COMBINATIONS OF 4-BROMO-2-(4-CHLOROPHENYL)-5-(TRIFLUOROMETHYL)- 1 H-PYRROLE-3-CARBONITRILE AND OXIDIZING AGENTS

[0001] The present invention relates to the use of combinations of 4-bromo-2-(4- chloro-phenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile and an oxidizing agent, for eradicating, eliminating or reducing aquatic organisms in ballast water or bilge water whereby 4-bromo-2-(4-chloro-phenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile and the oxidizing agent are in respective proportions to provide a synergistic effect against fouling organisms. Suitable oxidizing agents are e.g. bromine, chlorine, ozone, sodium hypochlorite, chlorine dioxide, hydrogen peroxide, potassium permanganate, potassium ferrate, peroxydisulfates such as ammonium peroxydisulfate, sodium peroxydisulfate, and potassium peroxydisulfate; peroxymonocarbonates such as calcium peroxycarbonate and sodium peroxycarbonate; peroxydicarbonates such as sodium peroxydicarbonate and potassium peroxydicarbonate; superoxides such as potassium superoxide and sodium superoxide; peroxides such as potassium peroxide, and sodium peroxide; and Fremy's salts such as potassium nitrosodisulfonate, sodium nitrosodisulfonate, and disodium nitrosodisulfonate.

[0002] The shipping industry is vital to the global marketplace, transporting cargo to and from all corners of the world. In addition to valuable cargo, however, ships may transport thousands of organisms in their ballast water. Ships pump ballast water into the bilge of the ship to provide stability and manoeuvrability during a voyage. Water is taken on at one port when cargo is unloaded and usually discharged at another port when the ship receives cargo. When ballast water is discharged in a remote port the concomitant introduction of non-indigenous organisms has the potential to shift the ecological balance of the local marine ecosystems. Governments and industry are moving to treat ballast water to prevent such undesired marine life exchange. Disinfection of ballast water presents unique challenges to conventional disinfection technologies owing to the large number density of organisms, the diversity of their composition, and the chemical and physical characteristics of ballast water. Ballast water treatment methods are typically categorized as either tanker-based or shore- based treatment technologies. Tanker-based treatment technologies comprise filtration, hydrocyclone treatment, ultra violet irradiation, thermal treatment, and the use of chemicals such as ozone, chlorine, hydrogen peroxide, and biocidal compounds.

[0003] Biocidal compounds for use in ballast water treatment should be effective in killing a broad range of marine life forms, have a quick decay rate, and degrade to non-toxic compounds.

[0004] 4-Bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile is disclosed in EP-0, 312,723 for controlling molluscs. Said compound can be represented by the formula :

[0005] EP-0,746,979 describes the use of 4-bromo-2-(4-chlorophenyl)-5-(trifluoro- methyl)-1 H-pyrrole-3-carbonitrile in antifoulant compositions which are applied to underwater surfaces in order to prevent the attachment of fouling organisms to said underwater surfaces.

[0006] 4-Bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile has a half-life of a few hours at a pH of 8.3 which is the pH of sea water before it degrades into non-toxic compounds. Its half-life increases to a couple of days at neutral and acidic pH.

[0007] It has now been found that a combination of 4-bromo-2-(4-chlorophenyl)-5- (trifluoromethyl)-i H-pyrrole-3-carbonitrile (often referred to as component (I)), or a salt thereof, together with one or more oxidizing agents (II) selected from bromine, chlorine, ozone, sodium hypochlorite, chlorine dioxide, hydrogen peroxide, potassium permanganate, potassium ferrate, ammonium peroxydisulfate, sodium peroxydisulfate, potassium peroxydisulfate, calcium peroxycarbonate, sodium peroxycarbonate, sodium peroxydicarbonate, potassium peroxydicarbonate, potassium superoxide, sodium superoxide, potassium peroxide, sodium peroxide, potassium nitrosodisulfonate, sodium nitrosodisulfonate, and disodium nitrosodisulfonate; whereby said component (I) and the oxidizing agent (II) are combined in certain ratios, has a synergistic effect on the control of fouling organisms. As used herein, "control" is defined to include the inhibition of attachment or settlement of fouling organisms to the surface of an object, the removal of fouling organisms that are attached to the surface of an object, and the growth of fouling organisms.

[0008] The present invention provides the use of a combination of 4-bromo-2-(4- chlorophenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile, or a salt thereof, as a component (I) together with an oxidizing agent (II) selected from bromine, chlorine, ozone, sodium hypochlorite, chlorine dioxide, hydrogen peroxide, potassium permanganate, potassium ferrate, ammonium peroxydisulfate, sodium peroxydisulfate, potassium peroxydisulfate, calcium peroxycarbonate, sodium peroxycarbonate, sodium peroxydicarbonate, potassium peroxydicarbonate, potassium superoxide, sodium superoxide, potassium peroxide, sodium peroxide, potassium nitrosodisulfonate, sodium nitrosodisulfonate, and disodium nitrosodisulfonate; whereby component (I) and the oxidizing agent (II) are in respective proportions to provide a synergistic effect against fouling organisms, for eliminating aquatic organisms in ballast water.

[0009] Wherever the term "4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1 H- pyrrole-3-carbonitrile" or component (I) is used throughout this text, it is meant to include said compound both in base or in salt form, the latter being obtained by reaction of the base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids, such as the hydrohalic acids, i.e. hydrofluoric, hydrochloric, hydrobromic and hydroiodic, sulfuric acid, nitric acid, phosphoric acid, phosphinic acid and the like; or organic acids, such as, for example, acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxopropanoic, ethanedioic, propanedioic, butanedioic, (Z)-2-butenedioic, (E)-2-butenedioic, 2-hydroxybutanedioic, 2,3-dihydroxybutanedioic, 2-hydroxy-1 ,2,3-propanetricarboxylic, methanesulfonic, ethanesulfonic, benzenesulfonic, 4-methyl-benzenesulfonic, cyclohexanesulfamic, 2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and the like acids. Said component (I) may also exist in the form of solvates, such as hydrates.

[0010] The ballast water to be disinfected using the combinations of the present invention includes fresh water, sea water and brackish water.

[0011] The combinations of the present invention can be used for disinfecting ballast water in tanker-based ballast water treatment systems or in a shore-based ballast water treatment systems.

[0012] The aquatic organisms that are commonly found in ballast water comprise phytoplankton (dinoflagellates and diatoms), crustaceans (crabs, shrimp, copepods, amphipods), rotifers, polychaetes, mollusks, fish, echinoderms, ctenophores, coelenterates, bacteria and viruses.

[0013] The combination of component (I) and an oxidizing agent (II) can be applied in the form of a composition wherein both said active ingredients are intimately admixed in order to ensure simultaneous administration to the materials to be protected. Application may also be in the form of two separate compositions each comprising respectively component (I) or an oxidizing agent (II) whereby said separate compositions are applied simultaneously in respective proportions to provide a synergistic effect against fouling organisms.

[0014] However the combination of component (I) and oxidizing agent (II) can also be a "sequential-combined" administration or application, i.e. component (I) and oxidizing agent (II) are administered or applied alternatively or sequentially in the same place in such a way that they will necessarily become admixed together at the site to be treated. This will be achieved namely if sequential administration or application takes place within a short period of time e.g. within less than 24 hours, preferably less than 15 hours, more preferably less than 3 hours. This alternative method can be carried out for instance by using a suitable single package comprising at least one container filled with a formulation comprising the active component (I) and at least one container filled with a formulation comprising an active component (II).

[0015] Component (I), i.e. 4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1 H-pyrrole- 3-carbonitrile, may be stored in the form of a concentrated water-dilutable formulation, so that it can easily be stored onboard a ship and used for disinfecting ballast water.

[0016] The oxidizing agent (II) may be stored in the form of a solid powder or concentrated water-dilutable formulation, so that it can easily be stored onboard a ship and used for disinfecting ballast water. The oxidizing agent potassium ferrate (K ₂ FeO ₄ ) is a solid that is only stable when kept dry. It decomposes to rust in acidic solution and is only stable in alkaline solution. Ferrates can be conveniently prepared by oxidizing an alkaline solution of an iron(lll) salt with concentrated chlorine bleach. Oxidizing agents (II) that are gaseous such as chlorine, chlorine dioxide or ozone may be stored in pressure cilinders or generated on-site and used immediately in the ballast water treatment system. On-site generation of chlorine dioxide may be done for example by reducing sodium chlorate in a strong acid solution with a suitable reducing agent (for example, hydrogen peroxide, sulfur dioxide, or hydrochloric acid). Alternatively chlorine dioxide can also be produced by electrolysis of an aqueous sodium chlorite solution.

[0017] In ballast water treatment, the amount of component (I) to be added is such that the concentration of said component (I) in the ballast water tank ranges from 0.01 ppm to 1000 ppm, preferably from 1 to 5 ppm. The amount of oxidizing agent (II) which is added is such a proportion that a synergistic effect against fouling organisms is obtained, in particular the ratio between component (I) and oxidizing agent (II) ranges from 20/80 to 1/99, more particular from 20/80 to 2.5/97.5, even more particular from 20/80 to 5/95.

[0018] Suitable formulations for use in the method of the invention can be water- dilutable liquid formulations which can be in the form of an emulsifiable concentrate (EC), a micro-emulsifiable concentrate (MEC) or a suspension concentrate (SC) containing a high proportion of the active ingredient 4-bromo-2-(4-chloro-phenyl)-5- (trifluoromethyl)-1 H-pyrrole-3-carbonitrile. An EC/MEC is a homogeneous liquid composition, usually containing the active ingredient dissolved in a substantially nonvolatile and water-immiscible organic solvent. An SC is a fine particle size dispersion of solid active ingredient in water. The concentrated formulations may contain 1 to 80% by weight of 4-bromo-2-(4-chloro-phenyl)-5-(trifluoromethyl)-1 H-pyrrole-3- carbonitrile.

[0019] The formulations for use in the method of the invention may also be in the form of a solid formulation for dispersion into water such as wettable powder (WP), water dispersible granules (WG) or water dispersible tablets (WT).

[0020] Suitable carriers for solid formulations, such as dusts, dispersable or flowable powders, are any dispersant that does not adversely affect the active ingredients, for example, clays (for example, kaolin, bentonite, acid clay, and the like), talcs (for example, talc powder, agalmatolite powder, and the like), silicas (for example, diatomaceous earth, silicic acid anhydride, mica powder, and the like), alumina, sulfur powder, activated charcoal, and the like.

[0021] Suitable solvents for the liquid formulations comprising 4-bromo-2-(4-chloro- phenyl)-5-(trifluoromethyl)-1 H-pyrrole-3-carbonitrile include methyl ketone, methyl isobutyl ketone, cyclohexanone, xylenes, toluene, chlorobenzene, paraffins, kerosene, white oil, alcohols (e.g. butanol), methylnaphthalene, trimethylbenzene, trichloroethylene, N-methyl-2-pyrrolidone, and tetrahydrofurfuryl alcohol (THFA).

[0022] The formulations for use in the method of the invention may further comprise suitable substances known in the art of formulation, such as, for example natural or regenerated mineral substances, solvents, dispersants, surfactants, wetting agents, binders, anti-freeze agents, anti-foaming agents, buffering agents, acidulants, alkalizing substances, corrosion inhibitors, water-repelling agents and other active ingredients. Suitable surfactants are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.

[0023] In a further embodiment, the combinations of the present invention are used for disinfecting ballast water treatment in combination with an existing method for ballast water treatment such as filtration, hydrocyclone treatment, ultra violet irradiation, and thermal treatment.

[0024] Composition examples Example 1 : liquid composition (all percentages in % w/w) component (I) 10 %

Soprophor 796/P® 15 % ethoxylated castor oil 24 % acetophenone as solvent up to 100 %

Soprophor 796/P® is a alpha-[2,4,6-tris[1 -(phenyl)ethyl]phenyl]-omega-hydroxy poly(olyethelene)poly(oxypropylene) copolymer comprising from 2 to 8 moles of poly(oxypropylene) units and 16 - 30 units of poly(oxyethylene) units marketed by Rhodia. Ethoxylated castor oils are available as Alkamuls® from Rhodia, for example, Alkamuls BR®, Alkamuls EL 620 LI®, Alkamuls OR 10®, Alkamuls OR 36®, and

Alkamuls OR 40®. A particular group of these ethoxylated castor oils are ethoxylated castor oils containing 12-40 ethoxy units.

The solvent acetophenone can be replaced by other suitable solvents such as benzyl alcohol, butyl lactate, dipropylene glycol n-butyl ether or methyl n-amyl ketone.

Other surfactants for use in these formulations are ethoxylated fatty alcohol, ethoxylated fatty acid and etho-propoxylated block copolymer.

[0025] Biological Experiments

Experiment 1 : poison plate assay

Name of the primary compound: 4-bromo-2-(4-chloro-phenyl)-5-(trifluoromethyl)-

1 H-pyrrole-3-carbonitrile as component (I)

Name of the combination partners: - hydrogen peroxide (35 %) as component (ll-a);

-sodium hypochlorite (14 %) as component (ll-b);

Stock solution: 32.000 and 8.000 ppm in DMSO

Test combinations: % product A + % product B

100 ++ O0

20 ++ 8800

15 ++ 8855

10 ++ 9900

5 ++ 9955

2.5 ++ 9977..55

0 ++ 110000

Concentrations of total single active ingredient in the toxicity tests : a series of concentrations increasing with steps of 1/3 : 0.08 - 0.1 1 - 0.15 - 0.20 - 0.27 - 0.35 - 0.47 - 0.63 - 0.84 - 1 .13 - 1.50 - 2.00 - 2.67 - 3.56 - 4.75 - 6.33 - 8.44 - 1 1.25 - 15.00 - 20.00 - 26.70 - 35.60 - 47.46 - 63.28 - 84.38 - 1 12.50 - 150.00 - 200.00 ppm.

Concentrations of total active ingredient in the combination tests against Artemia : a series of concentrations increasing with steps of 1/3 : 0.27 - 0.36 - 0.48 - 0.63 - 0.84 - 1 .13 - 1 .50 - 2.00 - 2.67 - 3.56 - 4.75 - 6.33 - 8.44 - 1 1.25 - 15.00 - 20.00 ppm.

Concentrations of total active ingredient in the combination tests against algae : a series of concentrations increasing with steps of 1/3 : 3.38 - 4.51 - 6.01 - 8.01 - 10.68 - 14.24 - 18.98 - 25.31 - 33.75 - 45.00 - 60.00 - 80.00 ppm.

Culture medium : algae: BG 1 1 liquid mineral medium

Artemia salina: artificial seawater

Experimental set up : 24-well plates

Species of algae : (1 ) : Chlorella vulgaris CCAP 21 1/12

(2) : Anabaena cylindrica CCAP 1403/2A (3) : Chlamydomonas sphagnophila CCAP 1 1 /36 E

Species of Artelmia : Artemia salina (EG Artemia Cysts, Great Salt Lake strain)

Inoculum : algae: 1990 μl of a 1/10 dilution in BG 1 1 of a two week old culture

Artemia: 1990 μl artificial seawater with 20 - 40 Artemia larvae (24 hours old)

Culture conditions : 21 ⁰ C, 65 % relative humidity, 1000 lux, 16 hour photoperiod

Evaluation: algae: after 3 weeks of exposure

Artemia: after 24 hours of exposure

Synergism between component (I) and one of the components (II) was determined by a commonly used and accepted method described by KuII F. C. et al. in Applied Microbiology, 9, 538-541 (1961 ) using the Synergy Index, which is calculated as follows for two compounds A and B:

Synergy Index (Sl)

wherein:

• Q _A is the concentration of compound A in ppm, acting alone, which produced an end point (e.g. MIC),

• Q _a is the concentration of compound A in ppm, in the mixture, which produced an end point (e.g. MIC),

• Q _B is the concentration of compound B in ppm, acting alone, which produced an end point (e.g. MIC),

• Qb is the concentration of compound B in ppm, in the mixture, which produced an end point (e.g. MIC).

MIC is the minimum inhibitory concentration, i.e. the lowest concentration of each test compound or mixture of test compounds sufficient to inhibit visible growth.

When the Synergy Index is greater than 1.0, antagonism is indicated. When the SI is equal to 1 .0, additivity is indicated. When the SI is less than 1.0, synergism is demonstrated.

When the Synergy Index is greater than 1.0, antagonism is indicated. When the SI is equal to 1 .0, additivity is indicated. When the SI is less than 1.0, synergism is demonstrated.

Table 1 : MIC-values (minimum inhibitory concentration in ppm) and synergy index of various active ingredients and their combination against algae

Table 2 : MIC-values (minimum inhibitory concentration in ppm) and synergy index of various active ingredients and their combination against Artemia salina

Experiment 2 : poison plate assay

Name of the primary compound: 4-bromo-2-(4-chloro-phenyl)-5-(trifluoromethyl)-

1 H-pyrrole-3-carbonitrile as component (I)

Name of the combination partners: - chlorine dioxide as component (ll-c);

- potassium peroxydisulfate as component (ll-d);

- potassium superoxide as component (ll-e);

- potassium peroxide as component (ll-f);

- potassium nitrosodisulfonate as component (ll-g);

- potassium (Vl) ferrate as component (ll-h);

Stock solution: 32.000 and 2500 ppm in DMSO

Test combinations: % product A + % product B

100 + 0

20 + 80

15 + 85

10 + 90

5 + 95

2. 5 + 97.5

0 + 100

Concentrations of total active ingredient in the combination tests against algae : a series of concentrations increasing with steps of 1/3 : 3.38 - 4.51 - 6.01 - 8.01 10.68 - 14.24 - 18.98 - 25.31 - 33.75 - 45.00 - 60.00 - 80.00 ppm.

Culture medium : algae: BG 1 1 liquid mineral medium

Experimental set up : 24-well plates

Species of algae : ( 1 ) : Chlorella vulgaris CCAP 21 1/12

(2) : Anabaena cylindrica CCAP 1403/2A

(3) : Chlamydomonas sphagnophila CCAP 11/36 E

(4) : Tribonema sp. CCAP 880/2

Inoculum : algae: 1990 μl of a 1/10 dilution in BG 1 1 of a two week old culture

Culture conditions : 21 ⁰ C, 65 % relative humidity, 1000 lux, 16 hour photoperiod

Evaluation: algae: after 3 weeks of exposure

Synergism between component (I) and one of the components (II) was determined using the Synergy Index as outlined above in Experiment 1 .

Table 3 : MIC-values (minimum inhibitory concentration in ppm) and synergy index of various active ingredients and their combination against algae

algae MIC-values synergy

Combination ratio ( I) to (II) species in ppm index

(I) + (N-C) (1 ) 100 + 0 107.0 -

(I) + (N-C) (1 ) 20 + 80 9.49 0.82

(I) + (N-C) (1 ) 15 + 85 7.12 0.65

(I) + (N-C) (1 ) 10 + 90 7.12 0.68

(l) + (N-C) (1 ) 5 f 95 9.49 0.95

(l) + (N-C) (1 ) 2.5 f 97.5 9.49 0.98

(I) + (N-C) (1 ) 0 + 100 9.49 -

(I) + (ll-d) (1 ) 100 + 0 107.0 -

(I) + (ll-d) (1 ) 20 + 80 45.0 0.68

(I) + (ll-d) (1 ) 15 + 85 45.0 0.70

(I) + (ll-d) (1) 10 + 90 45.0 0.72

(I) + (ll-d) (1 ) 5 f 95 45.0 0.73

(I) + (ll-d) (1 ) 2.5 f 97.5 45.0 0.74

(I) + (ll-d) (1 ) 0 4 100 60.0 -