Laboratory tests with organic compounds as larvicides for Culex quinquefasciatus say

UL^-ARY
TATE PLANT BOARD

January 1943 U. S.- E-585
n EPPARTMENT (
If TT
AGRICULTURE \
E"PEAU OF
SENTOMOLOGY A"D /
rLANT QUARANTINE


LABORATORY TESTS WITH ORGANIC COMP)UNrS [S
LARVICIDES FOR CUI Y OUQ "' TT'T.' SAY

By R. C. Bushland and W. V. King,
Division of Insects Affecting Man and Animals



One phase of the investigation on new chemicals for mosquito larvi-
cides consists in selecting toxic materials for field tests. During the
course of this study data have been accumulated on a variety of organic
compounds, and the results obtained with 114 of these materials are summa-
rized here. Although none of these compounds can be recomrmenided at present
for use in mosquito control, the data may be of interest to other workers on
organic insecticides.

The test insect was the southern house mosquito (Culex quinquefasciatus
Say). Eggs were obtained from oviposition tubs exposed on the laboratory
grounds at Orlando, Fla., and the larvae were reared indoors in battery jars.
For food they were given one of the proprietary dog biscuits, finely ground,
as employed by Crowell (3) in rearing Anopheles larvae.

The organic compounds used in these studies were either commercial
products of high purity or materials synthesized by chemists of the Division
of Insecticide Investigations. The latter were given "E" numbers. Patents
have been issued on some of these compounds. To disperse the test materials
in distilled water, the compounds were introduced in acetone solution. The
feN materials insoluble in acetone were finely ground and used as suspen-
sions. A maximum concentration of 2.5 ml. of acetone in 2.0 ml. of water
was used, since this concentration, as reported by Fink and coworkers (4)
and confirmed by the present writers, was har..less to the test larvae.

The testing procedure was a modification of methods described by Camp-
bell, Sullivan, and Smith (2); Fink, Smith, Vivian, and Claborn (4); and
Phillips and S.'ingle (5). When most of the larvae in a culture had reached
the fourth instar (usually 6 days after hatching), they w-ere collected in a
wire strainer and gently washed with distilled water. Larvae of uniform
size were then placed in groups of 50 in beakers containing 25 ml. of water.
A beaker of larvae was selected at r3ndo.:n, and the contents %.ere poured into
a larger beaker of distilled water, containing the material dispersed in
225 ml. Thus, a test consisted of 50 fourth instars in 250 ml. of distilled
water containing the desired quantity of test material.





- 2-


The test beakers were immersed in a constant-temrperature water bath
at 78 F., and after approximately 16 hours the first mortality readings
were taken. Those beakers in which larvae survived were replaced in the
water bath, and 24 hours later final readings were taken.

A large proportion of larvae killed by most organic insecticides sink
to the bottom of a test beaker, where they are easily counted, but sometimes
the dead or dying larvae float at the surface and may be difficult to
distinguish from those not fatally poisoned. Before mortality readings
were taken, the beakers were gently stirred to cause as many of the larvae
as possible to sink. Larvae at the bottom of the beaker and incapable of
rising to the surface were counted as dead. Sick larvae at the water surface
which were so helpless that they could not wriggle off when lifted on the
tip of a bent teasing needle were also considered as dead. Thus only larvae
capable of vigorous movement were counted as alive. With certain compounds
a few of the moribund larvae recovered during the second day.

For each culture of larvae used in these tests an untreated check
beaker was prepared. In the tests reported herein rarely was a dead larva
found in a check beaker. Such mortality could be attributed to injury from
washing or counting. Fourth instars can spend 2 days in distilled water
without food with negligible natural mortality. Each culture was further
tested against a median lethal concentration of a compound of established
toxicity, either phenothiazine or 4-(p-bromophenylazo)-m-cresol.

The highest concentration used routinely in these studies was 100 parts
per million (p.p.m.). Materials lethal at this concentration were retested
at successively lower concentrations until approximately the median lethal
concentration was attained. With materials of outstanding toxicity several
toxic concentrations were tested to obtain indications as to the slope of
the concentration-mortality curve. All materials were tested at least twice
to arrive at a concentration-mortality figure. For two or more tests at
a.1j concentration larvae from different cultures were used.

The data on 114 compounds are summarized in tables 1 to 5. The most
toxic compounds (table 1) are arranged in the approximate order of toxicity
(based on the 40-hour readings), while the others (tables 2 to 5) are listed
alphabetically, since the exact order of toxicity within tables could not
be established in a few tests. As the primary object of this study was to
select very toxic compounds, it was not considered practical to make direct
comparisons of the less toxic materials. Because of variation in resistance
between lots of larvae, a compound causing high mortality in tests against
twvo lots was not necessarily more toxic than another producing a considerably
lower kill at the same concentration against two different lots of larvae.

Unless otherwise indicated, all the compounds were soluble in water
at the concentrations used in the tests.






-3-


Phenothiazine, which was employed as a standard of comparison, was
reported by Fink and coworkers (4) to have caused 100-percent mortality
of Culex larvae at 1 p.p.m. In the tests carried out by the present
authors a culture was seldom so susceptible that all larvae were killed
at this concentration. In a typical series of tests against 56 lots of
100 larvae (in duplicate beakers of 50) each, phenothiazine c-uced a
mean mortality of 70.5 + 2.32 percent in 16 hours and 69.1 2.03 percent
in 40 hours, some of the larvae previously recorded as dead having re-
covered. Only 1 lot sho.'ed 100-percent mortality, but the least kill
was 40 percent. In these tests the lo..e=t concentration givir.g 100-
percent kill (minimum lethal concentration) averaged about 2 p.p.m.
while the median lethal concentration was about 0.75 p.p.m. Against
another series of 51 lots, 0.75 p.p.m. caused .Tean mortalities of 52.4
2.95 and 55.4 + 2.93 pere!-.t in 16 and 4j hours, respectively.

p-Bro.ohydrazobenzene was found to be more toxic than phenothiazine.
The medium lethal concentration for Culex quinu ifas-iatus la.r:.'. with
.this azo co:.pja.,d appears to be slightly moro than 1.0 p.p.m., and the
median lethal concentration bet.veen 0.5 and 0.75 p.p.m.

The 11 compounds of outstanding toxicity listed in table 1 represent
several classes in the aromatic series. Since this report deals with
large variety of compounds with but few representatives of any group,
little can be concluded regarding chemical constitution and toxicity.

In tests on screwworms (Cochliomyvia americana C.&P.) Bushland (1)
found quinoline, isoquinoline, 6-nitroquinoline, and 8-nitroquinoline to
be approximately as toxic as phenothiazine, 2,6-dimethylquinoline and
6-riethylquinoline to be somewhat less toxic, and 7-methylquinoline and
8-methylquinoline less effective than the 6-rrmethyl derivative but more
toxic than 8-hydroxyquinoline. These nine compounds were tested against
Culex larvae, and again all showed larvicidal quantities, but the order
of toxicity was very different. As mosquito larvicides 8-methylquino-
line ranked first and 7-methylquinoline second, both being outstanding-
ly toxic with median lethal concentrations approximately 1 and 2 p.p.m.,
respectively; 8-hydroxyquinoline was third, 100 p.p.m. killing all the
larvae. The remaining six compounds caused only partial kills of mos-
quito larvae at 100 p.p.m.

The results with quinoline derivatives against these two species of
dipterous larvae further illustrate the specificity of toxic action of
organic insecticides and add to the instances of unpredictable changes
in toxicity brought about by slight changes in the composition of the
molecule or in the position of substituents.

Among the materials of little toxicity the results with phenol and
the three cresols deserve notice, since these substances have been used
as the chief ingredients of mosquito larvicides. In additi:;, to the
tests reported in table 5, mixtures of the cresols were tested in both
tap and distilled water and found to be no more effective than the pure
isomers. In vie-., of these results the value of these substances in
mosquito larvicides is questionable.






-4-


Summary.--The results are given on 114 organic compounds that were
evaluated for toxicity to fourth instars of CuleX gquinguefasciatus Say.
The tests were conducted in beakers containing 50 laboratory-reared
larvae in 250 ml. of distilled water and the desired quantity of test
material. Test beakers were held at 78 F. and mortality readings were
made at intervals of approximately 16 and 40 hours. Materials found
lethal at a maximum concentration of 100 p.p.m. were tested at successively
lower concentrations until part of the larvae survived. Minimum lethal
and sublethal concentrations were retested against different cultures
of larvae.

Materials were considered to be of outstanding toxicity if they
caused 100-percent mortality at concentrations of 10 p.p.m. or less.
Eleven materials in this category were toxic in the following order:
p-bromohydrazobenzene, xanthene, p-thiocyanoiodobenzene, dibenzothio-
phene, p-thiocyanobromobenzene, 8-methylquinoline, 4-chloro-2-nitrodi-
phenylamine, dimethylacridan, thio-2-naphthyl methyl ether, 7-methylquino-
line, p-nitrobenzyl bromide. p-Bromohydrazobenzene was more toxic than
phenothiazine in these laboratory tests.

The order of toxicity to Culex larvae for quinoline and eight of its
derivatives was different from that reported for these substances against
screwworms.

Phenol and the three cresols failed to kill all larvae in 40 hours
at 100 p.p.m.

LITERATURE CITED

(1) Bushland, R. C.
1940. The toxicity of some organic compounds to young screw-
worms. Jour. Econ. Ent. 33: 669-676.

(2) Campbell, F. L., Sullivan, W. N., and Smith, C. R.
1933. The relative toxicity of nicotine, anabasine, methyl
anabasine, and lupinine for culicine mosquito larvae.
Jour. Econ. Ent. 26: 500-509, illus.

(3) Crowell, R. L.
1940. Insectary rearing of Anopheles quadrimaculatus.
(A preliminary report.) Amer. Jour. Hyg. 32, Sec. C:
12-20.

(4) Fink, D. E., Smith, L. E., Vivian, D. L., and Claborn, H. V.
1938. Toxicity tests with synthetic organic compounds against
culicine mosquito larvae. U. S. Bur. Ent. and Plant
Quar. E-425, 34 pp. [Processed.]

(5) Phillips, A. M., and Swingle, M. C.
1940. Rearing of mosquito larvae and effect of diet on their
resistance to rotenone and nicotine. Jour. Econ. Ent.
33: 172-176.






- 5 -


Table 1.-Compounds of outstanding toxicity (lethal at 10 p.p.m. or less)

I :Concen-: : Average mortality
Compound : No. :tration:Tests: After : After
: : : 16 hours : 40 hours


P.p.m. No. Percent


Percent


p-Bromohydrazobenzene
BrC 6H1NMHC6Hj


Xanthene
C6FI4CH2CgHOi



p-Thiocyanoiodobenzene
IC6HhSCN




Dibenzothiophene
C6H4SC6H4




p-Thiocyanobromobenzene
BrC6HR4SCN


8-Methylquinoline
CH3C9H6N






4-Chloro-2-nitrodiphenylamine
cO6HSCH (ClN)02


1207 1.25
1.0
.75
.5

1327 3.0
2.0
1.5
1.0

1396 2.5
2.0
1.5
1.0
.5

130 2.5
2.0
1.5
1.0
.5


1397


2.5
2.0
1.5
1.0
.5


Comm. 5.0
4.0
3.0
2.5
2.0
1.5
1.0
.5

ID .399 4.0
3.0
2.0
1.0


LIBRARY
STATE PLANT BOARD


99.5
93
88
25

98
92
77
50

100
98
86
46
6

100
99
85
49
9

100
97
68
29
7


100
99
92
36

100
98
92
so80


98
88
53
32


97
81
41
12


96
68
45
34


100
100
97
91
53
60
22

100
97
69
52


100
97
96
86
80
59
35
11





- 6 -


Table 1.--(Continued)


: 2 :Concen-: a Average mortality
Compound : No. :tration:Tests: After : After
: : :l6 hours: 40 hours

P.p.m. No. Percent Percent

Dimethylacridan 178 5.0 3 95 97
C C(CH )2C H 4.0 3 90 95
-6 4 3.0 3 76 79
2.0 3 59 66
1.0 3 15 31

Thlo-2-naphthyl methyl ether 1132 9.0 4 99 97
CH107 SCH3 4.0 2 92 87
3.0 2 15 13
2.0 1 0 0

7-Methylqulnoline Comm. 5.0 4 94 914
C3C9H6N 14.0 3 88 91
3.0 3 67 83
2.0 3 47 61
1.0 3 10 33
p-Nitrobenzyl bromide Comm. 5.0 4 92 96
NO2C6H4CH2Br 4.0 4 56 70
3.0 4 27 42
2.0 2 4 7






-7-


Table 2.--Compounds that killed all larvae at 20 p.p.m. but not at 10 p.p.m.

: E :Concen-: : Average mortality
Compound : No. :tration:Tests: After : After
: : 16 hours : 40 hours

P.p.m. No. Percent Percent

p-Aminohiphenyl 606 10 2 21 55
NH2C6 H4C6H5

2,5-Dibromo-o-cresol Comm. 10 3 39 71
Br 2C6H 2(CH3)OH 5 3 0 6

N-Phenylanthranilic acid Comm. 10 2 22 24
C 6H 5NHC 6H COOH
65 614
Xanthone Comm. 10 2 34 63
C6HWC(0)C6HO 5 2 0 12
6H4C 0) C040








Table 3.--Compound8 that killed all larvae at 50 p.p.m. but not at 20 p.p.m.

: E :Concen-: : Average mortality
Compound : No. :tration;:Tests: After : After
:_ l :16 hours; : 40 hours


P.p.m. No. Percent


Percent


o-Aminob iphenyl
NH C M17HO
2 64"6"5


o-Bromophenetole
BrC 6H4OC2H5

p-Bromophenetole
BrCA6HOCE5

3-Chloroac enaph then
C10H5(CH2)2C1



3,4-Dichloronitrobenzene
CI2C6H3N02

2,6-Dichloro-4-nitrophenol
C12C62(N02)OH


2,4-Dinitrobromobenzene
(NO2)2C6H3Br


2,4-Dinitrochlorobenzene
(N02)2C6H3Cl

2,4-Dinitrophenol acetate
CH 3C02C6H3(N02)2

Hexachlorethane
CCl 3CCl3


p-Nitrophenetole
N02C6H0C05

m-Nitrophenyliodochloride
(N02)C6H41C12

Phenazine oxide
CGH01NONC6H4


* Comm.


Comm.


Comm.


1176



Comm.


130o4


Comm.



Comm.


1537


20 2


20 3


20
10

20
10
5
2.5

20
10

20
10
5

20
10
5
20
10

20
10


Comm. 1/20
10
5


Comm.


1374


1538


20 2


2j Formed a colloidal precipitate which disappeared after a few hours.





-9-


Table 4.--Compounds that killed all larvae at 100 p.p.m. but not at 50 p.p.m.

: E :Concen-: : Average mortality"
Compound : No. ;tration:Tests; After ; After
:*I :: 16 hours : 40 hours


Bis-(l-aminophenyl) disulfide
(~2CH 002S2

o-Bromoanisole
BrC6H-OCH3

p-Bromoanisole
BrC6H1OCH3

2.4-Dimethylquinoline
(OH3)2C9 5H

o-Dlchlorobenzene
OCl2C6H

p-Dichlorobenzene
C2CO6H

alpha-Ethyl butyraldehyde
semicarbazone
(C2H5)2CHCHM CON2

8-Hydroxylquinoline
HOCgH6N


P.p.m. No. Percent

345 /5o 3 97
20 2 4


Comm.


Comm.

Comm.


Comm.


Comm.


1523


Comm. 1/50


Me thylphenylni tro soamine
C6H(cH 3 )iNO

o-Nitroanisole
NO2C6hOCH3

p-Nitroanisole
N0O2C6H10CH3

p-Nitrobenzonitrile
No2C6HOON

c-Nitrophenetole
NO2C64002oH

1,3,5-Trinitrobenzene
(iNo2)3C6H3


Comm.

Co.n.


Comm.
COO-B.


Comm.

Conmm.


Comm. 50


1/ Insoluble in water at this
_/ Tests in tap water.


concentration


Percent





- 10 -


Table 5.--Compounds that were not completely effective at 100 p.p.m.
(nontoxic and slightly toxic materials)
: E ;Concen-: : Average mortality
Compound : No. :tration:Tests: After : After
_:_:_: 16 hours: 40 hours


Acetonyl acetone disemicarbazone
(CH3 C(NHCONH2) 2CH2) 2

Anisalacetone
CH 3OCH4CHCHCOCH3

Anisaldehyde
CHo0C6H4CHO

Anisole
C6H5OCH3

Benzaldehyde semicarbazone
C 6H CHNNHCONH2

N-Ben zylpyromuc amide
(C4H30 )CONHCH2C605

Carvacrylpropionamide
C2H5COiNHC6H3(CH3 )(c3H7)

Cinchonine
C19H2220

Cinnamaldehyde semicarbazone
C6H5CHCHCHMHCONH2

o-Cresol
CH3C6 H40H

m-Cresol
CH3C oH40H


p-Cresol
CHR3C6H40H


P.p.m. No. Percent

1522 1/1ioo00 2 0

Comm. 100 2 64


Comm. 100 2 59


Comm. 100

1462 1/100oo


1335 100


1469


100


Comm. 1/100

1463 1/100

Comm. 200
100
50
Comm. 200
100
50
Comm. 200
100
50


100
79
15
100
51
9
100
37
9


Percent






- 11 -


Table 5.--(Continued)


: 9 :Concen-
: No. :tration


Compound


: : Average mortality
.:Tests: After : After
S : 16 hours 40 hours


P.p.m. No. Percent


Percent


Crotonaldehyde semicarbazone
CH CHCHCHNNHCONH

N-Cyclohexylpyromucamide
(C4H,30)CONXC6HII

Di-n-butylamine
(C 4R9)2NH

2,6-Dimethyl-4-heptanone
semicarbazone
((CH3 )2 CHCH2)2 CNNHCONH2

2,6-Dimethylquinoline
(CH3 )2C9H

2,4-Dinitroanisole
CH3OC6H3 (NO2)2

1,4-Diphenyleemicarbazlde
C 6H5NNMCONHC6H5

Di-henyl sulfoxide
(C6H5)2SO

Di-o-tolylthlourea
CS(NHC6H4CH3)2

2-Turanacrylamide
(C4H30)CH:CHCONH2

2-(D-Gluco-D-glucoheptohexa-
hydroxyhexyl) benzimidazole
CH2OH(CHOH) 5CNC 6HNH

l-Hydroxy-2-acetonaphthone
C oH6(OH)COCH3


p-Hydroxyacetophenone semi-
carbazone
HOC 61H1C(NNHCONH2 )CH3


150o4 100oo


1402 100


Co=m. 100


1505 100



Comm. 100
50

1535 100
50

1106 V100


Comm. 100
50

Comm. V100
V 50


1479


1423


100


100


1319 1V100
50
2120


1461


100






- 12 -


Table 5.--(Continued)


Compound


: No.


:Concen-:
:tration;Teetes:
* 0


Average mortality
After : After
16 hours: 40 hours


P.p.m. No. Percent


Indoleacetamide
C-mmNHCHCNHCOCH
'D" at 3

Indoleacetic acid
C6HNHCHCCH COOH

Indolebutyric acid
C eh1NHCHCC.HCCOOH

Iodoeobenzene
C6H5 I0

p-Iodosonitrobenzene
C6H4(N02)IO

Isoquinoline
CF6HCHNCHCH
LV -- i
p-Methylacetophenone semicarbazone
CH3C6H0C(CH3 )NNHCONH2

Methylanthranilic acid
CH3NHOC6H COOH

Methylindole butyrate
CI NHCHCC H COOCH
.64-_ 36 3
6-Methylquinoline
CH3C9H6N

3-Nitroacenaphthene
C10H5(CH2)202

p-Nitroacetanilide
CH 3CONHC6Ho02

p-Nitrolodoeobenzene acetate
(N02)C6H4l:(OCOCH3)2


comm.


Comm.


Comm.


163


100


100


100


100
50


1356 1/100


Comm.


1507


Comm.


Comm.


Comm.


100
50

100


100


100


100


1177 /lOO


1412


100


1424 -1/100
/ 50


Percent






- 13 -


Table 5.--(Continued)


I3 :Concen-: : Average mortality
Compound : No. :tration:Tests: After : After
: :,,,............ .l: 16 hours: 40 hours


p-Nitrophenyliodochloride
(NO2)COHIC12

p-Nitrophenylmercaptan
NO2C6H4SH

4-Nitrophthalimide
NO2C6H3(CO)2NH

6-Ni troquinoline
NO 2 C9 He

8-Nitroquinoline
NO2C9H6N

N-Ni tro sodicyclohexylamine
(c6H1) oNO


P. .m. No. Percent

1359 1/100 2 7


Comm. 1/100oo 2 46


1206


COmm.


Comm.


Percent


100 2


100
50
100
50


396 100oo


m-Phenetidine
C2H5OC6H24N2

p-Phenetidine
C2H5OC6H1N2

Phenetole
C 2H 5OC 6H5

Phenol
c6H5OH
6 5


- 1-Phenyleemicarbazide
()6H ?MBCONH1p
Phthalonitrile
C604(C1)2

Picric acid
(NO2)3C6H20H


1311


1342


Comm.


100 2


100
50


100 2


Comm.

Comm.


Comm.


Comm.


100

100


100


300
200
100


100
94
22


99.5
18

21






- 14 -


Table 5.--(Contiinued)


I iConcen-Z : Average mortality
Compound : No. :tration:Testo: After : After
1: : : 16 hours: 40 hours


P.p.m. No. Percent


Percent


Piperi dine-piperidyl
dithiocarbamate
(C H5 E10)C(S)SH(C5HN)

p-Propi onotoluide
C2H COHC6H.4CR3

Qninoline
CH1NCHCHCH

2,4,2', 214 '-Tetrabromodiphenylamine
Br2C6HR3HC 6H3Br2


1421


1263


Com.


100 2


100 2


100
50


1422 1/100


p-Tolyllactamide
CECHOHCOHC6BtCH
OR3 COCNHC6H14CH3
N-Xenylacetamide
CHR COMeCOH

N-Xenylbenzamide
C6 HCONHC6H4C6H5

N-Xenylformamide
HCONHC6H4C665

N-Xenylpropionamide
C2H5CONHC6H4C6H5

N-Xenylpyromucamide
(C4H30)CONHiO6H4C6H5

N-(2.4-Xylyl)benzamide
C6HCONHC6H3(CH3)2
N-(2,6-Xylyl)benzamide
C6 H CONHC6H3(CH3)2

N-(2.4-Xylyl)formamide
HCONHC6H3 (CH3)2


1431


100oo 3


1465 /100

1467 1/100

1464 1/100
1/ 50

1466 1/100

1465 1/100

1436 1/100
1/ 50

14358 1/100


1434


100oo 3





- 15 -


Table 5.--(Continued)
......:I :Concen-: : Average mortality
Compound. : No. :tration:Tests: After : After
: : : 16 hours: 40 hours
P.p.m. No. Percent Percent
N-(2.5-Xylyl)formamide 1435 100 3 0 1
HCONHC6H3 (CH3 )2
N-(2,4-Xylyl)Dropionamide 1439 100 3 0 0
C 2HCCONiHC6H3 (CH3 )2

N-(2,5-Xylyl)propionamide 14o0 100 3 0 0
C 2H 5CONHC 6H3 (CH3 ) 2
2 5 63 32
N-(2,6-Xylyl)oropionamide I4iI 100 2 0 0
C 2H5CONC6H3 (OCH3 )2

N-(2,4-Xylyl)pyro.mucamide 1442 100 2 3 15
(C4H30 )CONHC 6H3 (CH3 ) 2

N-(2,5-Xylyl)pyromucamide 1444 100 2 25 31
(C0H30)CONHC 6H3 (COH3 )2

N-(2,6-Xyyl)pyromucamide 1443 100 2 0 0
(C4H30 )CONHC6H3 (CH3) 2


I/ Insoluble in water at this concentration.




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