N-CYCLOBUTYL-THIAZOL-5-CARBOXAMIDES WITH NEMATICIDAL ACTIVITY
20200087271 · 2020-03-19
Assignee
Inventors
Cpc classification
C07D277/56
CHEMISTRY; METALLURGY
A01N43/72
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to compounds of the formula (I), in which the substituents are as defined in claim 1, which are suitable for use as nematicides.
##STR00001##
Claims
1. A compound of formula (I) ##STR00026## wherein B is phenyl which is unsubstituted or substituted by one or more R5; R1 is selected from CF.sub.3 and CHF.sub.2; R5 is independently selected from halogen, cyano, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, C1-C4-alkylsulfanyl, C1-C4-haloalkylsulfanyl, C1-C4-alkylsulfinyl, C1-C4-haloalkylsulfinyl, C1-C4-alkylsulfonyl, C1-C4-haloalkylsulfonyl, C2-C6-haloalkenyl, C2-C6 haloalkynyl, 5- or 6-membered heterocycle which is unsubstituted or substituted by one or more substituents R6 and C3-C6-cycloalkyl which is unsubstituted or substituted by one or more substituents R6; R6 is independently selected from halogen, cyano, C1-C4-alkyl, C1-C4-haloalkyl or C1-C4-alkoxycarbonyl; or a tautomer, stereoisomer, salt or N-oxide thereof.
2. The compound according to claim 1 wherein the compound is of formula (Ia) ##STR00027##
3. The compound according to claim 1 wherein B is phenyl which is unsubstituted or substituted by one or more R5; R1 is selected from CF.sub.3 and CHF.sub.2; R5 is independently selected from halogen, cyano, C1-C4-haloalkyl, C1-C4-haloalkoxy, C2-C6-haloalkenyl, 5- or 6-membered heterocycle or C3-C6-cycloalkyl wherein the heterocycle and the cycloalkyl are each optionally substituted by one or more substituents R6; R6 is independently selected from halogen, C1-C4-alkyl or C1-C4-haloalkyl.
4. The compound according to claim 1 wherein B is phenyl which is unsubstituted or substituted by one or more R5; R5 is independently selected from halogen, cyano, C1-C4-haloalkyl, C1-C4-haloalkoxy or C3-C6-cycloalkyl optionally substituted by one or more substituents R6; R6 is independently selected from halogen, C1-C4-alkyl or C1-C4-haloalkyl.
5. The compound according to claim 1 wherein B is phenyl which is unsubstituted or substituted by one or more R5; R1 is selected from CF.sub.3 and CHF.sub.2; R5 is independently selected from halogen or trifluoromethyl.
6. The compound according to claim 1 wherein B is R8 or R9; R8 represents ##STR00028## R9 represents ##STR00029## R10 is selected from fluoro, chloro, bromo, difluoromethyl, trifluoromethyl, difluoromethoxy and trifluoromethoxy; R11 is selected from fluoro, chloro, bromo and trifluoromethyl; R12 is selected from hydrogen, fluoro, chloro, bromo and trifluoromethyl.
7. The compound according to claim 1 wherein B is R8 or R9; R8 represents ##STR00030## R9 represents ##STR00031## R10 is chloro; R11 is fluoro, chloro or trifluoromethyl; R12 is hydrogen, chloro, fluoro or trifluoromethyl.
8. The compound according to claim 1 wherein the compound is selected from rac-4-(trifluoromethyl)-N-[(1,2 cis)-2-[2-(trifluoromethyl)phenyl]cyclobutyl]thiazole-5-carboxamide; rac-(4-(trifluoromethyl)-N-[(1,2 cis)-2-[4-(trifluoromethyl)phenyl]cyclobutyl]thiazole-5-carboxamide; rac-N-[(1,2 cis)-2-[4-(trifluoromethoxy)phenyl]cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide; N-[(1 S,2S)-2-(4-chlorophenyl)cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide; N-[(1 S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide; N-[(1 S,2S)-2-(2,4-difluorophenyl)cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide; N-[(1 S,2S)-2-(2,4-dichlorophenyl)cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide; 4-(difluoromethyl)-N-[(1S,2S)-2-(2,4-difluorophenyl)cyclobutyl]thiazole-5-carboxamide; and N-[(1 S,2S)-2-(2,4-dichlorophenyl)cyclobutyl]-4-(difluoromethyl)thiazole-5-carboxamide.
9. Pesticidal composition, which, in addition to comprising formulation adjuvants, comprises a nematicidal effective amount of a compound of the formula I according to claim 1.
10. A composition according to claim 9, which further comprises one or more insecticidally, acaricidally, nematicidally and/or fungicidally active agents.
11. Method of protecting crops of useful plants against damages caused by nematode pests, which comprises treating the plants or the locus thereof with a composition according to either claim 9.
12. Method of protecting plant propagation material against damages caused by nematode pests, which comprises treating this material with a composition according to claim 9
13. Method of controlling and preventing endo- and ectoparasitic nematode infestations and infections in warm-blooded animals, which comprises injecting, topically applying or orally administering a composition according to claim 9.
14. A compound of formula (II) ##STR00032## wherein B and R1 are as defined in claim 1; or a compound of formula (III) ##STR00033## wherein R1 is as defined in claim 1; or a compound of formula (IV) ##STR00034## wherein Xa represents halogen and R1 is as defined in claim 1; or a compound of formula (V) ##STR00035## wherein B and R1 are as defined in claim 1.
Description
EXAMPLES
[0170] The following compounds may be prepared according to the methods described herein or according to known methods. Mp means melting point in C., Rt means retention time. .sup.1H NMR measurements were recorded on a Brucker 400 MHz spectrometer, chemical shifts are given in ppm relevant to a TMS standard. Spectra measured in deuterated solvents as indicated.
Method LCMS
[0171] Spectra were recorded on a Mass Spectrometer from Waters (SQD or ZQ Single quadrupole mass spectrometer) equipped with an electrospray source (Polarity: positive or negative ions, Capillary: 3.00 kV, Cone range: 30-60 V, Extractor: 2.00 V, Source Temperature: 150 C., Desolvation Temperature: 350 C., Cone Gas Flow: 0 L/Hr, Desolvation Gas Flow: 650 L/Hr, Mass range: 100 to 900 Da) and an Acquity UPLC from Waters: Binary pump, heated column compartment and diode-array detector. Solvent degasser, binary pump, heated column compartment and diode-array detector. Column: Waters UPLC HSS T3, 1.8 m, 302.1 mm, Temp: 60 C., DAD Wavelength range (nm): 210 to 500, Solvent Gradient: A=water+5% MeOH+0.05% HCOOH, B=Acetonitrile+0.05% HCOOH: gradient: gradient: 0 min 0% B, 100% A; 1.2-1.5 min 100% B; Flow (ml/min) 0.85.
Example 5: Preparation of N-[(1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]-4-(trifluoromethyl)thiazole-5-carboxamide
[0172] ##STR00015##
[0173] To a stirred solution of (1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutanamine (HCl-salt) (3.80 g, 16.1 mmol) in ethyl acetate (40 mL) was added triethyl amine (5.60 mL) at 0 C. under an atmosphere of argon. Then 4-(trifluoromethyl)thiazole-5-carbonyl chloride (3.72 g, 16.9 mmol, freshly prepared analogous to WO2007036733, page 216)) was added dropwise keeping the temperature between 0-5 C. The reaction mixture was stirred for 45 mins. Water (10 ml) was added and the resulting two layers separated. The water layer was extracted with ethyl acetate (EA) and the combined organic phases were then washed with brine (50 ml), dried over Na.sub.2SO.sub.4, filtered and evaporated to give the crude product as brown gum. The crude product was then purified by flash chromatography (eluent: cyclohexane/EA 2:1) to give N-[(1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]-4-(trifluoromethyl)-thiazole-5-carboxamide (4.9 g) as pale yellow solid (m.p. 122-124 C.).
[0174] .sup.1H-NMR (CDCl.sub.3, in ppm): 2.0-2.10 (m, 1H), 2.28-2.50 (m, 2H); 2.55-2.67 (m, 1H); 4.19-4.32 (dd, 1H); 4.91-5.05 (m, 1H); 5.88 (brs, 1H); 7.03-7.10 (m, 1H); 7.14-7.20 (dd, 1H); 7.37-7.40 (dd, 1H); 8.76 (s, 1H).
[0175] LC-MS: 379 [M+1], Rt=1.0 min.
Preparation of (1 S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutanamine
[0176] ##STR00016##
[0177] A three-neck bottom flask was charged with N-[(1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]acetamide (10 g, 41.38 mmol) and acetic acid (12.6 mL) at 20 C. Then aqueous HCl (37 mass %, 51.4 mL) was slowly added. The reaction mixture was heated up to 110 C. and stirred for 24 hours. The reaction mixture was cooled down to 10 C. and treated with aqueous NaOH (30 mass %, 114 mL) to adjust the ph to 10. To the reaction mixture was added dichloromethane and the organic layer is separated. The aqueous layer was then extracted with dichloromethane (2, 50 mL). The combined organic layers were washed with water (100 mL), separated, dried and evaporated to give (1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutanamine (6.2 g).
[0178] .sup.1H-NMR (CDCl.sub.3, in ppm): 0.99 (brs, 2H); 1.53-1.71 (m, 1H); 2.05-2.16 (m, 1H); 2.19-2.38 (m, 2H); 3.79-3.91 (m, 2H); 6.94 (dt, 1H); 7.06 (dd, 1H); 7.21 (t, 1H).
Preparation of N-[(1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]acetamide
[0179] ##STR00017##
[0180] A 500 mL autoclave (flashed with argon) was charged with N-[2-(2-chloro-4-fluoro-phenyl)cyclobuten-1-yl]acetamide 37.0 g, 151 mmol) under argon. A separated round bottom flask was charged with degassed methanol (303 mL) under argon. Under an inert atmosphere was then added (1R)-1-[Bis(1,1-dimethylethyl)phosphino]-2-[(1R)-1-[bis(2-methyl phenyl)phosphino]-ethyl]-ferrocene (0.95 g, 1.66 mmol), bis(1,5-cyclooctadiene)rhodium trifluoromethanesulfonate (0.71 g, 1.51 mmol). This mixture was stirred at room temperature for 30 mins to give a clear reddish solution, which was transferred to the autoclave. The autoclave filled with the reaction mixture was sealed and subjected to hydrogenation keeping a pressure of 10 bar at 50 C. for 19 hours. The reaction mixture was concentrated to give a brownish gum. The crude product was purified by flash chromatography (eluent: cyclohexane/EA; gradient 1:3 to 1:6) to give N-[(1S,2S)-2-(2-chloro-4-fluoro-phenyl)cyclobutyl]-acetamide (34.8 g) as resin.
[0181] .sup.1H-NMR (CDCl.sub.3, in ppm): 1.76 (s, 3H); 1.88-1.99 (m, 1H); 2.21-2.30 (m, 2H); 2.44-2.55 (m, 1H); 4.10-4.20 (q, 1H); 4.85-4.95 (m, 1H); 7.0-7.10 (dt, 1H); 7.15-7.20 (dd, 1H); 7.35-7.40 (dd, 1H).
Preparation of N-[2-(2-chloro-4-fluoro-phenyl)cyclobuten-1-yl]acetamide
[0182] ##STR00018##
[0183] A three-necked round bottom flask was charged with 2-(2-chloro-4-fluoro-phenyl)-2-cyclopropyl-acet-aldehyde 144.5 g, 727.5 mmol) and chlorobenzene (1820 mL). Aluminum trichloride (155.2 g, 1164 mmol) was added portion wise. The resulting solution was heated to 40 C. for two hours whereupon the reaction mixture turned brownish. The reaction mixture was allowed to standing overnight at room temperature. Acetonitrile (268.8 g, 342 mL, 6548 mmol) was added slowly and the temperature raised to 37 C. In the next step acetyl chloride was added at 40 C. Finally, the reaction mixture was heated to 50 C. and stirred for additional 1.5 hours. The reaction mixture was cooled down to room temperature and then poured into an emulsion of aqueous NaOH (3600 mL), toluene (360 mL); addition of ice (350 g) was necessary keeping the temperature at 5 to 0 C. The organic phase was separated and the aqueous phase was extracted with toluene (2, 200 mL). The combined organic phases were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product, which is an orange solid, was washed with ethyl acetate; filtration of the resulting precipitate gave N-[2-(2-chloro-4-fluoro-phenyl)cyclobuten-1-yl]acetamide (70.6 g) as white powder.
[0184] .sup.1H-NMR (CDCl.sub.3, in ppm): 1.95-2.15 (2s, 3H); 2.72 (t, 1H); 2.97 (t, 1H); 7.05-7.15 (dt, 1H); 7.18-7.28 (dd, 1H); 7.38-7.48 (dd, 1H); 8.95 (brs, 1H).
Preparation of 2-(2-chloro-4-fluoro-phenyl)-2-cyclopropyl-acet-aldehyde
[0185] ##STR00019##
[0186] A solution of 2-(2-chloro-4-fluoro-phenyl)-2-cyclopropyl-acetonitrile (31.0 g, 158 mmol, prepared analogous to WO2013064520, page 91) in toluene (79 mL) was cooled to 0 C. Bis(isobutyl)aluminum hydride (174.0 mL, 1.0 mol/L) was added drop wise while keeping the temperature at 10 to 0 C. During the addition the color of the reaction mixture became pink. After 1.5 hours the reaction mixture was carefully (strongly exothermic!) quenched with aqueous HCl (37 mass %) at 0 C. The reaction mixture was allowed to warm to room temperature and diluted with ethyl acetate and water. The layers were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4, filtered and evaporated to afford 2-(2-chloro-4-fluoro-phenyl)-2-cyclopropyl-acet-aldehyde (31.2 g) as pale yellow oil. The crude product was used directly without further purification.
[0187] .sup.1H-NMR (CDCl.sub.3, in ppm): 1.28-1.35 (m, 2H); 1.58-1.65 (m, 2H); 6.87-6.93 (dt, 1H); 7.06-7.14 (m, 1H); 7.14-7.21 (m, 1H); 9.0 (s, 1H).
[0188] The compounds listed below in Table 1 may be prepared in a similar manner as Example 5 or may be prepared according to methods as disclosed in Schemes 1-9 of WO2015/003951 and/or according to preparation methods disclosed in WO2013/143811.
TABLE-US-00002 TABLE 1 Example Name LogP MP ( C.) 1 rac-4-(trifluoromethyl)-N-[(1,2 cis)-2-[2- 123-132 (trifluoromethyl)phenyl]cyclobutyl]thiazole-5- carboxamide 2 rac-(4-(trifluoromethyl)-N-[(1,2 cis)-2-[4- 130-133 (trifluoromethyl)phenyl]cyclobutyl]thiazole-5- carboxamide 3 rac-N-[(1,2 cis)-2-[4- 94-97 (trifluoromethoxy)phenyl]cyclobutyl]-4- (trifluoromethyl)thiazole-5-carboxamide 4 N-[(1S,2S)-2-(4-chlorophenyl)cyclobutyl]-4- 3.21 103-108 (trifluoromethyl)thiazole-5-carboxamide 5 N-[(1S,2S)-2-(2-chloro-4-fluoro- 2.92 122-124 phenyl)cyclobutyl]-4-(trifluoromethyl)thiazole- 5-carboxamide 6 N-[(1S,2S)-2-(2,4-difluorophenyl)cyclobutyl]- 2.71 4-(trifluoromethyl)thiazole-5-carboxamide 7 N-[(1S,2S)-2-(2,4-dichlorophenyl)cyclobutyl]- 3.44 120-124 4-(trifluoromethyl)thiazole-5-carboxamide 8 4-(difluoromethyl)-N-[(1S,2S)-2-(2,4- 2.40 difluorophenyl)cyclobutyl]thiazole-5- carboxamide 9 N-[(1S,2S)-2-(2,4-dichlorophenyl)cyclobutyl]- 3.25 4-(difluoromethyl)thiazole-5-carboxamide
BIOLOGICAL EXAMPLES
Meloidogyne Spp. (Root-Knot Nematode)
Nematicide, Contact Activity, Preventive.
[0189] Filter papers (9 cm4.5 cm) with a small pocket were placed into plastic pouches (12 cm6 cm). One cucumber cv. Toshka seed was placed in the centre of the filter paper pocket of all the pouches needed for a test. The cucumber seeds in the pouches were treated with test solutions at 200 ppm by pipetting the solution directly over the cucumber seed in the filter paper pocket in the pouch. Prior to application, the compound solution was prepared at twice the concentration required and the egg suspension is prepared with FORL nutrient solution with 3000 eggs/0.5 ml. After applying all the treatments, 3000 eggs (in 0.5 ml of FORL nutrient solution) were pipetted into the pouches. The pouches were incubated in a moist chamber for twelve days and watered regularly to maintain good filter paper moisture essential for the growing cucumber root system. After this period, the filter paper containing the germinated cucumber seedling was removed from the plastic pouch to assess the number of galls caused by Meloidogyne spp. per root system. Phytotoxicity was measured as a reduction of growth of the emerged cucumber seedling in comparison to the control.
[0190] The following compounds showed a greater than 80% reduction of galling compared to the untreated control:
1,3,4, 5, 6, 7, 8, 9.
[0191] Heterodera schachtii (Sugar beet cyst nematode), Nematicide, contact activity The tested application rate of each compound was 200 ppm. All solutions were brought to a concentration of 400 ppm, respectively, as they were subsequently diluted by adding the equivalent amount of water containing juvenile nematodes. After preparation of the suspensions, 1 ml of each suspension and concentration was transferred to 16-well assay plates with a total of three replicates per treatment. Approximately 500 juveniles of Heterodera schachtii were added in 1 ml of water to each well. Nematodes in water served as controls. The plates were placed in a dark box and stored at room temperature. Nematode paralysis was determined after 24 hours incubation at 25 C. in darkness. Nematodes that showed no movement were considered immotile.
[0192] The following compounds showed a greater than 75% nematode immobilization compared to the untreated control:
3, 4, 5, 6, 7, 8, 9.
Meloidogyne Spp. (Root-Knot Nematode)
Nematicide, Contact Activity, Preventive
[0193] Cucumber cv. Toshka seeds were sown directly into pots filled with a sandy substrate. Six days later pots were each treated with 5 ml of a WP10 suspension of the test compound. Hereafter, pots were inoculated with 3000 eggs of M. incognita. The trial was harvested fourteen days after trial application and inoculation. Root galling was assessed according to Zeck's gall index (Zeck W. M. (1971) Ein Bonitierungsschema zur Feldauswertung von Wurzelgallenbefall. Pflanzenschutznachrichten Bayer 24, 1: 144-147). Phytotoxicity was measured as a reduction of growth of the emerged cucumber seedling in comparison to the control.
[0194] The following compounds showed a greater than 80% reduction of galling compared to the untreated control:
1,2,3,4, 5, 6, 7, 8, 9.
Meloidogyne Spp. (Root-Knot Nematode)
Nematicide, Contact Activity, Preventive
[0195] Coated tomato cv. Roter Gnom seeds were sown 0.5 to 1 cm deep in 45 ml pots filled with field soil. Then pots were infested with nematodes by pipetting 2000 eggs of Meloidogyne spp. within a 2 ml suspension on top of the seed. The seed hole was filled with soil hereafter. Assessment of phytotoxicity (in %) and root galling occurred 28 days after inoculation. The roots were washed free of soil debris and the gall index was assessed according to Zeck 1971 on a scale from 0 to 7.
[0196] Seed treatment rate: 1 mg Al/seed
[0197] The following compounds showed a greater than 80% reduction of galling compared to the untreated control:
4, 5, 6, 8, 9.
Pratylenchus zeae (Corn Lesion Nematode)
Nematicide, Contact Activity, Preventive
[0198] Coated corn cv. LG4620 seeds were sown 1 cm deep into 45 ml pots with soil (7:3 w/wa mixture of 70% field soil and 30% quartz Sand). Two days after sowing the pots were infested with 1500 nematodes (all stages) of Pratylenchus zeae within a 2 ml suspension in two holes to the left and right of the seed hole. Assessment of phytotoxicity (in %) and nematode numbers within the root system occurred 7 days after inoculation. The upper plant part was cut off and the roots were washed free of soil debris. Nematodes within the roots were stained with acid fuchsin stain solution. Nematodes within the roots were quantified under a dissecting scope at 40.
[0199] Seed treatment rate: 1 mg Al/seed
[0200] The following compounds showed a greater than 80% reduction of nematode population compared to the untreated control:
5.
Heterodera schachtii (Sugar Beet Cyst Nematode)
Nematicide, Contact Activity, Preventive
[0201] Coated sugar beat cv. Impulse seeds were planted in 45 ml pots filled with field soil. Seven days after sowing pots were infested with 500 J2 of Heterodera schachtii within a 2 ml suspension in two holes to the left and right of the seedling. Assessment of nematode numbers per g of root occurred 10 days after inoculation. The upper plant part was cut off and the roots were washed free of soil debris. Nematodes within the roots were stained with acid fuchsin stain solution. Nematodes within the roots were quantified under a dissecting scope at 40.
[0202] Seed treatment rate: 0.6 mg Al/seed
[0203] The following compounds showed a greater than 80% reduction of nematode population compared to the untreated control:
5, 6, 8.
Biological Comparison Data:
[0204] Compound C is disclosed in WO2015/003951 as an example on page 125 (Table 61, entry 61.93). The activities of compound C to treat Meloidogyne spp. (root-knot nematode) on cucumber is compared with the activities of example 7 and example 9 (see Table 1) according to the present invention. The tests are carried out at different concentrations (ppm). It can be seen that example 7 and 9 according to the present invention have surprisingly improved activity in comparison with compound C.
a) Nematicidal Activity Against Meloidogyne Spp. (Root-Knot Nematode):
Contact Activity, Preventive.
[0205] Filter papers (9 cm4.5 cm) with a small pocket were placed into plastic pouches (12 cm6 cm). One cucumber cv. Toshka seed was placed in the centre of the filter paper pocket of all the pouches needed for a test. The cucumber seeds in the pouches were treated with test solutions at 200 ppm by pipetting the solution directly over the cucumber seed in the filter paper pocket in the pouch. Prior to application, the compound solution was prepared at twice the concentration required and the egg suspension is prepared with FORL nutrient solution with 3000 eggs/0.5 ml. After applying all the treatments, 3000 eggs (in 0.5 ml of FORL nutrient solution) were pipetted into the pouches. The pouches were incubated in a moist chamber for twelve days and watered regularly to maintain good filter paper moisture essential for the growing cucumber root system. After this period, the filter paper containing the germinated cucumber seedling was removed from the plastic pouch to assess the number of galls caused by Meloidogyne spp. per root system.
TABLE-US-00003 Concentration Compound Compound structure (ppm) Mortality (%) Example 7
b) Nematicidal Activity Against Meloidogyne Spp. (Root-Knot Nematode):
Contact Activity, Preventive
[0206] Cucumber cv. Toshka seeds were sown directly into pots filled with a sandy substrate. Six days later pots were each treated with 5 ml of a WP10 suspension of the test compound. Hereafter, pots were inoculated with 3000 eggs of M. incognita. The trial was harvested fourteen days after trial application and inoculation. Root galling was assessed according to Zeck's gall index (Zeck W. M. (1971) Ein Bonitierungsschema zur Feldauswertung von Wurzelgallenbefall. Pflanzenschutznachrichten Bayer 24, 1: 144-147). Phytotoxicity was measured as a reduction of growth of the emerged cucumber seedling in comparison to the control.
TABLE-US-00004 Concentration Compound Compound structure (ppm) Mortality (%) Example 7