 6 <10ppm in 2.7% EtOH impound: 1, 2, 4-Trihydroxyanthraquinone; Purpurin k Inhib. 55.7% 6 <10ppm in 2.7% EtOH impound: 1-Aminoanthraquinone diazonium salt i Inhib. 20.8% 6 <10ppm in 2.7% EtOH

2 EP 0 525 073 B1

2 2 • -Dimethyl- [1 , 1 • -bi anthracene 3-9, 9' ,10, 10' -tetrone ; 2, 2 '-Dimethyl -1, 1 '-bi anthraquinone 59.7% e <10ppm in 2.7% EtOH

3-(D-apio-beta-D-Furanosyloxy) -1, 8-dihydroxy-6-methyl- 9, 1 0-anthracenedione; Frangulin B 57.1% e <10ppm in 2.7% EtOH

2-Chloroanthraquinone (99%) 98.7% 6 «10ppm in 2.7% EtOH

1 , 5-Dichloroanthraquinone (96%) 100% 6 «10ppm in 2.7% EtOH

1,4,5, 6-Tetrachloroanthraquinone 50.6% e «10ppm in 2.7% EtOH

1- Chloroanthraquinone 20.8% 6 <10ppm in 2.7% EtOH

1, 8-Dichloroanthraquinone (97%) 3.9% e <10ppm in 2.7% EtOH

2- Bromo-3-methylanthraquinone 26.0% 6 «10ppm in 2.7% EtOH

I- (2, 2, 2-Trimethylpropionamido) anthraquinone 18.1% S <10ppm in 2.7% EtOH

!, 6-Bis [2- (dimethylamino) ethoxy] -9, 1 0-anthracenedione; rilorone analog R11,043DA )8.7% @ lOppm in 2.7% EtOH

!-Methyl-l-nitroanthraquinone >0% 6 «10ppm in 2.7% EtOH

3 EP 0 525 073 B1

1-Amino- 9, 10-dihydro-9, lO-dioxo-2-anthraeenesulf onic acid; l-Aminoanthraquinone-2-aulf onic acid 37.5% e <10ppm in 2.7% EtOH

9, 10-Dihydro-5-nitro-9, 10-dioxo-l-anthracenesulf onic acid; 5-Nitroanthraquinone-l-sulf onic acid 66% S lOppm in 2.7% EtOH

3-Chloro-9, 10-dihydro-9, 10-dioxo-2-anthracenecarboxylic acid; 2-Chloroanthraquinone-3-carboxylic acid 100% 6 lOppm in 2.7% EtOH

50/50 Mixture of Anthrarufin and Danthron CA Reg. No. : None 62.5% 6 5ppm in 2.7% EtOH

Anthraquinone 37.5% Q lOppm in 2.7% EtOH

1, 8-Dihydroxy-3- (hydroxymethyl ) -anthraquinone; Aloe- emodin 43.6% 6 lOppm in 2.7% EtOH

1, 16-Dichloro-6, 15-dihydro-5, 9, 14, 16-anthrazinetetrone; 7, 16-Dichloroindanthrone 43.8% G lOppm in 2.7% EtOH

1,2,3,4,5, 8-Hexahydroxyanthraquinone; Alizarin cyanin JO. 6% e lOppm in 2.7% EtOH

2,4,5, 7-Tetrabromo-l , 8-dihydroxy-9 , 1 0-anthracenedione ; 2,4,5, 7-Tetrabromochrysazin 10.6% 6 lOppm in 2.7% EtOH

!4 1,2, 7-Trihydroxyanthraquinone; Anthrapurpurin 37.5% e lOppm in 2.7% EtOH

1, 4, 5-Trihydroxy-2-methyl-9, 1 O-anthracenedione; Zslandicin 50% 6 lOppm in 2.7% EtOH l,4,5,7-Tetrahydroxy-2-methyl-9,10-anthracenedione; Catenarin 37.5% lOppm in 2.7% EtOH

L,e-Dihydroxy-3-methoxy-6-methyl-9,10-anthracenedione; 'hyscion (4.4% 6 lOppm in 2.7% EtOH

, 4,5, B-Tetrahydroxy-2-methyl-9, 1 0-anthracenedione; :ynodontin 4.4% 6 lOppm in 2.7% EtOH

, 5, 8-Trihydroxy-3-methyl-9, 1 0-anthracenedione; elminthosporin 1.3% G lOppm in 2.7% EtOH

-Hydroxy-2- [ (6-O-fi-D-xylopyranosyl-fi-D- Lucopyranosyl) oxy] -9, 10-anthracenedione; Ruberythric :id

J. 7% g lOppm in 2.7% EtOH

■Phenoxy quini«rin-3, 4 • -disulf onic acid, di K salt M% 6 lOppm in 2.7% EtOH

, -) -l-Acetoxy-8-hydroxy-l, 4,4a, 9a- trahydroanthraquinone .1% G lOppm in 2.7% EtOH j. -iinu.no-* 1 1«- 1 i aunet.nyj.ann.no) methyl J phenyl ] - amino-9, 10-anthracenedione; Basic Blue 47 37.2% e lOppm in 2.7% EtOH

1, 5-Bis (2-carboxyanilino) -9, 10-anthracenedione; Acridylic acid 33.5% 6 lOppmin 2.7% EtOH

1, 8-Dihydroxy-9-anthranol; 1, 8-Dihydroxyanthranol B8.3% 6 <10ppm in 2.7% EtOH

1, 2, 10-Anthracenetriol; Anthrarobin 44.4% 6 lOppm in 2.7% EtOH l-Amino-4-bromo-2-methylanthraquinone (99%) B% 6 <10ppm

I, 4-Diaminoanthraguinone (97%) 27.5% e lOppm in 2.7% EtOH

I, 6-Diaminoanthraquinone >7.2% e lOppm in 2.7% EtOH

.-Amino-4[4-[t4-chloro-6[[2,3 or 4 -sulf ophenyl] amino] - .,3, 5-triazin-2-yl] amino] -3-sulf ophenyl] amino] -9, 10- lihyro-9,10-dioxo-2-anthracenesulfonic acid; Procion >lue HB(S); Reactive blue 2; Cibacron blue 3G-A; lasilen blue E-3G 7.5% 6 lOppm nthraquinone-l,5-disulfonic acid di Na salt hydrate 95%) 7.5 % 6 lOppm

> cr U U/O D I

■ nuwuoijuiHuiir-i , B-aisuiionic scia a.1 jja sale % Inhib. 9.8% e lOppm

Compound: Anthraquinone-2-sulf onic acid sodium salt monohydrate % Inhib. 17% 6 lOppm

Compound: 1, 2-Bis [ (4-sulf ophenyl) amino] -4-hydroxy-anthraquinone; Alizarin blue black B % Inhib. 17.5% 6 lOppm impound: (3, 4-dihydroxy-2-anthraquinonyl) -methyliminodiacetic acid; 3-aminomethylizarin-N,N-diacetic acid; Alizarin complexone dihydrate k Inhib. 14.3% 6 <10ppm in 2.7% EtOH

Compound: l-Amino-4- [ [3- (ethenylsulf onyl ) phenyl ] -9, 1 0-dihydro- 9,10-dioxo]-2-anthracene sulfonic acid, monosodium salt; Acid blue 215 Inhib. 19.7% Q lOppm ompound: 1- (Methyl amino) anthraquinone (98%) Inhib. 19.1% @ <10ppm. ompound: 2, 2 ( (9, 10-Dihydro-5, 8 -di hydroxy- 9, 10-dioxo-l , 4- anthracendiyl )diimino] bis (5-methylbenzenesulf onic acid], di Na salt; Acid green 41 Inhib. 17.8% 6 lOppm

i ^,.lv -yx,iyUIO-,, J-«-aaoxo-i,4-anthracenediyl)- Liimino]bis[5-butylbenzenesulfonic acid]; Acid green 7 6.5% e lOppm EP 0 525 073 B1

1,1' -Xminobis [4-amino] 9, 10-anthracenedione, sulfonated; Acid black 48 13.8% 8 lOppm l-Amino-9, 10-dihydro-9, 10-dioxo-4- (phenylamino) -2- anthracenesulfonic acid, Na salt; Acid blue 25 26% 8 lOppm in 2.7% EtOH

4-[ [4- (Acetylamino) phenyl] amino] -l-amino-9, 10-dihydro- 9, 10-dioxo-2-anthracenesulfonic acid, Na salt; Acid blue 40 12.5% 8 lOppm l-Amino-9, 10-dihydro-9, 10-dioxo-4- [ [3 [ [2- (sulfoxy) ethyl] sulfonyl) phenyl] amino] -2-anthraeenesulf onic acid, disodium salt; Remazol Brilliant blue R; 12.4% 8 lOppm l-Amino-4 [ [3- [4, 6-dichloro-l, 3, 5-triazin-2-yl) amino] - 4-sulf ophenyl] amino] -9,10 dihydro-9, 10-dioxo-2- anthracenesulf onic acid; reactive blue 4; 7.3% 8 lOppm

1- (9, 10-Dihydro-9, lO-dioxo-l-anthracenyl-1, 2- hydrazinedisulfonic acid, di Na salt; (1- tathraquincnyl)-l,2-hydrazine disulfonic acid, di Na salt 10% 8 lOppm in 2.7% EtOH

9, 10-Dihydro-5, 6 -di hydroxy- 9, 10-dioxo-l- snthracenesulfonic acid; Alizarin-5-sulf onic acid 7.1% 8 <10ppm in 2.7% EtOH

!8 EP 0 525 073 B1

N- (4-cnloro-9, 10-dihydro-9, 10-dioxo-l- anthracenyl ) benzanu.de; l-Benzamido-4-chloroanthraquinone 8.6% 6 <10ppm in 2.7% EtOH l-Amino-4 -bromo-9, 10-dihydro-9, 10-dioxo-2- anthracenesulfonic acid, Na salt; l-Amino-4- bromoanthraquinone-2-sulf onic acid, Na salt 4.3% 6 lOppm in 2.7% EtOH l-Amino-9, 10-dihydro-4 [ [ (4 -methyl phenyl) - sulfonyl] amino] -9, 10-dioxo-2-anthracenesulf onic acid, Na salt; l-Amino-4- (p-toluenesulf onamido) - anthraquinone-2-sulf onic acid, Na salt 3.9% 6 lOppm in 2.7% EtOH

9, 10-Dihydro-9,10-dioxo-2, 3-anthracenedicarboxylic acid 10% e <10ppm in 2.7% EtOH

1,1 '-Iminobis (4-nitro-9, 10-anthracenedione) 5.7% 6 <10ppm in 2.7% EtOH

L-Amino-4 -chloro-2-methylanthraquinone 5.2% 6 <10ppm in 2.7% EtOH if 3-dimethyl-l, 4-dihydroxyanthraquinone; 2, 3- )imethylquinizarin j.7% G <10ppm in 2.7% EtOH

J-Methyl-1,3, 8-trihydroxyanthraquinone; Emodin 99% !.6% fi <10ppm in 2.7% EtOH

. , 4-Bis (methylamino) -anthraquinone 1.6% G <10ppm in 2.7% EtOH

9 n- \i-«mno-», xu-ainyaro-j-metnoxy-s, 10-dioxo-l - anthracenyl ) -4-methylbenzenesulf onamide; N- (4-Amino- 3-methoxyanthraquinone-l -yl ) -p-toluenesulf onamide ; 1- Amino-2-methoxy-4- (p-tolylsuf onamido) anthraquinone 4.3% 6 <10ppm in 2.7% EtOH

[1,1 '-BianthraceneJ-SfS'lClO'-tetrone; 1,1 •- Bi anthraquinone 12.9% 6 <10ppm in 2.7% EtOH

6, 7-Dichloro-l, 4-dihydroxyanthraquinone 97% 5.7% 6 <10ppm in 2.7% EtOH

2- [ [9,10-Dihydro-4- (methylamino) -9, 10-dioxo-l- inthracenyl ] amino] -5-methyl-benzenesulf onic acid, nonosodium salt; Alizarine astrol B-CF 2.6% 6 lOppm in 2.7% EtOH

>, 8-Diphenyl-anthra [2, 1-d: 6, 5-d' ] bisthiazole-6, 12- iione; Indanthrene yellow GCN •5.6% g lOppm in 2.7% EtOH

!-Methoxy-3-methyl-9, 10-anthracenedione i.0% e <10ppm in 2.7% EtOH

, 4-Bis [ (1-methylethyl) amino] -9, 10-anthracenedione; , 4-Di (isopropylamino) anthraquinone .0% 6 <10ppm in 2.7% EtOH

, 4-Bis [ (2,4, 6-triethylphenyl) amino] -9,10- nthracenedione; 1, 4-Bis (2, 4, 6-triethyl-

.3% G <10ppm in 2.7% EtOH — ■» ■» • —— i -w kuy^uunoaniniaquinone ; Disperse blue 3 €.4% 8 <10ppm in 2.7% EtOH

1 4 -Bis [ (4 , -methylphenyl ) amino} 9 , 1 0-anthracenedi one ; Solvent green 3 «.3% 6 <10ppnt in 2.7% EtOH

J-Amino-3-hydroxyanthraquinone LI. 4% 6 <10ppra in 2.7% EtOH

- (Bromothio) anthraquinone i.5% @ <10ppm in 2.7% EtOH

, 8-Bis (phenylmethoxy) -9, 10-anthracenedione; 1 , 6- ibenzyloxyanthraquinone .5% 8 <10ppm in 2.7% EtOH

-Amino-2- (2-aminoethylthio) -4-hydroxyanthraquinone 3.7% @ <10ppm in 2.7% EtOH

4-Bis (pentylamino) -9, 10-anthracenedione; Oil blue N 6% G <10ppm in 2.7% EtOH

■Amino-2-bromo-4-hydroxyanthraquinone 5% 6 <10ppni in 2.7% EtOH

Propionamidoanthraquinone .4% G <10ppm in 2.7% EtOH

4-Diamino-2f 3-bis (2-phenoxyethoxy) anthraquinone B% 8 <10ppm in 2.7% EtOH . j —— «r f * V J. — anthracenyljbenzamide; l-Benzamido-5- chl oroanthraquinone % Inhib. 10.4% e <10ppm in 2.7% EtOH

* — — w u % Inhib. 18.2% 6 <10ppm in 2.7% EtOH

anthracenediyl) ] bisbenzamide; 4,4- -Dibenzamido-1 , 1 ■ - dianthrimide k Inhib. 5.2% 8 <10ppm in 2.7% EtOH

impound: 1, 4, 5, 8-Tetraaminoanthraquinone; Disperse blue 1 i Inhib. 3.9% 6 <10ppm in 2.7% EtOH

Inhib. 10.4% 9 <10ppm in 2.7% EtOH

-~ '-cui^iaceneaisuifcnic acid, di Na salt; Anthraquinone-2, 7-disulf onic acid, di Na salt

Inhib. 9.1% e <10ppm 2.7% EtOH impound: 1,2, 3-trihydroxyanthraquinone; Anthragallol Inhib. 2.6% 6 <10ppm in 2.7% EtOH

>mpound: Carmine (Aluminum lake) Inhib. 7.8% 8 <10ppm in 2.7% EtOH impound: 9, 10-Dihydro-l, 4-dihydroxy-9, 10-dioxo-2- anthracenesulfonic acid Inhib. 7.8% 9 <10ppm in 2.7% EtOH EP 0 525 073 B1

2 -Amino- 3 -chl oroanthraquinone 3.9% e <10ppm in 2.7% EtOH

1- Anthraquinonesulf onic acid, Na salt 3.9% 6 <10ppm in 2.7% EtOH

2- tert-butylanthraquinone (98%) 5.2% 6 <10ppm in 2.7% EtOH

1 . 4- Dihydroxyanthraquinone 5.2% 6 <10ppm in 2.7% EtOH

1 . 5- Diamino-4 , 8-dihydroxyanthraguinone 2.6% 6 <10ppm in 2.7% EtOH

I -Hydroxy- 4- [ (4-methylphenyl ) amino] -9,10- snthracenedione; l-Hydroxy-4- (p-toluidino) - anthraquinone 3.9% 6 <10ppm in 2.7% EtOH

L , 4 -Dimethylanthraquinone (95%) >.2% G <10ppm in 2.7% EtOH

., 1 '-Xminobis-9, 10-anthracenedione; Dianthrimide !.6% 6 <10ppm in 2.7% EtOH

!- (Cyclopropylcarboxamido) anthraquinone !.6% 6 <10ppm in 2.7% EtOH

-Amino-2-methylanthraquinone; Disperse orange 11 .2% 6 <10ppm in 2.7% EtOH

3 cr U U/O D I

anthracenyl) amino] -5-methyl-benzenesulf onic acid, Na salt; Sol way purple R 9.1% e <10ppm in 2.7% EtOH

kU"»'UUflB: (9,10-Dihydro-5, e-dihydroxy-9, 10-dioxo-l, 4- anthracenediyl ) diimino] bis [ 5-methyl ] benzenesulf onic acid; alizarine viridine % Inhib. 2.6% 6 <10ppm in 2.7% EtOH

Compound: 1, 4-Bis (ethylamino) -9, 10-anthracenedione; Sudan blue % Inhib. 1.3% e <10ppm in 2.7% EtOH

Compound: 1, 4-Diamino-5-nitroanthraquinone % Inhib. 2.7-3% 6 <10ppm in 2.7% EtOH

impound: N-Benzyl-9, 10-dihydro-9, 10-dioxo-2-anthracenesulf onamide k Inhib. 3.9% 6 <10ppm in 2.7% EtOH

ninraqumones wnicn go not innipn suinae proauciion

The compounds listed in Table II were tested for inhibition of sulfide production as in Example 1 and w >und not to be effective inhibitors. The notation "<10 ppm" is as previously defined in Example 1.

iflnir TT impound: 3, 4-Dihydroxy-9, 10-dioxo-2-anthracenesulf onic acid; Alizarin red S monohydrate :A Reg. No. : 130-22-3 Inhib. 0 G lOppm ource: Aldrich tr u aza uii bi

«_u«ifr>ouna : h- i ih- (Acetyjjnetfiyianu.no ; phenyl ] amino] -l-amino-9, 10- dihydro-9,10-dioxo-2-anthracenesulfonic acid, Na salt; Acid blue 41 CA Reg. No.: 2666-17-3 % Inhib. 0 6 lOppm Source : Aldrich

Compound: 4, 8-Diamino-9, 10-dihydro-l, 5-dihydroxy-9, 10-dioxo- 2, 6-anthracenedisulfonic acid, di Na salt; Acid blue 45 CA Reg. No. : 2861-02-1 % Inhib. 0 6 lOppm Source: Aldrich

Compound 3 3 • - 9 : , [ ( , 1 O-Dihydro-9 , 1 0-dioxo-l , 4 -anthracenediyl ) adiimino]bis [2,4, 6-trimethylbenzenesulf onicacid] , di Na salt; Acid blue 80 :A Reg. No. : 4474-24-2 Inhib. 0 (3 lOppm Source: Aldrich impound: 7-B-D-Glucopyranosyl-9, 10-dihydro-3, 5,6,8- tetrahydroxy-l-methyl-9,10-dioxo-2- anthracenecarboxylic acid; Carminic acid :a Reg. No. : 1260-17-9 i Inhib. 0 6 <10ppm iource : Aldrich lompound: l-Amino-4 [ [3- [ [4-chloro-6- [ (3-sulf ophenyl) amino] - 1,3, 5-triazin-2-yl] amino] -4-sulf ophenyl] amino] -9,10- dihydro-9, 10-dioxo-2-anthracenesulf onic acid; Reactive blue 5 A Reg. No. : 16823-51-1 Inhib. 0 @ lOppm ource: Sigma

> I_ I U *J£-*J U f J U I

- -f — «jrwu.w-», *v-aj.ox©-j.-antnracenyi) - benzamide; l-Amino-4-benzamidoanthraquinone CA Reg. No. : 81-46-9 % Inhib. 0 6 <10ppm in 2.7% EtOH Source: Aldrich

Compound: l-Amino-4- (methylamino) anthraquinone CA Reg. No. : 1220-94-6 % Inhib. 0 6 <10ppm in 2.7% EtOH Source: Pfaltz l Bauer impound: 5-hydroxy-l, 4-bis [ (4-methylphenyl) amino] -9, 10- anthracenedione; Sudan green :A Reg. No. : 4392-68-1 I Inhib. 0 6 <10ppm in 2.7% EtOH iource: Pfaltz i Bauer ompound: l-Bromo-4- (methylamino) anthraquinone A Reg. Nc: 128-93-8 _. Inhib. 0 6 <10ppm in 2.7% EtOH ource: Kodak ompound: l , 4-Diamino-2-methoxyanthraquinone; Disperse red 11 * Reg. No. : 2872-4 8-2 Inhib. 0 6 <10ppm in 2.7% EtOH 5urce: Sigma impound: Indanthrene black (suspension or liquid); C.I. Vat green 9 i Reg. No. : 6369-65-9 Inhib. -6.5% 6 <10ppm/8% EtOH urce: Pfaltz £ Bauer EP 0 525 073 B1

Compound: N,N'-<9,10-Dihydro-9, 10-dioxo-l, 5-anthracenediyl) - bisbenzamide; 1, 5-Dibenzamidoanthraquinone CA Reg. No. : 82-18-8 % Inhib. 0 e <10ppm in 2.7% EtOH Source: K t K

Compound: Alizarin blue black BC CA Reg . No . : None % Inhib. 1.9% 6 <10ppm in 2.7% EtOH Source: K t K

Compound: 9, 10-Dihydro-3, 4, 6-trihydroxy-9, 10-dioxo-2- anthracenesulf onic acid; Alizarin rubinol CA Reg. No.: 83631-51-0 % Inhib. 0 6 <10ppm in 2.7% EtOH Source: K i K

Compound: 9, 10-Dihydro-l, 3, 4, 5, 7, 8-hexahydroxy-9, 10-dioxo-2, 6- anthracenedisulf onic acid; Acid alizarin blue BB CAReg. No.: 63631-52-1 % Inhib. 0 6 <10ppm in 2.7% EtOH Source: K & K

Compound: N, N ' - [Iminobis (9, 10-dihydro-9, 10-dioxo-5, 1- anthracenediyl) ] bisbenzamide; 4, 5 '-Dibenzamido-l, 1 ' - dianthrimide CA Reg. No. : 129-28-2 % Inhib. 0 6 <10ppm in 2.7% EtOH Source: Aldrich

Compound: 1, 4, 4a, 9a -Tetrahydroanthraquinone CA Reg. No.: 56136-14-2 % Inhib. 0-7% (> lOppm in 2.7% EtOH Source: Aldrich

EXAMPLE 3

Inhibition of sulfide formation by 1-amino-anthraquinone

These determinations were carried out in the AutoAnalyzer® as described in Example 1. The resultin data is shown in Table III.

37 EP 0 525 073 B1

TABLE III Compound % inhibition of sulfide formation Chlorhexidine 88.7 83 10 1-amino-anthraquinone biguanide 1-amino anthraquinone 72

The data show that the 1-amino-anthraquinone biguanide which was derived from a chlorhexidine deriv- 15 ative and 1 amino-anthraquinone, as well as 1-amino-anthraquinone itself, inhibited sulfide formation by De- sulfovibrio desulfuricans G1 00A.

EXAMPLE 4

20 Effect of 1 ,8-dihydroxyanthraquinone on growth of D. desulfuricans G100A

Four 10 ml culture tubes of BTZ-3 medium as defined in Example 1 supplemented with sodium lactate (40 mM) and sodium sulfate (30 mM) and 0.5 ml of reducing agent were inoculated with 100 ul of a fully grown suspension of desulfuricans G100A. The culture was allowed to grow and the optical density at 660 nm 25 was monitored until the cells entered into exponential growth (approx. 12-15 h, O.D. 660 = 0.1-0.2). At this time 100 ul of a 300 ppm solution of 1,8 dihydroxyanthraquinone, 1-chloranthraquinone or 3-chloro-2- anthraquinone carboxylic acid, respectively, was added to one of the tubes. The ethanol:water (80:20) solvent (1 00 ul) was added to the fourth tube to serve as an uninhibited control. The resulting growth after the addition of the preferred inhibitors in comparison to the untreated control is shown in Table IV. 30

TARTT. TV Addition l£ the. Bacterial Culture* 35 Hours of 1, 8-dihydroxy- 1-chloro- 3-3-chloro-2-anthra- OrPWth Solvent, anthraquinone anthraquinone gulOquinone carboxylic acid 0 .01 .01 .01 .01 40 10 .07 .12 .11 .10 12# .11 .20 .12 .16 14 .14 .19 .12 .21 45 18 .25 .16 .27 .36 20 .31 .16 .32 .38 24 .65 .16 .32 .36 29 1.0 .18 .30 .37 50 34 1.2 .18 .32 .37 62 1.2 .30 .26 .34 02 1.2 .28 .23 .25 55 optical density reading at 660 nm 3 ppm inhibitor added at 12 hours.

38 EP 0 525 073 B1

This example clearly shows that the addition of each of the three compounds to a final concentration of 3 ppm causes virtually complete cessation of growth of D. desulfuricans GIOOAfor at least 80 hours.

5 EXAMPLES 5-10

Effect of 1,8-dihydroxyanthraquinone on different species of sulfate-reducing bacteria

Different species of sulfate-reducer were grown as described in Example 1 and then 250 ^l of the grown 10 suspension was inoculated into BTZ-3 medium containing reducing agent (1.0 ml/10 ml culture) and 1,8-dihydroxyanthraquinone at 3 ppm. Growth was monitored as optical density at 660 nm for 40 hours. The resulting data is shown in Table V.

TABLE V Example Desulfovibrio sp Optical Density ft fifiOntn - AQ* +AQ** £ D. multisDirans *1 * ->r 3. D. desulfuricans filOOA !>1 «; ->n 1 D. desulfuricans Norway -?ft no £ D. salexigens >i =. p£ 2. D. desulfurirans 77774 A*. np 1£ D . vulgaris ,?o .05

30 * no 1,8-dihydroxyanthraquinone present ** 1,8-dihydroxyanthraquinone present

35 EXAMPLE 11

Effect of 1,8-dihydroxyanthraquinone on Thiobacillus denitrificans

Thiobacillus denitrificans was grown on BTZ-3 medium as defined in Example 1 (supplemented with re- 40 ducing agent) and with 30 mM sodium nitrate plus 30 mM sodium thiosulfate. Cultures were anaerobic under a nitrogen atmosphere. Thus growth conditions mimicked those for the sulfate-reducing bacteria as closely as possible. The 1 ,8-dihydroxyanthraquinone was added to the cultures at the indicated time and the growth monitored by observing the optical density at 660 nm. The results are shown below in Table VI.

39 EP 0 525 073 B1

TABLE YI

5 Hours Optical Density # of Optical Density # +3ppm 1,8- orowth Control dihvdroxvanrhracruinone 10 o .02 .02 12 .06 .06 15 .08 .08 19* .09 .09 15 34 .22 .22 36 .29 .25 38 .32 .27 20 43 .40 .32 55 1.5 1.5

* Time 25 of addition of 3 ppm of anthraquinone # Optical Density 6660 nm

Significantly, the inhibitor had no effect on the growth of T. denitrificans. This organism was chosen be- 30 cause its central metabolism involves sulfur compounds as is also the case with sulfate reducers. However, the Thiobacilli carry out the oxidation of sulfide rather than the reduction of sulfate, i.e., the reverse reaction of the sulfate reducers.

EXAMPLE 12 35 Effect of 1,8-dihydroxyanthraquinone on Escherichia coli

Another non-sulfate reducing organism, Escherichia coli strain MC1 06 was grown underaerobic conditions on BTZ-3 with 30 mM sodium succinate as sole carbon and electron source with oxygen as electron acceptor 40 or under anaerobic conditions with hydrogen (80% as electron donor) and carbon dioxide (20%) as gas phase and sodium fumarate (30 mM) as electron acceptor. Both media contained 0.2% yeast extract. The resulting data are shown in Table VII.

40 EP 0 525 073 B1

TABLE VTT s Aerobic growth with succinate

Ootical Densitv (3 660 nm

Hours Control +5 ppm , 0 0.01 0.01 5.5 0.17 0.24 6.5 0.37 0.39 15 7.5 0.38 0.40 24 1.50 1.50

Anaerobic 20 growth under Hydrogen/carbon dioxide

Hours Control +5 ppm J 0 0.0 0.01 25 -i r- _ _ _ 0.30 0.29 4-5 0.4 5 0.62 5.5 0.61 0.79 so 6-0 0.75 0.87

*1, 8-dihydroxyanthraquinone

35 Aerobic growth with succinate refers to growth on succinate as a carbon and electron source and oxygen as an electron acceptor. Anaerobic growth under hydrogen/carbon dioxide refers to growth with hydrogen as an electron donor and fumarate as an electron acceptor. Hydrogen consumption was observed in these cultures indicating that hydrogen was in fact used as an electron donor. 40 The data show virtually no inhibition of 1 ,8-dihydroxy-anthraquinone on the aerobic or anaerobic growth of E. coli.

EXAMPLE 13

45 Effect on 1,8-dihydroxyanthraquinone on D. desulfuricans

D. desulfuricans strain G100Awas grown using pyruvate fermentation in the absence of sulfate. The resulting data are shown in Table VIII.

41 EP 0 525 073 B1

TABLE VTTT

Hours of Optical Density # Optical Density # flrpwth Control +3pptti AnthraouinonP 10 0 .11 .1 2 .12 .12 3.5 .14 .14 6.5* .15 .16 15 8.5 .18 .16 10.7 .19 .17 25 .26 .22 20 32 .37 .34 45 .46 .45

* 25 Time of addition of 3 ppm 1,8-dihydroxyanthraquinone. # Optical Density 6660 nm

The anthraquinone did not affect fermentative growth of D. desulfuricans G100A. This shows that the inhibitory 30 effects of anthraquinones are specific for sulfate reduction. Pyruvate fermentation does not involve the same enzyme systems utilized by the sulfate reduction pathway.

EXAMPLE 14

35 Effect of 1,8-dihydroxyanthraquinone on sulfide production by crude enrichments from various naturally-oc- curing environments

All samples were grown on standard BTZ-3 medium (as defined in Example 1) under hydrogen (80%) carbon dioxide (20%) with 10 mM acetate and 0.1% yeast extract. Mud samples were inoculated into this medium 40 and allowed to grow for 24 h to enrich for sulfate reducing bacteria. A 1 ml aliquot of this enrichment was trans- ferred to fresh medium and the sulfide evolved was measured over a three day period. Sulfide was assayed as described bySiegal, Anal. Biochem. 11: 126-132, 1965, herein incorporated by reference. The resulting data are shown in Table IX.

42 EP 0 525 073 B1

TABLE IX s Sample Source umol sulfide/dav umol sulfide/dav* VS-A 43.0 -1.0 VS-AN 12.0 -4.0 VS-AN2 116.0 w 0.0 VF-A 24.0 -2.0 VF-A2 64.0 -11.0 VF-AN 67.0 2.8 15 WCC-A1 411.0 67.0 WCC-A2 388.0 49.0 WCC-AN1 345.0 80.0 20 WCC-AN2 288.0 37.0 WADS 106.7 71.0 SM-Lewes 6.4 4.5

VS « Valley Stream State Park. Long Island, NY VF - Valley Garden Park, DE WCC - White Clay Creek Preserve, PA 30 WADS - Wilmington anaerobic digestor, DE SM-Lewes « Lewes saltmarsh, DE A - aerobic samples 35 AN - anaerobic samples

♦Cultures contain 5 ppm 1,8-dihydroxyanthraquinone. 40

The data shows that the majority of enrichments from natural sources were inhibited by the 1,8-dihydroxyanthraquinone. The cultures derived from the Wilmington anaerobic digestor or the Lewes saltmarsh were the least affected while the freshwater pond sediments showed the greatest inhibition. 45 EXAMPLE 15

Effect of Anthraquinones on the Respiration Rate of D. desulfuricans 27774 so The respiration rate (rate of hydrogen gas utlized) by desulfuricans 27774 was measured with sulfate as the electron acceptor and hydrogen as the sole electron donor in the presence of each of three anthraquinones. The purpose was to show that respiration, an energy-yielding cellular reaction pathway, is sensitive to the anthraquinones, when sulfate is the electron acceptor used. Reaction mixtures had the following composition and the reactions were carried out in a Gilson respiromenter flask using standard manometric techni- 55 ques. 200 ul of bacterial suspension (40 mg/ml protein of bacteria) 1 00 ul of 1 M phosphate buffer of pH 7 250 ul of 100 mM electron acceptor anthraquinone: 2 ul of 300ppm soln (0.6 ppm final cone.) 43 EP 0 525 073 B1

10 ul of 300 ppm (3 ppm) 20 ul of 300 ppm (6ppm) DI water to 1.0 ml. The bacteria, water, buffer and inhibitor were incubated for 30 minutes under a 100% hydrogen atmosphere and then the electron acceptor was tipped into the main compartment and the reaction started. Gas uptake was measured over a period of 240 min and rates of gas uptake determined. The results are summarized below in Table X as percent of the rates of hydrogen gas uptake relative to those from mixtures incubated in the absence of anthraquinone.

TABLE X Concen- Gas Uptake (as percent of control) tration 1 Chloro- 1, 8-Dihydroxy- 3-Chloro-2-anthre- (ppm) anthraquinone anthraquinone quinone carboxylic acid 0 100 100 100 .6 74 SC 73 1.5 50 45 55 3 38 30 41

This experiment shows that at 3 ppm the three anthraquinone inhibitors gave a 59-70% inhibtion of control respiration rates. This both supports the result of Example 4 showing that 3 ppm inhibits bacterial growth and further demonstrates that the sulfate reduction process is directly affected.

EXAMPLES 16-19

Effect of Anthraquinones on Respiration Rates of D. desulfuricans 27774

The following experiments show the effect of the 1,8-dihydroxy-anthraquinone on the rate of hydrogen utilization by bacteria in the presence of different electron acceptors. The method used was identical to that of Example 10, and the results are summarized in Tables XI, XII, XIII and XIV.

EXAMPLE 16

TABLF. XT

Sulfite as electron acceptor (no sulfate present) with and without 3 ppm anthraquinone

Hvdrooen. ;p Minutes Control +3ppm 1,8-dihydroxyanthraquinone 0 0 0 6 7 7 "16 26 25 26 45 43 59 139 i4i 71 179 i83

44 cr U U/O D I

Fumarate as electron acceptor (no sulfate present) with and without 3 ppm anthraquinone

^jpprn it B-dihydroxyanthraquinone 0 0 0 10 9 e 25 23 25 40 47 48 55 70 74 70 95 101

Thiosulfate as electron acceptor (no sulfate present) with and without 3 ppm anthraquinone

jvdrno-gn. |11 U W ^ ^ n>ppm 1, B-dihydroxyanthraquinone 0 0 0 11 38 33 21 90 73 31 144 118 41 204 176 EP 0 525 073 B1

EXAMPLE 19

TABLE XTV

Sulfate as electron acceptor with and without 3 ppm anthraquinone

— Hvdrn?Pnr [p Minutes Control +3ppm 1, 8-dihydroxyanthraquinone 0 o o 10 15 3 25 44 14 40 76 21 20 55 107 28 70 152 38 85 208 45 100 25 266 56 115 336 74

Examples 11-14 show that the sulfate-reduction pathway is specifically affected. Alternate electron ac- 30 ceptors (i.e, sulfite, thiosulfate and fumarate, Examples 16-18) show comparable respiration rates with and without anthraquinone hence the anthraquinones exhibit specificity for sulfate reduction.

EXAMPLE 20

35 Effect of reduced anthraquinones on growth of GIOOAand BTZ-3 +30 mM sodium lactate + 30 mM sodium sulfate

Three anthraquinones, 1, 8-dihydroxy-, 1-chloro-, and 3-chloro-2-carboxy- were prepared as 1 ml of 1000 ppm solutions in acetone for the first two compounds or water for the third. These solutions were then treated 40 with 1 mg of the strong reducing agent, sodium dithionite. 50 ul of each solution (for a final culture concentration of 5 ppm) was used immediately to treat growing cultures of D. desulfuricans G100A in BTZ-3 medium as defined in Example 1. The compounds were added to the 10 ml cultures at 15 h of growth.

46 EP 0 525 073 B1

TABLE XV

Optical Density 0 660 nm 5 Hours 1,8 diOH 1-Chloro 3-Chloro-2-COOH Control 0 0.02 0.02 0.02 0.02 2 0.02 0.02 0.02 0.02 10 12 0.10 0.08 0.07 0.08 15 0.2 0.15 0.1 0.18 17 0.24 0.20 0.15 0.26 20 0.23 0.21 15 0.17 0.37 25 0.22 0.22 0.17 0.50 30 0.23 0.22 0.16 0.98 45 0.25 0.24 0.22 1.50 20

The data show that 5 ppm of any of the three preferred anthraquinones will inhibit the growth of D. desulfuricans G100Aeven when pre-reduced by dithionite to the reduced form.

25 EXAMPLE 21

Assay of sulfide inhibition by anthraquinones using resting cells of D. gigas.

Experiments in this example were carried out using resting cells of Desulfovibrio gigas rather than growing 30 cells as was the case for the autoanalyzer system employing the Desulfovibrio desulfuricans G1 00A strain. The reason forthis was that the D. gigas strain retained respiratory activity under resting cell conditions where- as the D. desulfuricans G100A strain did not. Thus the experiment examined a different organism, different test system, and a different physiological state for the phenomenon of anthraquinone inhibition of sulfide production by Desulfovibrio sp. Active compounds, as determined by the autoanalyzer, were evaluated in com- 35 parison with four additional compounds not evaluated in the autoanalyzer. The date confirmed the inhibitory properties of the 1,8-dihydroxyanthraquinone, the 1-chloroanthraquinone and demonstrated the inhibitory effect of four new compounds not screened by the autoanalyzer method: the 2-bromoanthraquinone (CA Reg. No. 572-83-8), 1-fluoroanthraquinone (CAReg. No. 569-06-2), 1-cyanoanthraquinone (CAReg. No. 38366- 32-4) and 2-trifluoromethylanthraquinone (CAReg. No. 362-21-0). 40 5 00 ml cultures of Desulfovibrio gigas were grown on standard BTZ-3 medium as previously described in Example 1 with 30 mM sodium lactate, 30 mM sodium sulfate and 30 mM sodium fumarate. The culture was harvested after 24 h of growth at 31 °C by centrifugation at 8000 rpm in a GSA Sorvall rotor for 20 minutes. The cell pellet was resuspended in 30 ml of incubation assay mixture of the following composition: 50 mM HEPES buffer pH 7 buffer, 2.5 mM sodium lactate, 2.5 mM sodium sulfate. This suspension was centrifuged 45 again at 10,000 rpm in a Sorvall SS-34 rotor for 5 minutes. The resulting cell pellet was resuspended in 5 ml of incubation assay buffer. Cell densities were generally near 10e10 cells/ml. The samples were assayed for production in the presence of anthraquinones. 200 ul of the cell suspension was added to 1.2 ml of incubation assay mixture in a 5 ml capacity test tube. The tube was gassed with hydrogen gas for 20 minutes then the anthraquinones were added as ethanolic solutions, the tubes were then so gassed with hydrogen again for 5 minutes. The reaction mixtures were then incubated for 3 hours at 31 °C. The reactions were stopped by the addition of 200 ul of 1N sodium hydroxide. After 20 minutes the alkaline mixtures were centrifuged in a table top microf uge for 4 minutes at 14,000 rpm to remove cells and cell debris. The supernatant was then assayed for sulfide using the ferric chloride-DPD reagent system as described by Siegel etal., Anal. Biochem., 11:126-132, 1965, herein incorporated by reference. The results are summarized 55 in Table XVI.

47 EP 0 525 073 B1

TABT.F. YVT PPM % Sulfide Production 1,8 diOH- 2-Br 1-C1 1-F1 1-CN 2 TriF 0 100 100 100 100 100 100 0.1 101 99 81 105 118 99 0.5 51 33 26 23 20 17 2 4 0.6 1.6 0.9 6.7 1.7 5 2.3 0.6 1.3 0.9 6.8 1.5

The halo-derivatives as well as the cyano and hydroxy compounds displayed inhibitory properties with 50% or more inhibition of sulfide production in the 0.1-0.5 ppm range.

Claims

1. A process for inhibiting sulfide production by sulfate-reducing bacteria comprising contacting the medium containing the sulfate-reducing bacteria with an anthraquinone compound which has an activity of at least 21 calculated according to formula (I): Activity = 28.1 + 2 (coeff.) (I) wherein 2 (coeff.) comprises the sum of the coefficient values for each individual coefficient R^ through R8 present in the anthraquinones represented by structure A:

or the corresponding derivative of structure (A).

2. A process of Claim 1 for inhibiting sulfide production by sulfate-reducing bacteria comprising contacting the medium containing the sulfate-reducing bacteria with one or more of the following compounds: N-[1-(9,10-dihydro-9,10-dioxo)anthracenyl]-N'-(1-methylethyl)imidodicarbonimidic diamide hydrochloride CA Reg. No.: none 1- Aminoanthraquinone CAReg. No.: 82-45-1 2- Aminoanthraquinone CAReg. No.: 117-79-3 1-Amino-4-hydroxyanthraquinone CAReg. No.: 116-85-8 1 ,2-Diaminoanthraquinone CAReg. No.: 1758-68-5 2,6-Dihydroxyanthraquinone; Anthraflavic acid CAReg. No.: 84-60-6 Anthraquinone-2-carboxylic acid CAReg. No.: 117-78-2 1 ,5-Dihydroxyanthraquinone; Anthraruf in CAReg. No.: 117-12-4 1 ,2-Dihydroxyanthraquinone; Alizarin

48 EP 0 525 073 B1

CAReg. No.: 72-48-0 2,2'-[(9,10-Dihydro-9,10-dioxo-1,5-anthracenediyl)diimino]bis[5-methylbenzenesulfonic acid], di Na salt; Alizarine violet 3R 5 CAReg. No.: 6408-63-5 1 ,2,5,8-Tetrahydroxyanthraquinone; Quinalizarin CAReg. No.: 81-61-8 4- Amino-9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, monosodium salt; Nuclear fast red w CAReg. No.: 6409-77-4 1 ,8-Dihydroxyanthraquinone; Danthron CAReg. No.: 117-10-2 2,2'-[(9,10-Dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-methylbenzenesulfonic acid],di Na salt; Acid green 25 15 CAReg. No.: 4403-90-1 1-Amino-2,4-dibromoanthraquinone CAReg. No.: 81-49-2 5- Chloro-1-anthraquinonylamine CAReg. No.: 117-11-3 20 2-Ethylanthraquinone CAReg. No.: 84-51-5 1- Hydroxyanthraquinone CAReg. No.: 129-43-1 2- (Hydroxymethyl)anthraquinone 25 CAReg. No.: 17241-59-7 1-Amino-4-methoxyanthraquinone CAReg. No.: 116-83-6 1-Amino-6,7-dichloroanthraquinone CAReg. No.: 5355-88-4 30 Benz[a]anthracene-7,12-dione CAReg. No.: 2498-66-0 1,8-Dihydroxy-3-methylanthraquinone; Chrysophanic acid CAReg. No.: 481-74-3 10-[(3-Amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- 35 (hydroxyacetyl)-1-methoxy-5,12-naphthacenedione hydrochloride; Adriamycin hydrochloride CAReg. No.: 23214-92-8 9,10-Dihydro-4,5-dihydroxy-9,10-dioxo-2-anthracenecarboxylic acid; Rhein CAReg. No.: 478-43-3 (8S-cis)-8-Acetyl-10[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8, 40 11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride; Daunomycin hydrochloride CAReg. No.: 20830-81-3 1 ,2,4-Trihydroxyanthraquinone; Purpurin CAReg. No.: 81-54-9 1-Aminoanthraquinone diazonium salt 45 CAReg. No.: 16048-40-1 2,2'-Dimethyl-[1,1'-bianthracene]-9,9',10,10'-tetrone; 2,2'-Dimethyl-1,1'-bianthraquinone CAReg. No.: 81-26-5 3- (D-apio-beta-D-Furanosyloxy)-1 ,8-dihydroxy-6-methyl-9,10-anthracenedione; Frangulin B CAReg. No.: 14101-04-3 so 2-Chloroanthraquinone CAReg. No.: 131-09-9 1 ,5-Dichloroanthraquinone CAReg. No.: 82-46-2 1,4,5,8-Tetrachloroanthraquinone 55 CAReg. No.: 81-58-3 1-Chloroanthraquinone CAReg. No.: 82-44-0 1 ,8-Dichloroanthraquinone CAReg. No.: 82-43-9 49 EP 0 525 073 B1

2-Bromo-3-methylathraquinone CAReg. No.: 84-44-6 2-(2,2,2-Tri methyl propionamido)anthraquinone 5 CAReg. No.: None 2,6-Bis[2-(dimethylamino)ethoxy]-9, 10-anthracenedione; Tilorone analog R11,043DA CAReg. No.: 66686-31-5 2- Methyl-1-nitroanthraquinone CAReg. No.: 129-15-7 10 1-Amino-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid; 1-Aminoanthraquinone-2-sulfonic acid CAReg. No.: 83-62-5 9,10-Dihydro-5-nitro-9,10-dioxo-1-anthracenesulfonic acid; 5-Nitroanthraquinone-1-sulfonic acid CAReg. No.: 82-50-8 3- Chloro-9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid; 2-Chloroanthraquinone-3-carboxylic 15 acid CAReg. No.: 84-32-2 50/50 Mixture of Anthrarufin and Danthron CAReg. No.: None Anthraquinone 20 CAReg. No.: 84-65-1 1 ,8-Dihydroxy-3-(hydroxymethyl)-anthraquinone; Aloe-emodin CAReg. No.: 481-72-1 7,16-Dichloro-6,15-dihydro-5,9,14,18-anthrazinetetrone; 7,16-Dichloroindanthrone CAReg. No.: 130-20-1 25 1,2,3,4,5,8-Hexahydroxyanthraquinone; Alizarin cyanin CAReg. No.: None 2,4,5,7-Tetrabromo-1,8-dihydroxy-9,1 0-anthracenedione; 2,4,5,7-Tetrabromochrysazin CAReg. No.: 17139-66-1 1 .2.7- Trihydroxyanthraquinone; Anthrapurpurin 30 CAReg. No.: 602-65-3 1,4,5-Trihydroxy-2-methyl-9,1 0-anthracenedione; Islandicin CAReg. No.: 476-56-2 1 .4.5.7- Tetrahydroxy-2-methyl-9,1 0-anthracenedione; Catenarin CAReg. No.: 476-46-0 35 1,8-Dihydroxy-3-methoxy-6-methyl-9,1 0-anthracenedione; Physcion CAReg. No.: 521-61-9 1 .4.5.8- Tetrahydroxy-2-methyl-9,1 0-anthracenedione; Cynodontin CAReg. No.: 476-43-7 1.5.8- Trihydroxy-3-methyl-9,1 0-anthracenedione; Helminthosporin 40 CAReg. No.: 518-80-9 1- Hydroxy-2-[6-0-p-D-xylopyranosyl-p-D-glycopyranosyl)oxy]-9,1 0-anthracenedione; Ruberythric acid CAReg. No.: 152-84-1 2- Phenoxy quinizarin-3,4'-disulfonic acid, di K salt CAReg. No.: None 45 (+,-)-1-Acetoxy-8-hydroxy-1 ,4,4a,9a-tetrahydroanthraquinone CAReg. No.: 73794-49-7 1-Amino-4[[4-[(dimethylamino)methyl]phenyl]amino-9,10-anthracenedione; Basic Blue 47 CAReg. No.: 12217-43-5 1.5- Bis(2-carboxyanilino)-9, 10-anthracenedione; Acridylic acid 50 CAReg. No.: 81-78-7 1 ,8-Dihydroxy-9-anthranol; 1 ,8-Dihydroxyanthranol CAReg. No.: 480-22-8 1,2,10-Anthracenetriol; Anthrarobin CAReg. No.: 577-33-3 55 1-Amino-4-bromo-2-methylanthranquinone CAReg. No.: 81-50-5 1 ,4-Diaminoanthraquinone CAReg. No.: 128-95-0 2.6- Diaminoanthraquinone 50 EP 0 525 073 B1

CAReg. No.: 131-14-6 1- Amino-4[4-[[4-chloro-6[[2,3, or 4-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]- 9,10-dihyro-9,10-dioxo-2-anthracenesulfonic acid; Reactive blue 2; Procion blue HB(S); Cibacron blue 5 3G-A; Basilen blue E-3G CAReg. No.: 12236-82-7 Anthraquinone-1,5-disulfonic acid, di Na salt hydrate (95%) CAReg. No.: 853-35-0 Anthraquinone-2,6-disulfonic acid, di Na salt w CAReg. No.: 84-50-4 Anthraquinone-2-sulfonic acid, sodium salt monohydrate CAReg. No.: 131-08-8 1,2-Bis[(4-sulfophenyl)amino]-4-hydroxyanthraquinone; Alizarin blue black B CAReg. No.: 1324-21-6 15 3-Aminomethylalizarin-N,N-diacetic acid CAReg. No.: 3952-78-1 2- Amino-4-[[3-(ethenylsulfonyl)phenyl]-9,10-dihydro-9,10-dioxo]-2-anthracene sulfonic acid, monosodium salt; Acid blue 215 CAReg. No.: 14541-90-3 20 1-(Methylamino)anthraquinone CAReg. No.: 82-38-2 2,2'-[(9,10-Dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracendiyl)diimino]bis[5-methylbenzenesulfonicacid], di Na salt; Acid green 41 CAReg. No.: 4430-16-4 25 2,2'-[(9,10-Dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-butylbenzenesulfonic acid]; Acid green 27 CAReg. No.: 6408-57-7 1,1'-lminobis[4-amino]9, 10-anthracenedione, sulfonated; Acid black 48 CAReg. No.: 1328-24-1 30 1-Amino-9,10-dihydro-9,10-dioxo-4-(phenylamino)-2-anthracenesulfonic acid, Na salt; Acid blue 25 CAReg. No.: 6408-78-2 4-[[4-(Acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid, Na salt; Acid blue 40 CAReg. No.: 6424-85-7 35 1-Amino-9,10-dihydro-9,10-dioxo-4-[[3[[2-(sulfoxy)ethyl]-sulfonyl]phenyl]amino]-2-anthracenesulfonicacid, disodium salt; Remazol Brilliant blue R; CAReg. No.: 2580-78-1 1-Amino-4[[3-[4,6-dichloro-1,3,5-triazin-2-yl)amino]-4-sulfophenyl]amino]-9,10 dihydro-9,10-dioxo-2-anthracenesulfonic acid; Reactive blue 4 40 CAReg. No.: 13324-20-4 1-(9,10-Dihydro-9,10-dioxo-1-anthracenyl-1,2-hydrazinedisulfonic acid, di Na salt; (1-Anthraquinonyl)- 1,2-hydrazine disulfonic acid, di Na salt CAReg. No.: 54345-83-4 9,10-Dihydro-5,6-dihydroxy-9,10-dioxo-1-anthracenesulfonic acid; Alizarin-5-sulfonic acid 45 CAReg. No.: 6373-42-8 N-(4-Chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide; 1-Benzamido-4-chloroanthraquinone CAReg. No.: 81-45-8 1-Amino-4-bromo-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid, Na salt; 1-Amino-4-bromoanthraquinone-2-sulfonic acid, Na salt 50 CAReg. No.: 6258-06-6 1-Amino-9,10-dihydro-4[[(4-methylphenyl)sulfonyl]amino-9,10-dioxo-2-anthracenesulfonic acid, Na salt; 1-Amino-4-(p-toluenesulfonamido)anthraquinone-2-sulfonic acid, Na salt CAReg. No.: 64981-00-6 9,10-Dihydro-9,10-dioxo-2,3-anthracenedicarboxylic acid 55 CAReg. No.: 27485-15-0 1,1'-lminobis(4-nitro-9,1 0-anthracenedione) CAReg. No.: 128-88-1 1 -Amino-4-chloro-2-met hylant hraqu inone CAReg. No.: 3225-97-6 51 EP 0 525 073 B1

2.3- dimethyl-1,4-dihydroxyanthraquinone; 2,3-Dimethylquinizarin CAReg. No.: 25060-18-8 6-Methyl-1 ,3,8-trihydroxyanthraquinone; Emodin 5 CAReg. No.: 518-82-1 1.4- Bis(methylamino)-anthraquinone CAReg. No.: 2475-44-7 N-(4-Amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthracenyl)-4-methylbenzenesulfonamide; N-(4-Amino- 3-methoxyanthraquinone-1-yl)-p-toluenesulfonamide; 1-Amino-2-methoxy-4-(p-tolysufonamido)anthra- 10 quinone CAReg. No.: 81-68-5 [1 , 1 '-Biant hracene]-9,9'1 0, 1 0'-tetrone; 1 , 1 '-Biant hraquinone CAReg. No.: 914-20-5 6.7- Dichloro-1,4-dihydroxyanthraquinone 15 CAReg. No.: 1225-15-6 2-[[9,10-Dihydro-4-(methylamino)-9,10-dioxo-1-anthracenyl]amino]-5-methyl-benzenesulfonicacid, monosodium salt; Alizarine astral B-CF CAReg. No.: 6408-51-1 2.8- Diphenyl-anthra[2,1-d:6,5-d']bisthiazole-6,12-dione; Indanthrene yellow GCN 20 CAReg. No.: 129-09-9 2-Methoxy-3-methyl-9, 10-anthracenedione CAReg. No.: 17241-42-8 1,4-Bis[(1-methylethyl)amino]-9,1 0-anthracenedione; 1,4-Di(isopropylamino)anthraquinone CAReg. No.: 14233-37-5 25 1 ,4-Bis[(2,4,6-triethylphenyl)amino]-9,1 0-anthracenedione; 1 ,4-Bis(2,4,6-triethylanilino)anthraquinone CAReg. No.: 116-74-5 1- (2-Hydroxyethyl)amino-4-methylaminoanthraquinone; Disperse blue 3 CAReg. No.: 2475-46-9 1,4-Bis[(4-methylphenyl)amino]9,10-anthracenedione; Solvent green 3 30 CAReg. No.: 128-80-3 2- Amino-3-hydroxyanthraquinone CAReg. No.: 117-77-1 1-(Bromothio)anthraquinone CAReg. No.: None 35 1,8-Bis(phenylmethoxy)-9,1 0-anthracenedione; 1,8-Dibenzyloxyanthraquinone CAReg. No.: 69595-66-0 1- Amino-2-(2-aminoethylthio)-4-hydroxyanthraquinone CAReg. No.: None 1,4-Bis(pentylamino)-9, 10-anthracenedione; Oil blue N 40 CAReg. No.: 2646-15-3 1 - Ami no-2- bromo-4- hyd roxyan t hraq u i none CAReg. No.: 116-82-5 2- Propionamidoanthraquinone CAReg. No.: None 45 1 ,4-Diamino-2,3-bis(2-phenoxyethoxy)anthraquinone CAReg. No.: 41313-11-5 N-(5-Chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide; 1-Benzamido-5-chloroant hraquinone CAReg. No.: 117-05-5 Anthraquinone-1-arsonic Acid 50 CAReg. No.: None N,N'-[lminobis(9,10-dihydro-9,10-dioxo-4,1-anthracenediyl)]-bisbenzamide; 4,4'-Dibenzamido-1,1'-dianthrimide CAReg. No.: 128-79-0 1,4,5,8-Tetraaminoanthraquinone; Disperse blue 1 55 CAReg. No.: 2475-45-8 2-Methylanthraquinone CAReg. No.: 84-54-8 9,10-Dihydro-9,10-dioxo-2,7-anthracenedisulfonic acid, di Na salt; Anthraquinone-2,7-disulfonic acid, di Na salt 52 EP 0 525 073 B1

CAReg. No.: 853-67-8 1 ,2,3-trihydroxyanthraquinone; Anthragallol CAReg. No.: 602-64-2 Carmine (Aluminum lake) CAReg. No.: 1390-65-4 9,10-Dihydro-1,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid CAReg. No.: 145-48-2 2-Amino-3-chloroanthraquinone CAReg. No.: 84-46-8 1- Anthraquinonesulfonic acid, Na salt CAReg. No.: 128-56-3 2- tert-butylanthraquinone CAReg. No.: 84-47-9 1 .4- Dihydroxyanthraquinone CAReg. No.: 81-64-1 1.5- Diamino-4,8-dihydroxyanthraquinone CAReg. No.: 145-49-3 1- Hydroxy-4-[(4-methylphenyl)amino]-9,1 0-anthracenedione; 1-Hydroxy-4-(p-toluidino)-anthraquinone CAReg. No.: 81-48-1 1 ,4-Dimethylanthraquinone CAReg. No.: 1519-36-4 1,1'-lminobis-9, 10-anthracenedione; Dianthrimide CAReg. No.: 82-22-4 2- (Cyclopropylcarboxamido)anthraquinone CAReg. No.: None 1- Amino-2-methylanthraquinone; Disperse orange 11 CAReg. No.: 82-28-0 2- [(9,10-Dihydro-4-hydroxy-9,10-dioxo-1-anthracenyl)amino]-5-methyl-benzenesulfonic acid, Na salt; Solway purple R CAReg. No.: 4430-18-6 2,2'-[(9,10-Dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-methyl]benzenesulfonic acid; alizarine viridine CAReg. No.: 4430-16-4 1,4-Bis(ethylamino)-9,10-anthracenedione; Sudan blue CAReg. No.: 6994-46-3 1,4-Diamino-5-nitroanthraquinone CAReg. No.: 82-33-7 N-Benzyl-9,10-dihydro-9,10-dioxo-2-anthracenesulfonamide CAReg. No.: None 2-Bromoanthraquinone CAReg. No.: 572-83-8 1-Fluoroanthraquinone CAReg. No.: 569-06-2 1- Cyanoanthraquinone CAReg. No.: 38366-32-4 2- Trifluoromethylanthraquinone CAReg. No.: 362-21-0

A process for inhibiting sulfide production by sulfate reducing bacteria comprising contacting the medium containing the sulfate-reducing bacteria with a mixture of at least two or more compounds listed in Claim 2.

The process of Claim 2 wherein the compound is 1,8-dihydroxyanthraquinone.

The process of Claim 2 wherein the compound is 1-chloroanthraquinone or 2-chloroant hraquinone.

The process of Claim 2 wherein the compound is 3-chloro-2-anthraquinone carboxylic acid.

53 EP 0 525 073 B1

7. The process of Claim 2 wherein the compound is 2-bromoanthraquinone.

8. The process of Claim 2 wherein the compound is 1-fluoroanthraquinone.

9. The process of Claim 2 wherein the compound is 1-cyanoanthraquinone.

10. The process of Claim 2 wherein the compound is 2-trifluoromethylanthraquinone.

11. The process of Claim 1 or 2 wherein the sulfate-reducing bacterium comprises one or more of Desulfo- vibrio desulfuricans, D. salexigens, D. vulgaris, D. multispirans, or gigas.

12. The process of Claim 1 or 2 wherein the compound is dissolved or suspended in a solvent prior to contact with the medium.

13. The process of Claim 12 wherein the solvent comprises one or more of water, ethanol, acetone, methanol, dimethylsulfoxide, dimethylformamide, or tetrahydrofuran, or other water-miscible solvents.

14. The process of Claim 1 or 2 wherein the compound is contacted with the medium at a concentration of at least 0.1 ppm.

15. The process of Claim 1 or 2 further comprising addition of a reducing agent to the medium containing the sulfate-reducing bacteria.

16. The process of Claim 15 wherein the reducing agent is sodium sulfide and is present in the medium at a concentration of from 2 to 4 mM.

17. The process of Claim 1 or 2 used for waste treatment.

18. The process of Claim 1 or 2 used to reduce the growth of sulfate-reducing bacteria.

Patentanspruche

1. Verfahren zur Hemmung der Sulfid-Produktion durch Sulfatreduzierende Bakterien, umfassend das In- Beruhrung-Bringen des die Sulfat-reduzierenden Bakterien enthaltenden Mediums miteinerAntrachinon- Verbindung, die eine Aktivitat von wenigstens 21 , berechnet nach der Formel (I), hat Aktivitat = 28,1 + 2 (Koeff.) (I), worin 2 (Koeff.) die Summe der Koeff izienten-Werte fur jeden einzelnen Koeff izienten F^ bis R8 ist, der in den Anthrachinonen der Struktur (A)

(A)

oder den entsprechenden Derivaten der Struktur (A) vorliegt.

2. Verfahren nach Anspruch 1 zur Hemmung der Sulfid-Produktion durch Sulfat-reduzierende Bakterien, umfassend das In-Beruhrung-Bringen des die Sulfat-reduzierenden Bakterien enthaltenden Mediums mit einer oder mehrerer der folgenden Verbindungen:

CA Reg. Nr. : N- [1- (9, 10-Dihydro-9, 10-dioxo) anthracen- Keine yl] -N' - (1-methylethyl) imidodicarbonimid- diamid-hydroxchlorid

54 EP 0 525 073 B1

CA Reg. Nr.

1 - Aminoanthrachinon 82-45-1

2 - Aminoanthrachinon 117-79-3 l-Amino-4 -hydroxyanthrachinon 116- 85-8

1 , 2 -Diaminoanthrachinon 1758-68-5 2, 6 -Dihydroxyanthrachinon; Anthraf lavin- 84-60-6 saure

Anthrachinon-2-carbonsaure 117- 78-2

1, 5 -Dihydroxyanthrachinon; Anthraruf in 117-12-4

1, 2 -Dihydroxyanthrachinon; Alizarin 72-48-0 ' 2 , 2 - [ (9 , 10-Dihydro-9, 10-dioxo-l, 5-anthra- 6408- 63-5 cendiyl) diimino] bis [5-methylbenzolsulf on - saure] , Di-Na-Salz; Alizarinviolett 3R 1,2,5, 8-Tetrahydroxyanthrachinon; Chin- 81-61-8 alizarin

4 - Amino- 9, 10 -dihydro- 1 , 3 -dihydroxy- 9 , 10- 6409- 77-4 dioxo- 2 - anthracensul f onsaure , Mononat r ium- Salz; Kernechtrot

1, 8 -Dihydroxyanthrachinon; Danthron 117-10-2 ' 2 , 2 - [ (9 , 10 -Dihydro- 9 , 10 -dioxo- 1 , 4 -anthra- 4403-90-1 cendiyl) diimino] bis [5-methylbenzolsulf onsaure] , Di-Na-Salz; Saure-Grun 25

1 - Amino- 2 , 4 -dibromanthrachinon 81-49-2 5 - Chlor- 1-anthrachinonylamin 117-11-3

2 - Ethylanthrachinon 84-51-5

1 - Hydroxyanthrachinon 129-43-1

2- (Hydroxymethyl) anthrachinon 17241-59-7

1 -Amino- 4 -me thoxyanthrachinon 116-83-6

1 -Amino- 6, 7-dichloranthrachinon 5355-88-4

Benz [a] anthracen-7, 12-dion 2498-66-0

55 tr u aza Ui o bi

CA Keg. Nr.

1, 8-Dihydroxy-3-methylanthrachinon; 481-74-3 Chrysophansaure

10- [ (3 -Amino- 2 , 3 , 6- tridesoxy-a-L-lyxohexo- 23214-92-8 ) pyranosyl oxy] -7, 8, 9, 10 - tetrahydro- 6 , 8, 11- trihydroxy-8- (hydroxyacetyl ) -1-methoxy- 5, 12-naphthacendion-hydrochlorid; Adria- mycin-hydrochlorid

9, 10-Dihydro-4, 5-dihydroxy-9 , 10-dioxo-2- 478-43-3 anthracencarbonsaure ; Rhein (8S-cis) -8 -Acetyl- 10- [3 -amino- 2 , 3 , 6-tri- 20830-81-3 desoxy-a-L-lyxohexopyranosyl) oxy] - 7,8,9, 10-tetrahydro-6, 8, 11-trihydroxy-l- nethoxy-5, 12-naphthacendion-hydrochlorid; Daunomycin-hydrochlorid

1,2,4-Trihydroxyanthrachinon; Purpurin 81-54-9

L-Aminoanthrachinon-diazonium-Salz 16 04 8-40-1

2,2' -Dimethyl- [1,1' -bianthracen] - 81-26-5 3, 9' , 10, 10' -tetron; 2,2' -Dimethyl- 1, 1' -bi- mthrachinon

I - (D-apio-iS-D-Furanosyloxy) -1, 8-dihydroxy- 14101-04-3 j-methyl-9 , 10-anthracendion; Frangulin B

!-Chloranthrachinon 131-09-9

5-Dichloranthrachinon 82-46-2

.,4,5, 8-Tetrachloranthrachinon 81-58-3

.-Chloranthrachinon 82-44-0

. , 8-Dichloranthrachinon 82-43-9

-Brom-3-methylanthrachinon 84-44-6

- (2, 2, 2-Trimethylpropionamido) anthra- Keine hinon

, 6-Bis [2- (dimethylamino) ethoxy] -9, 10- 66686-31-5 nthracendion; Tiloron-Analoges , R11,043DA -Methyl - 1 -nitroanthrachinon 12 9-15-7

> P 0 525 073 B1

ua Keg . in .

83-62-5 .-Amino- 9, 10-dihydro-9 , 10-dioxo-2-anthra- :ensulf onsaure ; 1 -Aminoanthrachinon - 2 - sulf onsaure 82-50-8 ), 10-Dihydro-5-nitro-9 , 10-dioxo-l-anthra- :ensulf onsaure ; 5 -Nitroanthrachinon- 1- 3ulf onsaure

J-Chlor-9, 10 -dihydro- 9, 10-dioxo-2-anthra- 84-32-2 ;encarbonsaure ; 2 -Chloranthrachinon- 3 - :arbonsaure

50/50-Mischung aus Anthrarufin und Keine Danthron

\nthrachinon 84-65-1

-anthra- 481-72-1 L , 8 -Dihydroxy-3 - (hydroxymethyl) ^hinon; Aloe-emodin

7, 16-Dichlor-6, 15 -dihydro- 5 , 9, 14, 18- 13 0-20-1 anthrazintetron; 7 , 16 -Dichlorindanthron

1,2,3,4,5, 8-Hexahydroxyanthrachinon; Keine Mizarincyanin 17139-66- 2,4,5, 7-Tetrabrom-l , 8 -dihydroxy-9 , 10- anthracendion; 2 , 4 , 5 , 7-Tetrabromchrysazin Anthra- 602-65-3 1 . 2 . 7- Trihydroxyanthrachinon; purpurin

1,4, 5-Trihydroxy-2-methyl-9, 10 -anthracen- 476-56-2 dion; Islandicin

1.4.5. 7- Tetrahydroxy-2-methyl-9, 10-anthra- 476-46-0 cendion; Catenarin

1, 8 -Dihydroxy-3 -methoxy- 6 -methyl- 9, 10- 521-61-9 anthracendion; Physcion

1.4.5. 8- Tetrahydroxy-2-methyl-9 , 10-anthra- 476-43-7 cendion; Cynodontin

1.5. 8- Trihydroxy-3-methyl-9, 10-anthracen- 518-80-9 dion; Helminthosporin

57 EP 0 525 073 B1

CA Reg. Nr.

1- Hydroxy-2- [6 -0-6-D-xylopyranosyl -S-D- 152-84-1 glycopyranosyl) oxy] -9, 10 -anthracendion; Ruberythrinsaure ' 2- Phenoxychinizarin-3 , 4 -disulf onsaure-di - Keine K-Salz

(+, -) -1-Acetoxy- 8 -hydroxy- 1, 4 , 4a, 9a-tetra- 73 794-4 9-7 hydroanthrachinon l-Amino-4-{4- [ (dimethylamino) methyl] - 12217-43-5 phenyl } amino- 9 , 10 -anthracendion; Basisch Blau 47

1 . 5 - Bis (2-carboxyanilino) -9, 10 -anthracen- 81-78-7 dion; Acridylsaure

1, 8-Dihydroxy-9-anthranol; 1 , 8 -Dihydroxy- 480-22-8 anthranol

1, 2, 10-Anthracentriol; Anthrarobin 577-33-3

1 - Amino -4 -brom- 2 -methylanthrachinon 81-50-5

1 , 4-Diaminoanthrachinon 128-95-0

2 . 6 - Diaminoanthrachinon 131-14-6 l-Amino-4- [4- [ [4-Chlor-6- [ [2, 3 oder 4- 12236-82-7 sulfophenyl] amino] -1,3, 5-triazin-2-yl] - amino] -3 -sulfophenyl] amino] - 9 , 10 -dihydro - 9 , 10 -dioxo- 2 -anthracensulf onsaure ; Reaktivblau 2; Procion-Blau HB(S); Ciba- cron Blau 3G-A) ; Basilen Blau E-3G

Anthrachinon-1, 5 -disulf onsaure, Di-Na- 853-35-0 Salz-hydrat (95 %)

Anthrachinon-2 , 6 -disulf onsaure, Di-Na-Salz 84-50-4 Anthrachinon-2-sulfonsaure, Natrium-Salz- 131-08-8 monohydrat

1, 2 -Bis [ (4-sulfonylphenyl) amino] -4- 13 24-21-6 hydroxyanthrachinon; Alizarinblauschwarz B

3 - Aminomethylalizarin-N, N-diessigsaure 3952-78-1

58 cr u ozo u/o d i

ca Keg. Nr.

2-Amino-4-{ [3- (Ethenylsulf onyl ) phenyl] - 14541-90-3 9, 10 -dihydro- 9, 10-dioxo} -2-anthracensulf onsaure, Mono-Natrium-Salz ; Saure-Blau 215

1- (Methylamino) anthrachinon 82-38-2 2, 2' - [ (9, 10-Dihydro-5, 8 -dihydroxy- 9 , 10 - 4430-16-4 dioxo-1, 4 -anthracendiyl) diimino] -bis [5- methylbenzolsulf onsaure] , Di -Na-Salz, • Saure-Griin 41

2,2' - [ (9, 10-Dihydro-9, 10-dioxo-l, 4- 6408-57-7 anthracendiyl) diimino] -bis [5-butylbenzol- sulf onsaure] ; Saure-Griin 27

1,1' -Imino-bis [4 -amino] -9, 10 -anthracen- 1328-24-1 dion, sulfoniert; Saure-Schwarz 48 L-Amino-9, 10 -dihydro-9 , 10-dioxo-4- (phenyl- 6408-78-2 amino) - 2 -anthracensulf onsaure, Na-Salz ; 3aure-Blau 25

I- { [4- (Acetylamino) phenyl] amino} -1-amino- 6424-85-7 3,10 -dihydro-9 , 10-dioxo-2 -anthracensulf onsaure, Na-Salz; Saure-Blau 40

.-Amino-9, 10 -dihydro- 9 , 10 -dioxo-4 - [ [3- [ [2- 2580-78-1 sul : foxy) ethyl ] sul f onyl ] phenyl ] amino] - 2 - tnthracensul f onsaure , Di -Nat rium- Sal z ; ■emazol Brilliantblau R

-Amino-4- [ [3- [4 6 , -dichlor- 1 , 3 , 5 - triazin- 13324-20-4 -yl] amino] -4 -sulfophenyl] amino-9, 10-di- ydro -9,10- dioxo - 2 - ant hracensul f onsaure ; eaktivblau 4

- (9, 10-Dihydro-9, 10-dioxo-l-anthracenyl) - 54345-83-4 , 2 -hydrazindisulf onsaure, Di-Na-Salz; (1- nthrachinonyl ) - 1 2 -hydrazindisulf , onsaure , i-Na-Salz

10-Dihydro-5, 6 , -dihydroxy- 9 , 10-dioxo-l- 6373-42-8 nthracensulf onsaure ; Alizarin- 5 - sulf on- iure EP 0 525 073 B1

CA Reg. Nr.

N- (4-Chlor-9, 10 -dihydro- 9, 10-dioxo-l- 81-45-8 anthracenyl) benzamid; l-Benzamido-4 -chlor- ant hrachinon l-Amino-4-brom-9 , 10 -dihydro- 9 , 10-dioxo-2- 6258-06-6 anthracensulf onsaure , Na-Salz; l-Amino-4 - brora- ant hrachinon- 2 - sul f onsaure , Na - Sal z l-Amino-9, 10 -dihydro -4- [ [ (4-methylphenyl) - 64981-00-6 sulf onyl] amino-9 , 10-dioxo-2-anthracen- sulfonsaure, Na-Salz; l-Amino-4- (p-toluolsulf onamido) anthrachinon-2 - sulf onsaure , Na-Salz

9, 10-Dihydro-9, 10-dioxo-2 , 3 -anthracendi- 27485-15-0 carbonsaure

1, 1' -Iminobis (4-nitro-9, 10 -anthracendion) 128-88-1

1- Amino-4 -chlor2 -methylanthrachinon 3225-97-6

25060-18-8 2 , 3- Dimethyl -1 , 4 -dihydroxyant hrachinon; 2 . 3 - Dimethylchinizarin

6-Methyl-l, 3 , 8 - trihydroxyanthrachinon; 518-82-1 Emodin

1. 4- Bis (Methylamino) -anthrachinon 2475-44-7

N- (4 -Amino- 9 , 10 -dihydro- 3 -methoxy-9, 10 -di- 81-68-5 oxo-1- anthracenyl) -4 -methylbenzolsulf onamid; N- (4 -Amino-3 -methoxyanthrachinon- 1 - yl) -p-toluolsulf onamid; l-Amino-2-methoxy- 4- (p-toluolsulf onamido) anthrachinon

[1,1' -Bianthracen] -9, 9' , 10, 10' -tetron; 914-20-5 1,1' -Bianthrachinon

6. 7- Dichlor-l, 4 -dihydroxyanthrachinon 1225-15-6

2- [ [9, 10-Dihydro-4- (methylamino) -9, 10-di- 6408-51-1 oxo-1 -anthracenyl] amino] -5-methylbenzolsulf onsaure, Mono-Natrium-Salz ; Alizarin- astrol B-CF

2. 8- Diphenylanthra [2 , 1-d: 6, 5-d' ] bis- 129-09-9 thiazol-6 , 12-dion; Indanthren-Gelb GCN

60 EP 0 525 073 B1

CA Reg. I

2-Methoxy-3-methyl-9, 10 -anthracendion 17241-42-

1, 4-Bis [ (1-methylethyl) amino] -9, 10-anthra- 14233-37- cendion; 1 , 4 -Di (isopropylamino) anthrachinon

1, 4-Bis [(2,4, 6-triethylphenyl) amino] -9, 10- 116-74-5 anthracendion; 1, 4-Bis (2,4, 6-triethylanilino) anthrachinon

1- ( 2 -Hydroxy ethyl) amino -4 -methylamino- 2475-46-S anthrachinon; Dispersblau 3

1, 4-Bis [ (4-methylphenyl) amino] -9, 10- 128-80-3 anthracendion; Losungsmittel-Griin 3

2 - Amino- 3 - hydroxyanthrachinon 117-77-1

1- (Bromthio) anthrachinon Keine

1, 8 -Bis (phenylmethoxy) -9, 10 -anthracendion; 69595-66- 1, 8-Dibenzyloxyanthrachinon

1 -Amino- 2- (2 -aminoethylthio) -4-hydroxy- Keine anthrachinon

1, 4-Bis (pentylamino) -9, 10 -anthracendion; 2646-15-3 6l-Blau N

1 - Amino - 2 - brom - 4 - hydroxyant hrachinon 116-82-5

2- Propionamidoanthrachinon Keine

1, 4-Diamino-2, 3-bis (2 -phenoxyethoxy ) - 41313-11- ant hrachinon

NT- (5-Chlor-9, 10 -dihydro-9 , 10-dioxo-l- 117-05-5 anthracenyl) benzamid; l-Benzamido-5-chloranthrachinon tothrachinon- 1 - arsonsaure Keine

Sf,N' [Iminobis (9, 10-dihydro- 9 , 10-dioxo-4, 1- 128-79-0 anthracendiyl ) ] bisbenzamid; 4,4' -Dibenz - ' amido- 1 , 1 dianthrimid

L , 4 , 5 , 8-Tetraaminoanthrachinon; Dispers- 2475-45-8 Dlau 1

»1 la Keg. JMr.

2-Methylanthrachinon 84-54-8

9, 10-Dihydro-9, 10-dioxo-2, 7-anthracendi- 853-67-8 sulf onsaure, Di-Na-Salz; Anthrachinon- 2 , 7- disulf onsaure, Di-Na-Salz

1, 2 , 3-Trihydroxyanthrachinon; Anthragallol 602-64-2

Carmin (Aluminium- Farblack) 1390-65-4

9, 10 -Dihydro -1,4 -dihydroxy- 9, 10-dioxo-2- 145-48-2 ant hracensulf onsaure

2 -Amino- 3 -chloranthrachinon 84-46-8

1- Ant hrachinonsulf onsaure, Na-Salz 128-56-3

2- tert-Butylanthrachinon 84-47-9

L , 4 -Dihydroxyanthrachinon 81-64-1

L, 5-Diamino-4, 8 -dihydroxyanthrachinon 145-49-3

L -Hydroxy- 4- [ (4 -methylphenyl) amino] -9, 10- 81-48-1 mthracendion; l-Hydroxy-4- (p- toluidino) - mthrachinon

., 4-Dimethylanthrachinon 1519-36-4

-, 1' -Iminobis-9, 10 -anthracendion; Di- 82-22-4 mthrimid

I- (Cyclopropylcarboxamido) anthrachinon Keine

-Amino-2-methylanthrachinon; Dispers- 82-28-0 'range 11

[ - (9, 10 -Dihydro-4 -hydroxy- 9, 10-dioxo-l- 4430-18-6 nthracenyl) amino] -5-methylbenzolsulf on- aure, Na-Salz; Solvay-Purpur R

2' - [ (9, 10-Dihydro-5, , 8 -dihydroxy- 9 , 10- 4430-16-4 ioxo-1, 4 -anthracendiyl) diimino] -bis [5- ethyl]benzolsulf onsaure; Alizarin-Viridin 4-Bis , (ethylamino) -9, 10 -anthracendion; 6994-46-3 udanblau

, 4-Diamino-5-nitroanthrachinon 82-33-7 EP 0 525 073 B1

CA Reg. Nr. :

N-Benzyl-9, 10 -dihydro- 9 , 10-dioxo-2-anthra- Keine censul f onaraid

2 -Bromant hrachinon 572-83-8

1 - Fluoranthrachinon 569-06-2

1 - Cyananthrachinon 38366-32-4

2 -Trif luormethylanthrachinon 362-21-0

3. Verfahren zur Hemmung der Sulfid-Produktion durch Sulfatreduzierende Bakterien, umfassend das In- Beruhrung-Bringen des die Sulfat-reduzierenden Bakterien enthaltenden Mediums mit einer Mischung aus wenigstens zweien oder mehreren der in Anspruch 1 aufgefuhrten Verbindungen.

4. Verfahren nach Anspruch 2, worin die Verbindung 1,8-Dihydroxyanthrachinon ist.

5. Verfahren nach Anspruch 2, worin die Verbindung 1-Chloranthrachinon oder 2-Chloranthrachinon ist.

6. Verfahren nach Anspruch 2, worin die Verbindung 3-Chlor-2-anthrachinoncarbonsaure ist.

7. Verfahren nach Anspruch 2, worin die Verbindung 2-Bromanthrachinon ist.

8. Verfahren nach Anspruch 2, worin die Verbindung 1 -Fluoranthrachinon ist.

9. Verfahren nach Anspruch 2, worin die Verbindung 1 -Cyananthrachinon ist.

10. Verfahren nach Anspruch 2, worin die Verbindung 2-Trif luormethylanthrachinon ist.

11. Verfahren nach Anspruch 1 oder 2, worin das Sulfatreduzierende Bacterium eines oder mehrere von Desulfo- vibrio desulfuricans, D. salexigens, D. vulgaris, D. multispirans oder D. gigas ist.

12. Verfahren nach Anspruch 1 oder 2, worin die Verbindung vor der Beruhrung mit dem Medium in einem Losungsmittel gelost oder suspendiert wird.

13. Verfahren nach Anspruch 12, worin das Losungsmittel einen oder mehrere der Stoffe Wasser, Ethanol, Aceton, Methanol, Dimethylsulfoxid, Dimethylformamid oder Tetraf uran oderandere, mit Wasser misch- bare Losungsmittel umfalit.

14. Verfahren nach Anspruch 1 oder 2, worin die Verbindung mit dem Medium in einer Konzentration von wenigstens 0,1 ppm in Beruhrung gebracht wird.

15. Verfahren nach Anspruch 1 oder 2, weiterhin umfassend das Hinzufugen eines reduzierenden Mittels zu dem die Sulfatreduzierenden Bakterien enthaltenden Medium.

16. Verfahren nach Anspruch 15, worin das reduzierende Mittel Natriumsulfid ist und in dem Medium in einer Konzentration von 2 bis 4 mM vorliegt.

17. Verfahren nach Anspruch 1 oder 2, eingesetzt zur Abfall-Behandlung.

18. Verfahren nach Anspruch 1 oder 2, eingesetzt zur Verminderung des Wachstums Sulfat-reduzierender Bakterien.

63 EP 0 525 073 B1

Revendications

1. Un procede pour inhiber la production de sulfures par des bacteries sulfato-reductrices, comprenant la mise en contact du milieu contenant les bacteries sulfato-reductrices avec un compose d'anthraquinone ayant une activite d'au moins 21 calculee selon la formule (I) Activite = 28,1+2 (coeff.) (I) oil 2 (coeff.) represents la somme des valeurs de coefficient pour chaque coefficient individuel R^ a R$ present dans les anthraquinones representees par la structure A : 10

(A) 15

ou le derive correspondant de structure (A). 20 2. Un procede de la revendication 1 pour inhiber la production de sulfures par des bacteries sulfato-reductrices, comprenant la mise en contact du milieu contenant les bacteries sulfato-reductrices avec un ou plusieurs des composes suivants : Chlorhydrate de diamide N-[1-(9,10-dihydro-9,10-dioxo)anthracenyl]-N'-(1-methylethyl)imidodicarboni- 25 midique N° d'Enregistrement au CA : neant 1- aminoanthraquinone N° d'Enregistrement au CA : 82-45-1 2- aminoanthraquinone 30 N° d'Enregistrement au CA : 117-79-3 1 -am i no-4- hyd roxyan t hraq u i none N° d'Enregistrement au CA : 116-85-8 1 ,2-diaminoanthraquinone N° d'Enregistrement au CA : 1 758-68-5 35 2,6-dihydroxyanthraquinone ; acide anthraflavique N° d'Enregistrement au CA : 84-60-6 Acide ant hraqu inone-2-carboxyl ique N° d'Enregistrement au CA : 117-78-2 1,5-dihydroxyanthraquinone ; anthrarufine 40 N° d'Enregistrement au CA : 117-12-4 1,2-dihydroxyanthraquinone ; alizarine N° d'Enregistrement au CA : 72-48-0 2,2'-[(9,10-dihydro-9,10-dioxo-1,5-anthracenediyl)diimino]bis[acide 5-methyl benzene- sulfonique], sel disodique ; violet d'alizarine 3R 45 N° d'Enregistrement au CA : 6408-63-5 1,2,5,8-tetrahydroxyanthraquinone ; quinalizarine N° d'Enregistrement au CA : 81-61-8 Acide 4-amino-9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracene-sulfonique, sel monosodique ; rou- ge solide nucleaire 50 N° d'Enregistrement au CA : 6409-77-4 1,8-dihydroxyanthraquinone ; dantrone N° d'Enregistrement au CA : 117-10-2 2,2'-[9,10-dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[acide 5-methylbenzene-sulfonique], sel disodique ; vert acide 25 55 N° d'Enregistrement au CA : 4403-90-1 1-amino-2,4-dibromoanthraquinone N° d'Enregistrement au CA : 81-49-2 5-chloro-1 -anthraquinonylamine N° d'Enregistrement au CA : 117-11-3

64 EP 0 525 073 B1

2-ethylanthraquinone N° d'Enregistrement au CA : 84-51-5 1- hydroxyanthraquinone 5 N° d'Enregistrement au CA : 129-43-1 2- (hydroxymethyl)anthraquinone N° d'Enregistrement au CA : 17241-59-7 1- amino-4-methoxyanthraquinone N° d'Enregistrement au CA : 116-83-6 10 1-amino-6,7-dichloroanthraquinone N° d'Enregistrement au CA : 5355-88-4 Benz[a]ant hracene-7, 1 2-d ione N° d'Enregistrement au CA : 2498-66-0 1,8-dihydroxy-3-methylanthraquinone ; acide chrysophanique 15 N° d'Enregistrement au CA : 481-74-3 Chlorhydrate de 10-[(3-amino-2,3,6-tridesoxy-alpha-L-lyxo-hexopyrannosyl)oxy]-7,8,9,10-tetrahydro- 6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphtacenedione ; chlorhydrate de doxorubicine N° d'Enregistrement au CA : 23214-92-8 Acide 9,10-dihydro-4,5-dihydroxy-9,10-dioxo-2-anthracene-carboxylique ; rheine 20 N° d'Enregistrement au CA : 478-43-3 Chlorhydrate de (8S-cis)-8-acetyl-10-[(3-amino-2,3,6-tridesoxy-alpha-L-lyxo-hexopyrannosyl)oxy]- 7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphtacenedione ; chlorhydrate de daunorubici- ne N° d'Enregistrement au CA : 20830-81-3 25 1,2,4-trihydroxyanthraquinone ; purpurine N° d'Enregistrement au CA : 81-54-9 Sel de diazonium de 1-aminoanthraquinone N° d'Enregistrement au CA : 16048-40-1 2,2'-dimethyl-[1,1'-bianthracene]-9,9',10,10'-tetrone ; 2,2'-dimethyl-1,1'-bianthraquinone 30 N° d'Enregistrement au CA : 81-26-5 3- (D-apio-beta-D-furannosyloxy)-1,8-dihydroxy-6-methyl-9,10-anthracenedione ; franguline B N° d'Enregistrement au CA : 14101-04-3 2- chloroanthraquinone N° d'Enregistrement au CA : 131-09-9 35 1,5-dichloroanthraquinone N° d'Enregistrement au CA : 82-46-2 1,4,5,8-tetrachloroanthraquinone N° d'Enregistrement au CA : 81-58-3 1- chloroanthraquinone 40 N° d'Enregistrement au CA : 82-44-0 1 ,8-dichloroanthraquinone N° d'Enregistrement au CA : 82-43-9 2- bromo-3-methylanthraquinone N° d'Enregistrement au CA : 84-44-6 45 2-(2,2,2-trimethylpropionamido)anthraquinone N° d'Enregistrement au CA : neant 2,6-bis[2-(dimethylamino)ethoxy]-9,10-anthracenedione ; analogue de tilorone R11, 043DA N° d'Enregistrement au CA : 66686-31-5 2-methyl-1-nitroanthraquinone so N° d'Enregistrement au CA : 129-15-7 Acide 1-amino-9,10-dihydro-9,10-dioxo-2-anthracene-sulfonique ; acide 1-aminoanthraquinone-2-sulfonique N° d'Enregistrement au CA : 83-62-5 Acide 9,10-dihydro-5-nitro-9,10-dioxo-1-anthracene-sulfonique ; acide 5-nitroanthraquinone-1-sulfoni- 55 que N° d'Enregistrement au CA : 82-50-8 Acide 3-chloro-9,10-dihydro-9,10-dioxo-2-anthracene-carboxylique ; acide 2-chloroanthraquinone-3- carboxylique N° d'Enregistrement au CA : 84-32-2 65 EP 0 525 073 B1

Melange a 50/50 d'anthraruf ine et de dantrone N° d'Enregistrement au CA : neant Anthraquinone 5 N° d'Enregistrement au CA : 84-65-1 1,8-dihydroxy-3-(hydroxymethyl)anthraquinone ; aloemodine N° d'Enregistrement au CA : 481-72-1 7,16-dichloro-6,15-dihydro-5,9,14,18-anthrazinetetrone ; 7,16-dichloro-indanthrone N° d'Enregistrement au CA : 1 30-20-1 10 1,2,3,4,5,8-hexahydroxyanthraquinone ; alizarine cyanine N° d'Enregistrement au CA : neant 2,4,5,7-tetrabromo-1 ,8-dihydroxy-9,1 0-anthracenedione ; 2,4,5,7-tetrabromochrysazine N° d'Enregistrement au CA : 17139-66-1 1.2.7- trihydroxyanthraquinone ; anthrapurpurine 15 N° d'Enregistrement au CA : 602-65-3 1,4,5-trihydroxy-2-methyl-9, 10-anthracenedione ; islandicine N° d'Enregistrement au CA : 476-56-2 1.4.5.7- tetrahydroxy-2-methyl-9,10-anthracenedione ; catenarine N° d'Enregistrement au CA : 476-46-0 20 1,8-dihydroxy-3-methoxy-6-methyl-9, 10-anthracenedione ; physcion N° d'Enregistrement au CA : 521-61-9 1.4.5.8- tetrahydroxy-2-methyl-9,1 0-anthracenedione ; cynodontine N° d'Enregistrement au CA : 476-43-7 1 .5.8- trihydroxy-3-methyl-9,1 0-anthracenedione ; helminthosporine 25 N° d'Enregistrement au CA : 518-80-9 1-hydroxy-2-[6-0-p-D-xylopyrannosyl-p-D-glycopyrannosyl)oxy]-9, 10-anthracenedione ; acide rube- rythrique N° d'Enregistrement au CA : 1 52-84-1 Acide 2-phenoxyquinizarine-3,4'-disulfonique, sel dipotassique 30 N° d'Enregistrement au CA : neant (+,-)-1-acetoxy-8-hydroxy-1,4,4a,9a-tetrahydroanthraquinone N° d'Enregistrement au CA : 73794-49-7 1-amino-4-[4-[(dimethylamino)methyl]phenyl]amino-9,10-anthracenedione ; bleu basique 47 N° d'Enregistrement au CA : 12217-43-5 35 1,5-bis(2-carboxyanilino)-9,10-anthracenedione ; acide acridylique N° d'Enregistrement au CA : 81-78-7 1,8-dihydroxy-9-anthranol ; 1,8-dihydroxyanthranol N° d'Enregistrement au CA : 480-22-8 1,2,10-anthracenetriol ; anthrarobine 40 N° d'Enregistrement au CA : 577-33-3 1-amino-4-bromo-2-methylanthraquinone N° d'Enregistrement au CA : 81-50-5 1 ,4-diaminoanthraquinone N° d'Enregistrement au CA : 128-95-0 45 2,6-diaminoanthraquinone N° d'Enregistrement au CA : 131-14-6 Acide 1-amino-4-[4-[[4-chloro-6-[[2,3- ou 4-sulfophenyl]amino]-1 ,3,5-triazine-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-2-anthracene-sulfonique ; bleu reactif 2 : bleu Procion HB (S) ; bleu Cibacron 3G-A ; bleu Basilen E-3G 50 N° d'Enregistrement au CA : 12236-82-7 Acide anthraquinone-1,5-disulfonique, sel disodique hydrate (95 %) N° d'Enregistrement au CA : 853-35-0 Acide anthraquinone-2,6-disulfonique, sel disodique N° d'Enregistrement au CA : 84-50-4 55 Acide anthraquinone-2-sulfonique, sel sodique monohydrate N° d'Enregistrement au CA : 131-08-8 1,2-bis[(4-sulfophenyl)amino]-4-hydroxyanthraquinone ; noir-bleu d'alizarine B N° d'Enregistrement au CA : 1324-21-6 Acide 3-aminomethylalizarine-N,N-diacetique 66 EP 0 525 073 B1

N° d'Enregistrement au CA : 3952-78-1 Acide 2-amino-4-[[3-(ethenylsulfonyl)phenyl]-9,10-dihydro-9,10-dioxo]-2-anthracene-sulfonique, sel monosodique ; bleu acide 215 5 N° d'Enregistrement au CA : 14541-90-3 1-(methylamino)anthraquinone N° d'Enregistrement au CA : 82-38-2 2,2'-[(9,10-dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracenediyl)diimino]bis[acide 5-methylbenzenesul- fonique], sel disodique : vert acide 41 w N° d'Enregistrement au CA : 4430-16-4 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthracenediyl)diimino]bis[acide 5-butylbenzene- sulfonique] ; vert acide 27 N° d'Enregistrement au CA : 6408-57-7 1,1'-iminobis[4amino]-9,10-anthracenedione sulfonee ; noir acide 48 15 N° d'Enregistrement au CA : 1328-24-1 acide 1-amino-9,10-dihydro-9,10-dioxo-4-(phenylamino)-2-anthracene-sulfonique, sel de Na ; bleu acide 25 N° d'Enregistrement au CA : 6408-78-2 Acide 4-[[4-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxo-2-anthracene-sulfonique, sel 20 de Na ; bleu acide 40 N° d'Enregistrement au CA : 6424-85-7 Acide 1-amino-9,10-dihydro-9,10-dioxo-4-[[3-[[2-(sulfoxy)ethyl]sulfonyl]phenyl]amino]-2-anthracene- sulfonique, sel disodique ; Bleu brillant Remazol R N° d'Enregistrement au CA : 2580-78-1 25 Acide 1-amino-4-[[3-[4,6-dichloro-1,3,5-triazine-2-yl)amino]-4-sulfophenyl]amino]-9,10-dihydro-9,10- dioxo-2-anthracene-sulfonique ; bleu reactif 4 N° d'Enregistrement au CA : 1 3324-20-4 Acide 1-(9,10-dihydro-9,10-dioxo-1-anthracenyl-1,2-hydrazine-disulfonique, sel disodique ; acide (1- anthraquinonyl)-1,2-hydrazine-disulfonique, sel disodique 30 N° d'Enregistrement au CA : 54345-83-4 Acide 9,10-dihydro-5,6-dihydroxy-9,10-dioxo-1-anthracene-sulfonique ; acide alizarine-5-sulfonique N° d'Enregistrement au CA : 6373-42-8 N-(4-chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide ; 1-benzamido-4-chloroanthraquinone N° d'Enregistrement au CA : 81-45-8 35 Acide1-amino-4-bromo-9,10-dihydro-9,10-dioxo-2-anthracene-sulfonique, sel de Na ; acide 1-amino-4- bromoanthraquinone-2-sulfonique, sel de Na N° d'Enregistrement au CA : 6258-06-6 Acide 1-amino-9,10-dihydro-4-[(4-methylphenyl)sulfonyl]amino-9,10-dioxo-2-anthracene-sulfonique, sel de Na ; acide 1-amino-4-(p-toluene-sulfonamido)anthraquinone-2-sulfonique, sel de Na 40 N° d'Enregistrement au CA : 64981-00-6 Acide9,10-dihydro-9,10-dioxo-2,3-anthracene-dicarboxylique N° d'Enregistrement au CA : 27485-15-0 1,1'-iminobis(4-nitro-9, 10-anthracenedione) N° d'Enregistrement au CA : 128-88-1 45 1-amino-4-chloro-2-methylanthraquinone N° d'Enregistrement au CA : 3225-97-6 2.3- dimethyl-1,4-dihydroxyanthraquinone ; 2,3-dimethylquinizarine N° d'Enregistrement au CA : 25060-18-8 6-methyl-1 ,3,8-trihydroxyanthraquinone; emodine so N° d'Enregistrement au CA : 518-82-1 1.4- bis (methylamino) anthraquinone N° d'Enregistrement au CA : 2475-44-7 N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthracenyl)-4-methylbenzene-sulfonamide ; N-(4-amino- 3-methoxyanthraquinone-1-yl)-p-toluene-sulfonamide ;1-amino-2-methoxy-4-(p-tolysulfonamido)anthra- 55 quinone N° d'Enregistrement au CA : 81-68-5 [1,1'-bianthracene]-9,9',10,10'-tetrone ; 1,1'-bianthraquinone N° d'Enregistrement au CA : 914-20-5 6,7-dichloro-1,4-dihydroxyanthraquinone 67 EP 0 525 073 B1

N° d'Enregistrement au CA : 1225-15-6 Acide 2-[[9,10-dihydro-4-(methylamino)-9,10-dioxo-1-anthracenyl]amino]-5-methylbenzene-sulfonique, sel monosodique ; alizarine astral B-CF 5 N° d'Enregistrement au CA : 6408-51-1 2,8-diphenyl-anthra[2,1-d:6,5-d']bisthiazole-6,12-dione ; jaune d'indanthrene GCN N° d'Enregistrement au CA : 129-09-9 2-methoxy-3-methyl-9, 10-anthracenedione N° d'Enregistrement au CA : 17241-42-8 10 1,4-bis[(1-methylethyl)amino]-9,10-anthracenedione ; 1,4-di(isopropylamino)anthraquinone N° d'Enregistrement au CA : 14233-37-5 1,4-bis[2,4,6-triethylphenyl)amino]-9, 10-anthracenedione ; 1,4-bis(2,4,6-triethylanilino)anthraquinone N° d'Enregistrement au CA : 116-74-5 1- (2-hydroxyethyl)amino-4-methylaminoanthraquinone; bleu disperse 3 15 N° d'Enregistrement au CA : 2475-46-9 1,4-bis[(4-methylphenyl)amino]-9,10-anthracenedione ; vert au solvant 3 N° d'Enregistrement au CA : 128-80-3 2- amino-3-hydroxyanthraquinone N° d'Enregistrement au CA : 117-77-1 20 1-(bromothio)anthraquinone N° d'Enregistrement au CA : neant 1,8-bis(phenylmethoxy)-9,1 0-anthracenedione ; 1,8-dibenzyloxyanthraquinone N° d'Enregistrement au CA : 69595-66-0 1-amino-2-(2-aminoethylthio)-4-hydroxyanthraquinone 25 N° d'Enregistrement au CA : neant 1,4-bis(pentylamino)-9,10-anthracenedione ; bleu a I'huile N N° d'Enregistrement au CA : 2646-1 5-3 1- amino-2-bromo-4-hydroxyanthraquinone N° d'Enregistrement au CA : 116-82-5 30 2-propionamidoanthraquinone N° d'Enregistrement au CA : neant 1,4-diamino-2,3-bis(2-phenoxyethoxy)anthraquinone N° d'Enregistrement au CA : 41313-11-5 N-(5-chloro-9,10-dihydro-9,10-dioxo-1-anthracenyl)benzamide ; 1-benzamido-5-chloroanthraquinone 35 N° d'Enregistrement au CA : 117-05-5 Acide anthraquinone-1-arsonique N° d'Enregistrement au CA : neant N,N'-[iminobis(9,10-dihydro-9,10-dioxo-4,1-anthracenediyl)]bisbenzamide ; 4,4'-dibenzamido-1,1'- dianthrimide 40 N° d'Enregistrement au CA : 128-79-0 1,4,5,8-tetraaminoanthraquinone ; bleu disperse 1 N° d'Enregistrement au CA : 2475-45-8 2- methylanthraquinone N° d'Enregistrement au CA : 84-54-8 45 Acide 9,10-dihydro-9,10-dioxo-2,7-anthracene-disulfonique, sel disodique ; acide anthraquinone-2,7-disulfonique, sel disodique N° d'Enregistrement au CA : 853-67-8 1,2,3-trihydroxyanthraquinone ; anthragallol N° d'Enregistrement au CA : 602-64-2 so Carmin (laque d'aluminium) N° d'Enregistrement au CA : 1 390-65-4 Acide 9,10-dihydro-1,4-dihydroxy-9,10-dioxo-2-anthracene-sulfonique N° d'Enregistrement au CA : 145-48-2 2-amino-3-chloroanthraquinone 55 N° d'Enregistrement au CA : 84-46-8 Acide 1-anthraquinone-sulfonique, sel de Na N° d'Enregistrement au CA : 128-56-3 2-ferf-butylanthraquinone N° d'Enregistrement au CA : 84-47-9 68 EP 0 525 073 B1

1 .4- dihydroxyanthraquinone N° d'Enregistrement au CA : 81-64-1 1.5- diamino-4,8-dihydroxyanthraquinone 5 N° d'Enregistrement au CA : 145-49-3 1- hydroxy-4-[(4-methylphenyl)amino]-9, 10-anthracenedione ; 1-hydroxy-4-(p-toluidino)anthraquinone N° d'Enregistrement au CA : 81-48-1 1,4-dimethylanthraquinone N° d'Enregistrement au CA : 151 9-36-4 10 1,1'-iminobis-9,10-anthracenedione ; dianthrimide N° d'Enregistrement au CA : 82-22-4 2- (cyclopropylcarboxamido)anthraquinone N° d'Enregistrement au CA : neant 1- amino-2-methylanthraquinone ; orange disperse 11 15 N° d'Enregistrement au CA : 82-28-0 Acide 2-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthracenyl)amino]-5-methylbenzene-sulfonique, sel de Na ; pourpre Solway R N° d'Enregistrement au CA : 4430-1 8-6 Acide 2,2'-[(9,10-dihydro-5,8-dihydroxy-9,10-dioxo-1,4-anthracenediyl)diimino]bis[5-methyl]benzene- 20 sulfonique ; alizarine viridine N° d'Enregistrement au CA : 4430-1 6-4 1,4-bis(ethylamino)-9,10-anthracenedione : bleu Soudan N° d'Enregistrement au CA : 6994-46-3 1,4-diamino-5-nitroanthraquinone 25 N° d'Enregistrement au CA : 82-33-7 N-benzyl-9,10-dihydro-9,10-dioxo-2-anthracene-sulfonamide N° d'Enregistrement au CA : neant 2- bromoanthraquinone N° d'Enregistrement au CA : 572-83-8 30 1-fluoroanthraquinone N° d'Enregistrement au CA : 569-06-2 1- cyanoanthraquinone N° d'Enregistrement au CA : 38366-32-4 2- trifluoromethylanthraquinone 35 N° d'Enregistrement au CA : 362-21-0.

3. Un procede pour inhiber la production de sulfures par des bacteries sulfato-reductrices, comprenant la mise en contact du milieu contenant les bacteries sulfato-reductrices avec un melange d'au moins deux ou plusieurs des composes enumeres dans la revendication 2. 40 4. Le procede de la revendication 2, dans lequel le compose est la 1,8-dihydroxyanthraquinone.

5. Le procede de la revendication 2, dans lequel le compose est la 1-chloroant hraquinone ou la 2-chloroanthraquinone. 45 6. Le procede de la revendication 2, dans lequel le compose est I'acide 3-chloro-2-anthraquinone-carboxylique.

7. Le procede de la revendication 2, dans lequel le compose est la 2-bromoanthraquinone. so 8. Le procede de la revendication 2, dans lequel le compose est la 1-fluoroanthraquinone.

9. Le procede de la revendication 2, dans lequel le compose est la 1-cyanoanthraquinone.

10. Le procede de la revendication 2, dans lequel le compose est la 2-trifluoromethylanthraquinone. 55 11. Le procede de la revendication 1 ou 2, dans lequel la bacterie sulfato-reductrice comprend I'un ou plusieurs de Desulfovibrio desulfuricans, D. salexigens, D. vulgaris, D. multispirans et D. gigas.

12. Le procede de la revendication 1 ou 2, dans lequel le compose est dissous ou mis en suspension dans

69 EP 0 525 073 B1

un solvant avant le contact avec le milieu.

13. Le procede de la revendication 12, dans lequel le solvant comprend un ou plusieurs de I'eau, I'ethanol, I'acetone, le methanol, le dimethylsulfoxyde, le dimethylformamide et le tetrahydrofuranne, ou d'autres solvants miscibles a I'eau.

14. Le procede de la revendication 1 ou 2, dans lequel le compose est mis en contact avec le milieu a une concentration d'au moins 0,1 ppm.

15. Le procede de la revendication 1 ou 2, comprenant de plus I'addition d'un agent reducteurau milieu contenant les bacteries sulfato-reductrices.

16. Le procede de la revendication 15, dans lequel I'agent reducteurest le sulfure de sodium et il est present dans le milieu a une concentration 2 a 4mM.

17. Le procede de la revendication 1 ou 2, utilise pour le traitement de dechets.

18. Le procede de la revendication 1 ou 2, utilise pour reduire la croissance de bacteries sulfato-reductrices.

70 US. 1

GAS (3) 10T 10T 10T OQOO OQOO OOOO B_ CELL SUSPENSION (1) AUTO INHIBITOR -f SAMPLER

b_ SUBSTRATE (5)

ACID (7) 01 OOOO OOOO

3AS MEMBRANE (8) SURFACTANT (9)

)PD (10)

FERRIC :hloride (11)

(tCORDER