INVESTIGATIONS IN FISH CONTROL
100. Observations on the Effects of Irrigation Water Containing 3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants
101. Residues of Malachite Green in Muscle, Eggs, and Fry of Treated Atlantic Salmon and Chinook Salmon
102. Effects of Water Temperature, Hardness, and pH on the Toxicity of Benzocaine to Eleven Freshwater Fishes
OCH 3
CH 2
UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE
Investigations in Fish Control, published by the Fish and Wildlife Service, include reports on the results of work at the National Fisheries Research Center at La Crosse, Wisconsin, and reports of other studies related to fish control and acute toxicity of piscicides. Though each report is regarded as a separate publication, several may be issued under a single cover, for economy. See inside back cover for list of current issues.
Copies of this publication may be obtained from the Publications Unit, U.S. Fish and Wildlife Service, 1849 C Street, N.W, Mail Stop 130 ARLSQ, Washington, DC 20240, or may be purchased from the National Technical Information Service (NTIS), 5285 Port Royal Road, Springfield, VA 22161.
ISSN 0565-0704
INVESTIGATIONS IN FISH CONTROL
100. Observations on the Effects of Irrigation Water Containing 3-Trifluoromethyl-4-Nitrophenol (TFM) on Plants
By Philip A. Gilderhus
101. Residues of Malachite Green in Muscle, Eggs, and Fry of Treated Atlantic Salmon and Chinook Salmon
By J. L. Alien
102. Effects of Water Temperature, Hardness, and pH on the Toxicity of Benzocaine to Eleven Freshwater Fishes
By Terry D. Bills, George E. Howe, and Leif L. Marking
U.S. Fish and Wildlife Service National Fisheries Research Center La Crosse
P.O. Box 818 La Crosse, Wisconsin 54602
1990
Observations on the Effects of Irrigation Water Containing 3-Trifluoromethyl-4-Nitrophenol (TFM)
on Plants
by
Philip A. Gilderhus
U.S. Fish and Wildlife Service National Fisheries Research Center La Crosse
P.O. Box 818 La Crosse, Wisconsin 54602
ABSTRACT. Because concerns have been expressed about the effects of irrigating truck-garden crops with water from a stream treated with the lampricide 3-trifluoromethyl-4-nitrophenol (TFM), I conducted studies on the effects of TFM on young plants of common vegetables and fruits. Plants established in horticultural flats were irrigated for 12 h with water containing 10 mg/L of TFM and later compared with plants irrigated for a similar period with untreated water. Lettuce, radish, sweet corn, and potato plants were virtually unaffected. Green bean and tomato plants developed brown or dead spots on many leaves but growth rates and survival were not affected. Cucumber and cantaloupe plants were severely damaged; some were killed and about 40% of the leaves on surviving plants were dead or dying. Two weeks after treatment, the mean weight of surviving treated cantaloupe plants was significantly less than that of control plants.
The parasitic sea lamprey (Petromyzon marinus) is con trolled in the Great Lakes by treating more than 400 nurs ery streams with the lampricide 3-trifluoromethyl-4-nitro- phenol (TFM) to kill the larvae. Some of these tributaries flow through agricultural land, where the stream water is used to irrigate truck-garden crops. Farmers have been reluctant to forego irrigation while a stream is being treated, without evidence that TFM would damage their crop. Lack of irrigation for even 1 day reportedly can affect crop growth, delay marketing, and prevent a farmer from getting full market price for a product. To date, sea lamprey control agencies have had no specific informa tion to give to farmers about potential damage to their crops from using TFM-treated irrigation water.
It is known that TFM has adverse effects on aquatic plants ranging from reduced production after exposure to 10 mg/L for 1 h to plants becoming limp and cyanotic after exposure to 20 mg/L for 3 h (National Research Council of Canada 1985). No studies of the effects of TFM on terres trial plants have been published. In response to requests from control agents for information on how TFM affects terrestrial plants, I conducted studies at the National Fish eries Research Center, La Crosse, Wisconsin, to determine
if irrigation with water containing TFM is deleterious to selected truck-garden plants.
Methods and Materials
Plants of eight common vegetables and fruits were raised in horticultural flats filled with sandy loam soil. Lettuce, radish, green bean, cucumber, and cantaloupe plants were started from seed in flats 53 X 27 cm and 6 cm deep. Corn and potatoes, which need deeper soil, were planted in flats 36 X 33 cm and 15 cm deep. Seedling tomato plants were transplanted from a garden plot into flats that were divided into sections 8 cm square and 6 cm deep. The plants were allowed to grow until they were well established (25 to 35 days after planting). Their sizes and the numbers used are listed in the Table.
The studies were conducted in a series of vertical- walled 0.005-ha concrete ponds (Figure). Two ponds held treatment water and the plants were kept in two unfilled ponds. For treatment, the plants were placed on racks with an oscillating sprinkler between the racks at the same height. A submersible pump transferred fresh water or
1
INVESTIGATIOS IN FISH CONTROL 100
Table. Size and number of plants used and effects of irrigating them with water containing TFM.
Plant
LettuceRadishGreen beanSweet cornPotatoTomatoCucumberCantaloupe
"At time of treatment.bSee text for details.
Height ofplants (cm)a
8-108-108-10 '
25-3025-3015-1813-1813-18
Plantsper pan
15151516469
15
Pans pertreatment
33233332
Damage fromTFM exposureb
NoneNoneLeaf spottingNoneNoneLeaf spottingSevereSevere
water containing TFM (field grade; 39.9% active ingre dient) from the sump of the reservoir pond to the sprinkler (Figure). The spray crossed each rack twice per minute. Treatments were designed to simulate the maximum expo sure likely to occur; the plants were sprayed with water containing 10 mg/L of TFM (calculated as the sodium salt) for 12 h. Controls were sprayed with clear water during the same period. Except during treatment, the plant flats were kept on the floor of the pond and covered with perforated aluminum screen to partly shield them from direct sun light. Plants were watered daily before and after treatment, except when there was adequate rain. The water was ap plied from above the plants with a fine-spray watering can.
Mean weights of treated and control plants of green beans and cantaloupe were compared by use of the Stu dent's t test.
Results
Lettuce, radish, sweet corn, and potato plants showed no adverse effects from the application of TFM. Green bean and tomato plants showed some damage.
At the time of treatment, the green bean plants were 8 to 10 cm high, each with two large unifoliate leaves. By 6 days after the TFM treatment, 70% of the leaves had brown spots of dead tissue that covered as much as 50% of individual leaves (average, about 15%). After 12 days, the dead spots on treated leaves were about the same as after 6 days, and all treated plants were growing and adding new trifoliate leaves at the same rate as the controls. Fifteen days after the first treatment, the bean plants were sub jected to a second treatment to provide data on more advanced plants. The results were about the same as in the first treatment; about 90% of the new leaves sustained
some damage (brown, dead spots) but all plants continued to grow. Two weeks after the second treatment, the plants were cut off at the ground and weighed to the nearest 0.1 g. Mean weights of treated and control plants (N = 16) did not differ significantly.
Two days after treatment, 50% of the leaves on treated tomato plants had numerous small brown spots; 4 to 5 days after treatment, the tomato plants began to look healthier and began developing new leaves; and 8 days after treat ment, the treated plants were growing and blooming at about the same rate as the controls.
Cucumber and cantaloupe plants sustained severe dam age from irrigation with water containing TFM. Some damage to cucumber plants was apparent 20 h after treat ment, as evidenced by brown spots on some of the leaves. Two days after treatment, 60-70% of the leaves had large brown spots of dead tissue. At 4 days after treatment, five plants were dead and the brown areas on live plants were drying out. By 8 days after treatment, 40% of the leaves on surviving treated plants were dead or dying, but the plants continued to grow at their tips and produced blossoms. Overall, cucumbers sustained major damage; 20% of the plants were killed and 40% of the leaves were severely damaged on surviving plants.
Cantaloupe plants were vigorous and healthy at the time of treatment. At 6 days after treatment, 75% of the leaves on treated plants showed damage (brown spots), two plants were dead, and two were nearly dead. After 2 weeks all of the treated plants looked unhealthy; 40% of the leaves showed damage and new leaves were small and pale. The control plants were growing well, appeared healthier, and had added large, dark green leaves. At 2 weeks after treatment, the mean weight (N= 16) of treated plants (10.8 g) was 33% less than that of controls (16.2 g). The difference was highly significant (P < 0.01).
OBSERVATIONS ON THE EFFECTS OF IRRIGATION WATER
SUMPTFM RESERVOIR
RACKS
REGULAR POSITION OF PLANTS
DRY POND
SPRINKLERDRY POND
WATER RESERVOIR
Figure. Schematic diagram of 0.005-ha ponds used for irrigating plants with water containing TFM.
Discussion
The treatment regimen used in these studies represented the maximum exposure that might occur when plants are irrigated with water from streams treated with TFM, and the length of treatment probably exceeded normal irriga tion periods. Irrigation for a shorter period or at a lower concentration of TFM would probably show propor tionately less effect on plants.
Although green beans and tomatoes showed no appar ent growth inhibition, there was sufficient damage to indi vidual leaves to indicate that exposure of these plants to TFM should be avoided. The severe damage sustained by cucumber and cantaloupe plants indicated that these plants and perhaps all plants of the family Cucurbitaceae should not be irrigated with water containing TFM. Be cause there is a wide variation in effects between types of plants, the best policy for types that have not been tested is to avoid irrigating them with water containing TFM.
Reference
National Research Council of Canada, Panel on TFM and Bayer 73. 1985. TFM and Bayer 73: Lampricides in the aquatic environment. National Research Council of Canada, Ottawa. Publication NRCC-22488. 184pp.
Gild
erhu
s, P
hilip
A.
1990
. O
bser
vati
ons
on t
he E
ffec
ts o
f Ir
riga
tion
Wat
er
Con
tain
ing
3-T
rifl
uoro
met
hyl-
4-N
itro
phen
ol (
TF
M)
on P
lant
s. U
.S.
Fish
W
ildl.
Serv
., In
vest
. F
ish
Con
trol
100
. 3
pp.
The
eff
ects
of 3
-tri
fluo
rom
ethy
l-4-
nitr
ophe
nol (
TFM
) on
you
ng p
lant
s of
com
m
on v
eget
able
s an
d fr
uits
wer
e st
udie
d. P
lant
s es
tabl
ishe
d in
hor
ticul
tura
l fl
ats
wer
e ir
riga
ted
for
12 h
with
wat
er c
onta
inin
g 10
mg/
L o
f TFM
and
late
r com
pare
d w
ith p
lant
s ir
riga
ted
for
a si
mila
r pe
riod
with
unt
reat
ed w
ater
. L
ettu
ce,
radi
sh,
swee
t cor
n, a
nd p
otat
o pl
ants
wer
e vi
rtua
lly u
naff
ecte
d. G
reen
bea
n an
d to
mat
o pl
ants
dev
elop
ed b
row
n or
dea
d sp
ots
on m
any
leav
es b
ut g
row
th r
ates
and
su
rviv
al w
ere
not
affe
cted
. C
ucum
ber
and
cant
alou
pe p
lant
s w
ere
seve
rely
da
mag
ed.
Key
wor
ds:
Lam
pric
ides
, T
FM,
irri
gatio
n, t
erre
stri
al p
lant
s.
_
Gild
erhu
s, P
hilip
A.
1990
. O
bser
vati
ons
on t
he E
ffec
ts o
f Ir
riga
tion
Wat
er
Con
tain
ing
3-T
rifl
uoro
met
hyl-
4-N
itro
phen
ol (
TF
M)
on P
lant
s. U
.S.
Fish
W
ildl.
Serv
., In
vest
. Fis
h C
ontr
ol 1
00.
3 pp
.
The
eff
ects
of 3
-tri
fluo
rom
ethy
l-4-
nitr
ophe
nol (
TFM
) on
you
ng p
lant
s of
com
m
on v
eget
able
s an
d fr
uits
wer
e st
udie
d. P
lant
s es
tabl
ishe
d in
hor
ticul
tura
l fl
ats
wer
e ir
riga
ted
for
12 h
with
wat
er c
onta
inin
g 10
mg/
L o
f TFM
and
late
r com
pare
d w
ith p
lant
s ir
riga
ted
for
a si
mila
r pe
riod
with
unt
reat
ed w
ater
. L
ettu
ce,
radi
sh,
swee
t cor
n, a
nd p
otat
o pl
ants
wer
e vi
rtua
lly u
naff
ecte
d. G
reen
bea
n an
d to
mat
o pl
ants
dev
elop
ed b
row
n or
dea
d sp
ots
on m
any
leav
es b
ut g
row
th r
ates
and
su
rviv
al w
ere
not
affe
cted
. C
ucum
ber
and
cant
alou
pe p
lant
s w
ere
seve
rely
da
mag
ed.
Key
wor
ds:
Lam
pric
ides
, T
FM,
irri
gatio
n, t
erre
stri
al p
lant
s.
Gil
derh
us,
Phi
lip
A.
1990
. O
bser
vati
ons
on t
he E
ffec
ts o
f Ir
riga
tion
Wat
er C
onta
inin
g 3-
Tri
- fl
uoro
met
hyl-
4-N
itro
phen
ol (T
FM
) on
Pla
nts.
U.S
. Fis
h W
ildl.
Serv
., In
vest
. Fis
h C
ontr
ol 1
00. 3
pp.
The
eff
ects
of
3-tr
iflu
orom
ethy
l-4-
nitr
ophe
nol
(TFM
) on
you
ng p
lant
s of
com
mon
veg
etab
les
and
frui
ts w
ere
stud
ied.
Pla
nts
esta
blis
hed
in h
ortic
ultu
ral
flat
s w
ere
irri
gate
d fo
r 12
h w
ith w
ater
con
tain
ing
10 m
g/L
of
TFM
and
lat
er c
ompa
red
with
pla
nts
irri
gate
d fo
r a
sim
ilar
peri
od w
ith u
ntre
ated
wat
er.
Let
tuce
, ra
dish
, sw
eet c
orn,
and
pot
ato
plan
ts w
ere
virt
ually
una
ffec
ted.
Gre
en b
ean
and
tom
ato
plan
ts
deve
lope
d br
own
or d
ead
spot
s on
man
y le
aves
but
gro
wth
rate
s an
d su
rviv
al w
ere
not a
ffec
ted.
Cuc
umbe
r an
d ca
ntal
oupe
pla
nts
wer
e se
vere
ly d
amag
ed.
Key
wor
ds:
Lam
pric
ides
, T
FM,
irri
gatio
n, t
erre
stri
al p
lant
s.
Gil
derh
us,
Phi
lip
A.
1990
. O
bser
vati
ons
on t
he E
ffec
ts o
f Ir
riga
tion
Wat
er C
onta
inin
g 3-
Tri
- fl
uoro
met
hyl-
4-N
itro
phen
ol (T
FM
) on
Pla
nts.
U.S
. Fis
h W
ildl.
Serv
.Jnv
est.
Fis
h C
ontr
ol 1
00. 3
pp.
The
eff
ects
of
3-tr
iflu
orom
ethy
l-4-
nitr
ophe
nol
(TFM
) on
you
ng p
lant
s of
com
mon
veg
etab
les
and
frui
ts w
ere
stud
ied.
Pla
nts
esta
blis
hed
in h
ortic
ultu
ral
flat
s w
ere
irri
gate
d fo
r 12
h w
ith w
ater
con
tain
ing
10 m
g/L
of
TFM
and
lat
er c
ompa
red
with
pla
nts
irri
gate
d fo
r a
sim
ilar
peri
od w
ith u
ntre
ated
wat
er.
Let
tuce
, ra
dish
, sw
eet c
orn,
and
pot
ato
plan
ts w
ere
virt
ually
una
ffec
ted.
Gre
en b
ean
and
tom
ato
plan
ts
deve
lope
d br
own
or d
ead
spot
s on
man
y le
aves
but
gro
wth
rate
s an
d su
rviv
al w
ere
not a
ffec
ted.
Cuc
umbe
r an
d ca
ntal
oupe
pla
nts
wer
e se
vere
ly d
amag
ed.
Key
wor
ds:
Lam
pric
ides
, TFM
, ir
riga
tion,
ter
rest
rial
pla
nts.
Residues of Malachite Green in Muscle, Eggs, and Fry of Treated Atlantic Salmon and Chinook Salmon
by
J. L. Alien
U.S. Fish and Wildlife Service National Fisheries Research Center La Crosse
P.O. Box 818 La Crosse, Wisconsin 54602
ABSTRACT. Residues of malachite green in muscle, eggs, and fry of Atlantic salmon (Salmo salar) and chinook salmon (Oncorhynchus tshawytscha) were determined by colorimetric analysis after the fish had been routinely treated with the chemical at fish hatcheries (10-47 times, 1 ppm for 1 h). The concentration of residues in fish muscle generally depended on the elapsed time since the last treatment; concentrations were usually highest (about 1.0 to 2.5 ug/g) in fish sampled 1 or 2 days after the last treatment and had declined somewhat to values as low as 0.33 ug/g after 18-41 days. Residues in eggs taken from adults that had been treated with malachite green were about 0.1 to 4.2 ug/g in Atlantic salmon and 0.1 to 1.0 ug/g in chinook salmon; there was little relation between the residue concentrations in the eggs and the elapsed time since the last treatment. Residues of malachite green in fry newly hatched from eggs of treated chinook salmon ranged from 0.14 to 1.16 ug/g.
Malachite green has been used extensively in fish cul ture (usually in the zinc-free oxalate form) for the control of fungal infections and external parasites (Nelson 1974; Alderman 1985). It is the most effective antifungal treat ment used in aquaculture (Meyer and Hoffman 1976; Bai ley 1983; Alderman 1985). It has never been registered for use on food fish (such use was banned in 1978) because of potential health risks; use is currently limited to the treat ment of nonfood fish (e.g., salmon brood stock) under an Investigational New Animal Drug Application issued by the U.S. Food and Drug Administration.
The potential threat of malachite green to human health was first pointed out by Werth and Boiteux (1958), who described marked increases in the incidence of internal tumors in the progeny of laboratory rats fed malachite green. Meyer and Jorgenson (1983) reported a significant increase in gross abnormalities in rainbow trout (On corhynchus mykiss) hatched from eggs treated with mal achite green at concentrations of 1 mg/L for 1 h daily (30 applications); 3 mg/L for 1 h every other day (15 applica tions), and 5 mg/L for 1 h weekly (5 applications). They also reported that malachite green produced significant teratological effects at all levels of treatment when it was administered orally to New Zealand white rabbits at doses
as low as 5 mg/kg body weight. Although a different delivery system and a higher dose rate were used in the rabbits than are normally used in fish culture, the existence of a serious potential hazard is obvious.
Information on residues of malachite green that might occur in fish flesh must be determined as part of the evaluation of its use. Poe and Wilson (1983), who re ported the presence of malachite green in channel cat fish (Ictalurus punctatus) after treatment, wrote that a greenish color appeared on the surface of the catfish muscle tissue after frozen storage for 13 to 60 days; and T. D. Bills (personal communication) indicated that a solution of 85% ethyl alcohol, 10% formalin, and 5% acetic acid (AFA) that was used to preserve fish exposed to malachite green developed a bluish color. Thus, the presence of malachite green can be determined by colori metric analysis.
Alien and Hunn (1986) reported preliminary results of malachite green analysis after residues were extracted with AFA from muscle tissue of rainbow trout and muscle tissue and eggs of Pacific salmon (Oncorhynchus sp.). Colorimetric analysis showed that muscle of rainbow trout exposed to 0.1 mg/L of malachite green for 24 h at 14° C contained 1.87 |lg/g of malachite green; the residue
1
INVESTIGATIONS IN FISH CONTROL 101
declined to 0.22 [ig/g in muscle after 6 days of withdrawal in fresh water. Residues in eggs from these fish ranged from 0.1 to 4.1 ppm. In the present study, I used AFA extraction and colorimetric analysis to examine adult salmonids treated with malachite green to obtain further data on residues of malachite green in the muscle tissue and eggs and, especially, newly hatched fry. The analyses were done on fish from national fish hatcheries treated and sampled under hatchery conditions.
Materials and MethodsAdult Atlantic salmon (Salmo salar} at the Berkshire
(Massachusetts) National Fish Hatchery (NFH) were treated on 5 consecutive days each week 20 to 47 times with 1 ppm of malachite green for 1 h. Samples of muscle tissue and eggs were collected from 1 to 41 days after the last treatment of these fish, as well as from un treated adults (to determine background readings of mal achite green).
Adult chinook salmon (Oncorhynchus tshawytscha) from the Warm Springs (Oregon) NFH and Winthrop (Washington) NFH were also treated on 5 consecutive days each week with 1 ppm of malachite green for 1 h. The fish at Warm Springs NFH were treated 27 to 30 times and samples of muscle tissue were collected 1, 6, 12, or 18 days after the last treatment; those at Winthrop NFH received 10 treatments at various intervals and were sampled 8 days after the last treatment. At both hatch eries, eggs were collected at fertilization (0 h) and 24 h after fertilization, and fry were sampled 24 h after hatch ing. Muscle tissue, eggs, and fry from untreated adults were collected at Warm Springs NFH to provide back ground values.
Samples of fish tissue, eggs, and fry from the several hatcheries were frozen and shipped to the La Crosse (Wis consin) National Fisheries Research Center for analysis. I extracted 10-g samples (wet weight) with 25 mL of AFA at room temperature in the dark for 24 h, centrifuged samples at about 2,000 rpm, and filtered them through a glass fiber filter. The absorbance of sample extracts at 615 nm was determined on a Beckman DU6 spectrophotometer and compared to the absorbance of malachite green oxalate standards in AFA. Residue concentrations are reported as fig/g malachite green oxalate.
Results and DiscussionThe presence of malachite green residues in the fish
eggs and fry was readily apparent from the blue-green
color of the tissue extracts. Because fish and eggs have a naturally occurring yellowish color, malachite green resi dues are seen as green by the human eye. The presence of naturally occurring colors also produced a low back ground reading in the untreated fish, eggs, and fry, but too few untreated samples were tested to establish a useful mean background reading for subtraction from the sample residue concentrations. Background color readings in the muscle from two untreated adult Atlantic salmon were 0.09 to 0.15 |j,g/g (malachite green equivalents) and 0.10 \ig/g in the eggs of one (Table 1). The residue concen trations reported here include the background values.
Residues reported as malachite green in Atlantic sal mon showed no relation between the number of treatments received and the concentration of residue in the muscle (Table 1). Residues ranged from 0.33 to 2.54 jig/g. The residue concentrations were lowest (0.33, 0.35 fig/g) in muscle from two adults that received the fewest treatments (20) and were sampled 24 and 33 days after the last treat ment; however, the residue concentration in the muscle of one other adult was 2.54 [ig/g after 20 treatments. Eggs from treated fish contained 0.21 to 4.16 |J,g/g of residual malachite green.
Chinook salmon from Warm Springs NFH tested for malachite green residues had received 27 to 30 treatments of 1.0 ppm for 1 h and were sampled at 1, 6, 12, and 18 days after the last treatment. Background color readings were 0.32 and 0.35 |j,g/g in muscle tissue, 0.03 to 0.08 fig/g in eggs, and 0.22 [ig/g in fry (Table 2). The mean concentration of malachite green in muscle of the treated fish ranged from 0.473 to 1.08 fig/g. Residues in the mus cle were slightly higher in fish sampled at 1 day after the last treatment than in those sampled after 6-18 days.
The mean concentration of malachite green in eggs taken from adults after different frequencies of treatment ranged from 1.16 to 1.86 |J,g/g. In eggs sampled 24 h after fertilization, the average levels ranged from 0.84 to 1.54 \ig/g. In fry from treated adults, average residues ranged from 0.97 to 1.16 |U.g/g.
The mean concentration of malachite green in muscle of adult salmon from Winthrop NFH that had been treated 10 times at 1.0 ppm for 1 h was 0.43 [ig/g (Table 2). The mean concentration in eggs was 0.173 \ig/g at collection and 0.113 \ig/g in 24 h; the average concentration in the fry was 0.14 [ig/g.
In general, malachite green seemed to be readily taken up by adult salmon treated with it and was incorporated into their eggs before spawning. Residues in the fry hatched from eggs of treated fish indicated that little of the chemical was lost during incubation.
RESIDUES OF MALACHITE GREEN
Table 1. Residues of malachite green oxalate (\ig/g) in muscle and eggs of Atlantic salmon subjected to 20 to 47 1-h treatments with 1 ppm of malachite green at the Berkshire National Fish Hatchery. Each row of data represents one fish.
Time after last treatment (days)
No treatmentNo treatment
11123567
243341
Number of treatments at 1 ppm
00
4245472045204747202045
Sample
Muscle
0.150.091.662.13
2.54
1.110.642.220.330.350.91
Eggs
0.10
3.153.264.162.513.600.722.673.282.29
0.21
Table 2. Mean residues (SD in parentheses) of malachite green ([Lg/g) in muscle, eggs, and fry from treated (1 ppmfor 1 h) and untreated chinook salmon at two national fish hatcheries (NFH).
Time from last treatment (days)
Warm Springs NFH1
1
6
6
12
18
No treatmentWinthrop NFH
8
Number of
fish
3
3
3
3
3
3
2
3
Number of
treatments at 1 ppm
27
30
29
30
30
30
0
10
Sample
Muscle
0.997(0.197)1.08
(0.159)0.75
(0.14)0.473
(0.186)0.577
(0.228)0.743
(0.104)0.335a
0.43(0.044)
Eggs (Oh)
1.34(0.835)1.82
(0.300)1.86
(0.681)1.47
(0.941)1.16a
1.69(0.662)0.08a
0.173(0.055)
Eggs (24 h)
0.843(0.401)1.40
(0.332)1.45
(0.561)0.973
(0.601)1.24
(1.06)1.54
(0.611)0.03a
0.113(0.04)
Fry(24 h)
0.970(0.423)1.04
(0.091)1.00
(0.246)1.16s
1.01(0.946)1.03
(0.375)0.22"
0.14(0.01)
"Average for two fish. bRepresents one fish.
INVESTIGATIONS IN FISH CONTROL 101
ReferencesAlderman, D. J. 1985. Malachite green: a review. J. Fish Dis.
8:289-298. Alien, J. L., and J. B. Hunn. 1986. Fate and distribution studies of
some drugs used in aquaculture. Vet. Hum. Toxicol. 28:21-24. Bailey, T. A. 1983. Method for in vitro screening of aquatic
fungicides. J. Fish Dis. 6:91-100. Meyer, F. P., and G. L. Hoffman. 1976. Parasites and diseases of
warrnwater fishes. U.S. Fish Wildl. Serv., Resour. Publ. 127.20pp.
Meyer, F. P., and T. A. Jorgenson. 1983. Teratological and other effects of malachite green on development of rainbow trout and rabbits. Trans. Am. Fish. Soc. 112:818-824.
Nelson, N. C. 1974. A review of the literature on the use of malachite green in fisheries. National Technical Information Service, Springfield, Va. PB-235-450/AS. 79 pp.
Poe, W. E., and R. P. Wilson. 1983. Adsorption of malachite green by channel catfish. Prog. Fish-Cult. 45:228-229.
Werth, V. G., and A. Boiteux. 1958. Disturbances of the heredi tary pattern and production of tumors by experimental tissue anoxia. Arzneim.-Forsch. 8:735-744.
Alie
n, J.
L.
1990
. Res
idue
s of M
alac
hite
Gre
en in
Mus
cle,
Egg
s, a
nd F
ry fr
om
Tre
ated
Atla
ntic
Sal
mon
and
Chi
nook
Sal
mon
. U
.S.
Fish
Wild
l. Se
rv.,
Inve
st. F
ish
Con
trol
101
. 4 p
p.
Res
idue
s of
mal
achi
te g
reen
in m
uscl
e, e
ggs,
and
fry
of A
tlant
ic s
alm
on (S
alm
o sa
lar)
and
Chi
nook
sal
mon
(O
ncor
hync
hus
tsha
wyt
scha
) w
ere
dete
rmin
ed b
y co
lori
met
ric
anal
ysis
aft
er th
e fi
sh h
ad b
een
rout
inel
y tr
eate
d w
ith th
e ch
emic
al a
t fi
sh h
atch
erie
s (1
0--4
7 tim
es,
1 pp
m f
or 1
h).
The
con
cent
ratio
n of
resi
dues
in f
ish
mus
cle
gene
rally
dep
ende
d on
the
elap
sed
time
sinc
e th
e la
st t
reat
men
t; co
ncen
tr
atio
ns w
ere
usua
lly h
ighe
st (
abou
t 1 .
0 to
2.5
(ig
/g)
in f
ish
sam
pled
1 o
r 2
days
af
ter
the
last
trea
tmen
t and
had
dec
lined
som
ewha
t to
valu
es a
s lo
w a
s 0.
33 |J
.g/g
af
ter
18-4
1 da
ys.
Res
idue
s in
egg
s ta
ken
from
adu
lts t
hat h
ad b
een
trea
ted
with
m
alac
hite
gre
en w
ere
abou
t 0.1
to
4.2
[ig/
g in
Alta
ntic
sal
mon
and
0.1
to
1 .0
(ig/
g in
chi
nook
sal
mon
; th
ere
was
littl
e re
latio
n be
twee
n th
e re
sidu
e co
ncen
trat
ions
in
the
eggs
and
the
elap
sed
time
sinc
e th
e la
st tr
eatm
ent.
Res
idue
s of
mal
achi
te g
reen
in
new
ly h
atch
ed f
ry r
ange
d fr
om 0
.14
to 1
.1
Key
wor
ds:
Mal
achi
te g
reen
, res
idue
s, A
tlant
ic s
alm
on, C
hino
ok s
alm
on, s
alm
on
eggs
, fry
, col
orim
etri
c an
alys
is.
Alie
n, J.
L.
1990
. Res
idue
s of M
alac
hite
Gre
en in
Mus
cle,
Egg
s, a
nd F
ry fr
om
Tre
ated
Atla
ntic
Sal
mon
and
Chi
nook
Sal
mon
. U
.S.
Fish
Wild
l. Se
rv.,
Inve
st. F
ish
Con
trol
101
. 4 p
p.
Res
idue
s of
mal
achi
te g
reen
in m
uscl
e, e
ggs,
and
fry
of A
tlant
ic s
alm
on (
Salm
o sa
lar)
and
chi
nook
sal
mon
(O
ncor
hync
hus
tsha
wyt
scha
) w
ere
dete
rmin
ed b
y co
lori
met
ric
anal
ysis
aft
er th
e fi
sh h
ad b
een
rout
inel
y tr
eate
d w
ith th
e ch
emic
al a
t fi
sh h
atch
erie
s (1
0--4
7 tim
es,
1 ppm
for
1 h
). T
he c
once
ntra
tion
of re
sidu
es in
fis
h m
uscl
e ge
nera
lly d
epen
ded
on t
he e
laps
ed ti
me
sinc
e th
e la
st tr
eatm
ent;
conc
en
trat
ions
wer
e us
ually
hig
hest
(ab
out
1.0
to 2
.5 (
J,g/g
) in
fis
h sa
mpl
ed 1
or
2 da
ys
afte
r th
e la
st tr
eatm
ent a
nd h
ad d
eclin
ed s
omew
hat t
o va
lues
as
low
as
0.33
[ig
/g
afte
r 18
-41
days
. R
esid
ues
in e
ggs
take
n fr
om a
dults
tha
t had
bee
n tr
eate
d w
ith
mal
achi
te g
reen
wer
e ab
out 0
.1 t
o 4.
2 [l
g/g
in A
ltant
ic s
alm
on a
nd 0
.1 t
o 1.
0 [l
g/g
in c
hino
ok s
alm
on;
ther
e w
as li
ttle
rela
tion
betw
een
the
resi
due
conc
entr
atio
ns in
th
e eg
gs a
nd th
e el
apse
d tim
e si
nce
the
last
trea
tmen
t. R
esid
ues
of m
alac
hite
gre
en
in n
ewly
hat
ched
fry
ran
ged
from
0.1
4 to
1.1
6 [l
g/g.
Key
wor
ds:
Mal
achi
te g
reen
, res
idue
s, A
tlant
ic s
alm
on, C
hino
ok s
alm
on, s
alm
on
eggs
, fr
y, c
olor
imet
ric
anal
ysis
.
Alie
n, J
. L.
199
0. R
esid
ues
of M
alac
hite
Gre
en i
n M
uscl
e, E
ggs,
and
Fry
fro
m T
reat
ed A
tlant
ic
Salm
on a
nd C
hino
ok S
alm
on. U
.S. F
ish
Wild
l. Se
rv.,
Inve
st. F
ish
Con
trol
101
.4 p
p.
Res
idue
s of
mal
achi
te g
reen
in
mus
cle,
egg
s, a
nd f
ry o
f A
tlant
ic s
alm
on (
Salm
o sa
lar)
and
chi
nook
sa
lmon
(O
ncor
hync
hus
tsha
wyt
scha
) w
ere
dete
rmin
ed b
y co
lori
met
ric
anal
ysis
aft
er th
e fis
h ha
d be
en
rout
inel
y tr
eate
d w
ith th
e ch
emic
al a
t fis
h ha
tche
ries
(10
-47
times
, 1
ppm
for
1 h)
. The
con
cent
ratio
n of
re
sidu
es in
fis
h m
uscl
e ge
nera
lly d
epen
ded
on th
e el
apse
d tim
e si
nce
the
last
trea
tmen
t; co
ncen
trat
ions
w
ere
usua
lly h
ighe
st (
abou
t 1 .
0 to
2.5
^g/
g) in
fis
h sa
mpl
ed 1
or
2 da
ys a
fter
the
last
trea
tmen
t and
had
de
clin
ed s
omew
hat t
o va
lues
as
low
as 0
.33 ̂
.g/g
aft
er 1
8-41
day
s. R
esid
ues i
n eg
gs ta
ken
from
adu
lts th
at
had
been
trea
ted
with
mal
achi
te g
reen
wer
e ab
out 0
. 1 t
o 4.
2 ^.
g/g
in A
ltant
ic s
alm
on a
nd 0
. 1 t
o 1 .
0 ^.
g/g
in
chin
ook
salm
on;
ther
e w
as l
ittle
rel
atio
n be
twee
n th
e re
sidu
e co
ncen
trat
ions
in th
e eg
gs a
nd th
e el
apse
d tim
e si
nce
the
last
tre
atm
ent.
Res
idue
s of
mal
achi
te g
reen
in
new
ly h
atch
ed f
ry r
ange
d fr
om 0
.14
to
Alie
n, J
. L.
199
0. R
esid
ues
of M
alac
hite
Gre
en i
n M
uscl
e, E
ggs,
and
Fry
fro
m T
reat
ed A
tlant
ic
Salm
on a
nd C
hino
ok S
alm
on. U
.S. F
ish
Wild
l. Se
rv.,
Inve
st. F
ish
Con
trol
101
. 4 p
p.
Res
idue
s of
mal
achi
te g
reen
in
mus
cle,
egg
s, a
nd f
ry o
f A
tlant
ic s
alm
on (
Salm
o sa
lar)
and
chi
nook
sa
lmon
(O
ncor
hync
hus
tsha
wyt
scha
) w
ere
dete
rmin
ed b
y co
lori
met
ric
anal
ysis
aft
er th
e fis
h ha
d be
en
rout
inel
y tr
eate
d w
ith th
e ch
emic
al a
t fis
h ha
tche
ries
(10
-47
times
, 1
ppm
for
1 h)
. The
con
cent
ratio
n of
re
sidu
es in
fis
h m
uscl
e ge
nera
lly d
epen
ded
on th
e el
apse
d tim
e si
nce
the
last
trea
tmen
t; co
ncen
trat
ions
w
ere
usua
lly h
ighe
st (
abou
t 1 .
0 to
2.5
Hg/
g) in
fis
h sa
mpl
ed 1
or
2 da
ys a
fter
the
last
trea
tmen
t and
had
de
clin
ed so
mew
hat t
o va
lues
as
low
as 0
.33
^.g/
g af
ter
1 8-4
1 da
ys. R
esid
ues
in e
ggs t
aken
from
adu
lts th
at
had
been
trea
ted
with
mal
achi
te g
reen
wer
e ab
out 0
. 1 t
o 4.
2 ^.
g/g
in A
ltant
ic s
alm
on a
nd 0
. 1 t
o 1 .
0 ^.
g/g
in
chin
ook
salm
on;
ther
e w
as l
ittle
rel
atio
n be
twee
n th
e re
sidu
e co
ncen
trat
ions
in th
e eg
gs a
nd th
e el
apse
d tim
e si
nce
the
last
tre
atm
ent.
Res
idue
s of
mal
achi
te g
reen
in
new
ly h
atch
ed f
ry r
ange
d fr
om 0
.14
to
Key
wor
ds:
Mal
achi
te g
reen
, res
idue
s, A
tlant
ic s
alm
on, C
hino
ok s
alm
on, s
alm
on e
ggs,
fry,
col
orim
etri
c an
alys
is.
Key
wor
ds:
Mal
achi
te g
reen
, res
idue
s, A
tlant
ic s
alm
on, C
hino
ok s
alm
on, s
alm
on e
ggs,
fry,
col
orim
etri
c an
alys
is.
Effects of Water Temperature, Hardness, and pH on the Toxicity of Benzocaine to Eleven Freshwater Fishes
by
Terry D. Bills, George E. Howe, and Leif L. Marking
U.S. Fish and Wildlife Service National Fisheries Research Center La Crosse
P.O. Box 818 La Crosse, Wisconsin 54602
ABSTRACT. The toxicity of benzocaine (ethyl 4-aminobenzoate; 98%) to eleven freshwater fishes was evaluated under various physical and chemical conditions. Under standard test conditions (pH 7.8, 12° C, soft water), the 24-h LC50 (mg/L) was 17.2 for lake sturgeon (Acipenserfulvescens), 22.5 for rainbow trout (Oncorhynchus mykiss), 34.0 for muskellunge (Esox masquinongy), 24.0 for northern pike (Esox lucius), 19.0 for common carp (Cyprinus carpio), 25.9 for fathead minnow (Pimephales promelas), 28.0 for channel catfish (Ictaluruspunctatus), 28.1 for striped bass (Morone saxatilis), 21.9 for green sunfish (Lepomis cyanellus), 21.9 for bluegill (L. macrochirus), and 22.0 for walleye (Stizostedion vitreum). Tests conducted at three water temperatures, four hardnesses, and three pH's showed that none of these variables influenced toxicity. The higher temperatures tested (to 22° C) did not increase the rate of toxicosis. Rainbow trout survived exposure to the recommended use concentra tion of 25 mg/L for 15 min, but did not survive exposure to 75 or 125 mg/L. Users of benzocaine should be cautioned that overdoses may lead to undue stress or mortality in exposed fish. Comparison of benzocaine concentrations by high performance liquid chromatography at 0 and 96 h showed no degradation of the compound during the exposure period.
Anesthetics are used for a number of fishery applica tions, ranging from mild sedation for transport to total anesthetization for marking, tagging, spawn-taking, and surgical procedures. Although many chemicals have been used to anesthetize fish (McFarland 1959; Bell 1967), tricaine methanesulfonate (MS-222) is the only chemical registered for use on fish in the United States. The label for MS-222 forbids anesthetized fish to be released to the wild or used as food until after a 21-day withdrawal. Salmon anesthetized with MS-222 and killed during spawning must be discarded rather than used as human or animal food (Gilderhus 1989). Because of the withdrawal period for MS-222, there is a need for an alternate anesthetic.
Many substances have been tested as potential anesthet ics for fish (McFarland 1959; Bell 1967; Gilderhus et al. 1973; Dawson and Gilderhus 1979). McErlean (1967) first suggested that ethyl-p-aminobenzoate be used as an anesthetic for cold-blooded vertebrates. The 183 fishery workers who responded to a survey conducted by Marking and Meyer (1985) used a total of 11 different chemicals to anesthetize fish. Gilderhus and Marking (1987) identified
benzocaine, from a group of 16 anesthetics, as a possible candidate for use in fisheries. Chemically, it is similar to MS-222, differing only by the position of a single substi- tuent: the amino group is in the meta position in MS-222 and in the para position on benzocaine. Also, because benzocaine is widely used in human over-the-counter drug preparations, its registration for fishery use may be easier and less costly than for other candidate anesthetics (Gil derhus 1989).
Previous studies of benzocaine (Dawson and Gilderhus 1979; Gilderhus 1989) determined the compound's effec tiveness as a fish anesthetic but provided little information on its toxicity to fish. Standardized toxicity information is required to satisfy requirements of the U.S. Food and Drug Administration for minor use of animal drugs. The pur pose of our study was to determine the toxicity of benzo caine to representative coldwater and warmwater fish in laboratory tests; to evaluate the effects of water tempera ture, hardness, and pH on toxicity; and to determine the safety to fish of treatment concentrations that were 3 and 5 times the effective rate for trout.
1
INVESTIGATIONS IN FISH CONTROL 102
Materials and Methods
Static test procedures used in this study followed those prescribed by the Committee on Methods for Acute Tox- icity Tests with Aquatic Organisms (1975), ASTM Com mittee E-35 on Pesticides (1980), and the U.S. Department of Agriculture (1986). We exposed 20 fish to each concen tration of benzocaine in glass jars containing 15 L of oxygen-saturated test water. Reconstituted test waters were prepared according to standardized procedures to produce the desired water quality.
The solutions were adjusted to a selected pH (± 0.2 unit) with chemical buffers (Committee on Methods for Acute Toxicity Tests with Aquatic Organisms 1975), before each test and at 24-h intervals, as needed. Tem peratures were regulated by immersing the test jars in constant-temperature water baths. To assess the effects of water hardness, we buffered the test solutions to a constant pH with sodium bicarbonate, using the procedure of Marking (1975).
The test species were lake sturgeon (Acipenser ful- vescens), rainbow trout (Oncorhynchus my kiss), mus- kellunge (Esox masquinongy), northern pike (Esox lu- cius\ common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), channel catfish (Ictalurus punc- tatus), striped bass (Morone saxatilis), green sunfish (Le- pomis cyanellus), bluegill (L. macrochirus), and walleye (Stizostedion vitreum). They were obtained from a State or Federal fish hatchery or produced at the National Fisheries Research Center, La Crosse, Wisconsin, and were main tained according to the standard procedures for handling experimental fish. The fish were acclimated to the desired water chemistries and temperatures for 24 h before each test. Mortalities were recorded at 1,3, 6, and 12 h on the first day of exposure and daily thereafter for 96 h. The methods of Litchfield and Wilcoxon (1949) were used to compute the LC50's (concentration causing 50% mortal ity) and 95% confidence intervals.
Three species (rainbow trout, channel catfish, and blue- gill) were used in tests to determine the effects of water temperature, hardness, and pH on the toxicity of benzo caine. In tests to determine safe use pattern levels, we exposed groups of 300 rainbow trout to benzocaine at the prescribed effective treatment concentration of 25 mg/L for 15 min (Gilderhus 1989) and to 3 and 5 times this effective level (U.S. Department of Agriculture 1986). We observed the fish for a 14-day postexposure period for unusual behavior or delayed mortality, using the criteria set forth by Lennon and Walker (1964).
Benzocaine (ethyl 4-aminobenzoate, 98%; Aldrich Chemical Company, Milwaukee, Wisconsin) was dis solved in ethanol to make stock solutions, and aliquots of
these solutions were pipetted into test vessels to reach the desired test concentrations.
Benzocaine concentrations in water samples were quantified at 0 and 96 h by high performance liquid chro- matography (HPLC). Quantification equipment and con ditions were as follows: Waters HPLC system, consisting of a 71 OB WISP autosampler, a 510 variable speed pump, a 481 spectrophotometer detector, and a 730 data module integrator. A Micro Pak, C 18 , reverse-phase column (30 cm X 4 mm) was used with a mobile phase consisting of 70% HPLC methanol, 26% HPLC water, and 4% acetic acid. Flow rate for the mobile phase was 2.0 mL/min and the detector was set at a wavelength of 286 nm. This method resulted in a retention time of about 3.6 min. To quantify peak areas, we used the Waters External Standard Quantification program.
Results
Toxicity to Eleven Species of Fish
Benzocaine was toxic to all species exposed in soft water at 12° C; the 24-h LC50's ranged from 17.2 mg/L for lake sturgeon to 34.0 mg/L for muskellunge (Table 1). Coldwater and warmwater species responded similarly; the 24-h LC50 was 22.5 mg/L for rainbow trout and 21.9 mg/L for bluegills. The toxicity of benzocaine did not increase significantly with longer exposures; the LC50's changed little between 1 and 24 h for the species exposed.
Influence of Temperature, Water Hardness, andpH
The toxicity of benzocaine was not significantly affected by any of the water characteristics tested. In creased water temperature, which increases the metabolic rate of poikilotherms, usually increases the rate of uptake of toxic chemicals and results in greater mortality. How ever, the toxicity of benzocaine was not affected by changes in water temperature in these exposures. For ex ample, with rainbow trout, the 24-h LC50 was 17.0 mg/L in water at 7° C and 20.5 mg/L in water at 17° C (Tables 2, 3, and 4). The 24-h LC50's for channel catfish were 28.0 mg/L at 12° C and 27.9 mg/L at 22° C.
Likewise, neither water hardness nor pH affected tox icity. For example, in channel catfish, the 24-h LC50 was 30.2 mg/L in very soft water (10-12 mg/L, total hardness as milligrams per liter CaCO3 ) and 30.0 mg/L in very hard water (300-320 mg/L total hardness). The 24-h LC50's for channel catfish were 30.1 mg/L in acidic water (pH 6.5) and 29.0 mg/L in alkaline water (pH 9.5).
The HPLC analysis of benzocaine concentrations in water from randomly selected test vessels agreed closely
EFFECTS OF WATER TEMPERATURE, HARDNESS, AND pH
Table 1. Toxicity (LC50 in mgIL, and 95% confidence interval) ofbenzocaine to eleven species offish in soft waterat 12°C.
Species
Lake sturgeon3
Rainbow trout
Northern pike
Muskellunge3
Fathead minnow
Common carp
Channel catfish
Striped bassa
Bluegill
Green sunfish
Walleye3
1
28.025.3-31.0
27.026.7-28.4
35.028.0-43.7
34.029.5-39.1
35.033.0-37.2
22.621.2-24.1
36.033.1-39.1
32.927.6-39.2
26.522.6-31.0
26.525.0-28.1
37.732.4-43.9
3
24.220.0-29.2
24.223.3-25.1
35.028.0-43.7
34.029.5-39.5
29.027.2-30.9
21.920.7-23.2
35.032.5-37.7
28.122.7-34.8
22.820.4-25.4
25.022.8-27.4
25.922.1-30.4
Duration of exposure (hours)
6 12
20.5 18.4-22.8
23.0 22.521.8-24.2 21.3-23.8
35.0 27.528.0-43.7 26.1-29.0
34.0 34.029.5-39.1 29.5-39.1
26.0 25.924.8-27.4 24.9-26.9
21.9 19.320.7-23.2 18.6-20.0
29.5 29.027.8-31.3 27.2-30.9
28.1 28.122.7-34.8 22.7-34.8
22.8 21.020.4-25.4 19.5-22.6
23.0 22.021.8-24.3 20.8-23.3
22.0 22.019.7-24.6 19.7-24.6
24
17.215.5-19.1
22.521.3-23.8
24.021.6-26.6
34.029.5-39.1
25.924.9-26.9
19.017.9-20.1
28.026.7-29.3
28.122.7-34.8
21.920.3-23.6
21.920.6-23.2
22.019.7-24.6
96
17.215.5-19.1
11.010.2-11.8
20.018.1-22.0
30.026.7-33.7
25.924.9-26.9
19.017.9-20.1
18.512.3-14.8
28.122.7-24.8
17.015.8-18.3
20.219.0-21.4
"Ten fish per test concentration; 20 for other species.
Table 2. Toxicity (LC50 in mg/L, and 95% confidence interval) of benzocaine to rainbow trout in water of differenttemperatures, hardnesses, and pH's.
Temperature(°C)
7
12
17
12
12
12
12
12
12
12
Hardness pH
Soft 7.8
Soft 7.8
Soft 7.8
Very soft 8.2
Soft 8.2
Hard 8.2
Very hard 8.2
Soft 6.5
Soft 8.5
Soft 9.5
1
23.021.8-24.3
27.025.7-28.4
26.023.6-26.4
31.028.4-33.8
28.026.2-29.9
24.021.5-26.8
30.027.6-32.6
27.024.9-29.2
35.532.5-38.8
31.028.5-33.7
Duration of exposure (hours)
3 6 12
19.8 19.0 17.018.8-20.9 18.0-20.1 15.7-18.4
24.2 23.0 22.523.3-25.1 21.8-24.2 21.3-23.8
23.8 22.0 22.022.7-24.9 20.7-23.3 20.7-23.3
26.8 24.0 24.024.7-29.1 21.4-26.9 21.4-26.9
26.0 24.0 24.023.7-28.5 21.4-26.9 21.4-26.9
24.0 24.0 24.021.5-26.8 21.5-26.8 21.5-26.8
27.0 27.0 27.024.9-29.2 24.9-29.2 24.9-29.2
24.0 24.0 24.021.4-26.9 21.4-26.9 21.4-26.9
30.4 29.0 24.027.9-33.1 26.8-31.4 21.4-26.9
24.2 24.2 21.021.5-27.2 21.5-27.2 19.7-22.4
24
17.015.7-18.4
22.521.3-25.8
20.519.2-21.9
22.020.3-23.9
24.021.4-26.9
24.021.5-26.8
23.221.1-25.5
21.019.5-22.6
22.020.4-23.8
19.017.3-20.8
96
13.312.3-14.3
11.010.2-11.8
7.205.92-9.76
8.607.98-9.26
7.506.53-8.62
9.708.90-10.6
23.221.1-25.5
INVESTIGATIONS IN FISH CONTROL 102
Table 3. Toxicity (LC50 in mg/L, and 95% confidence interval) of benzocaine to blue gills in water of differenttemperatures, hardnesses, andpH's.
Temperature(°Q
12
17
22
12
12
12
12
12
12
12
Table 4.
Hardness
Soft
Soft
Soft
Very soft
Soft
Hard
Very hard
Soft
Soft
Soft
Toxicity (LC50 in
PH
7.8
7.8
7.8
8.2
8.2
8.2
8.2
6.5
8.5
9.5
Duration of exposure (hours)
1
26.522.6-31.0
27.020.7-35.2
27.024.8-29.4
29.027.4-30.7
28.526.6-30.5
29.526.7-32.5
29.027.2-30.9
25.523.4-27.8
29.026.1-32.2
25.022.2-28.1
3
22.820.4-25.4
25.022.4-27.9
22.019.5-24.8
27.025.4-28.7
25.023.8-26.2
24.522.6-26.5
28.026.7-29.4
22.020.2-24.0
25.023.0-27.1
23.521.2-26.1
6
22.820.4-25.4
25.022.4-27.9
21.019.5-22.6
25.022.7-27.6
23.021.8-24.3
23.021.8-24.3
25.624.3-27.0
21.220.1-22.3
23.021.3-24.8
23.020.8-25.4
mg/L, and 95% confidence interval) ofbenzocaine
12
21.019.5-22.6
25.022.4-27.9
18.616.9-20.4
22.321.1-23.6
22.921.7-24.2
23.021.6-24.5
24.323.4-25.3
21.220.1-22.3
21.519.9-23.2
22.520.8-24.3
24
21.920.3-23.6
23.021.1-25.1
17.816.5-19.2
22.321.1-23.6
22.020.8-23.3
23.021.6-24.5
21.319.8-22.9
21.220.1-22.3
20.619.3-22.0
22.520.8-24.3
96
17.015.8-18.3
18.016.2-20.0
9.207.48-11.3
16.014.6-17.5
17.015.8-18.3
18.016.9-19.2
15.113.6-16.7
19.017.4-20.8
19.518.6-20.4
22.020.7-23.3
to channel catfish in water of, differenttemperatures, hardnesses, andpH's.
Duration of exposure (hours)Temperature
(°C)
12
17
22
12
12
12
12
12
12
12
Hardness
Soft
Soft
Soft
Very soft
Soft
Hard
Very hard
Soft
Soft
Soft
pH
7.8
7.8
7.8
8.2
8.2
8.2
8.2
6.5
8.5
9.5
1
36.033.1-39.1
34.532.4-36.7
34.031.3-36.9
49.044.8-53.4
48.043.9-52.3
49.043.4-55.3
49.044.8-53.5
49.044.8-53.5
48.043.9-52.5
42.039.2-45.0
3
35.032.5-37.7
33.031.2-34.9
33.031.2-34.9
35.032.9-37.3
34.732.6-37.0
33.031.2-34.9
34.732.6-36.9
38.034.8-41.5
35.533.0-38.2
35.132.6-37.8
6
29.527.8-31.3
33.031.2-34.9
30.028.3-31.2
34.031.9-36.2
34.732.6-37.0
33.031.2-34.9
34.732.6-36.9
32.030.5-33.6
35.032.9-37.3
33.031.2-34.9
12
29.027.2-30.9
31.029.4-32.7
28.026.7-29.4
31.029.5-32.5
30.228.7-31.8
30.028.4-31.7
30.028.2-31.9
31.029.5-32.6
31.530.0-33.1
29.027.2-30.9
24
28.026.7-29.3
29.026.9-31.2
27.926.5-29.3
30.028.2-31.9
30.228.7-31.8
30.028.2-31.9
29.427.6-31.3
30.128.5-31.8
31.029.3-32.7
29.027.2-30.9
96
13.512.3-14.8
16.014.1-18.2
13.811.3-16.8
20.017.9-22.5
15.813.7-18.2
28.026.6-29.4
15.013.8-16.3
27.526.1-29.0
28.026.2-30.0
26.023.7-28.5
EFFECTS OF WATER TEMPERATURE, HARDNESS, AND pH
with the calculated concentrations (Table 5). At the begin ning of the exposures, concentrations in the water samples were within 5% of the calculated values. Analysis of the same vessels 96 h later showed little, if any, change in the benzocaine concentration. Also, water temperature, hard ness, and pH did not affect the rate of degradation of benzocaine over the 96-h period.
Use Pattern Exposure
Rainbow trout exposed to benzocaine at the use pattern concentration of 25 mg/L for 15 min recovered from the anesthesia within 15 min or less and responded much like control fish during the 14-day postexposure period. How ever, all fish exposed to 3 or 5 times the effective concen tration died within 30 min.
Discussion
The use pattern concentration of 25 mg/L was chosen to represent the level that was effective for anesthetizing small salmonids (Gilderhus 1989). At that level, fish were effectively anesthetized in 3 min or less, recovered from anesthesia in less than 5 min, and survived 15 min of exposure. In our use pattern exposures, the fish were also exposed for 15 min, which is about 5 times the duration necessary for effective anesthesia. Although the X3 and X5 concentrations were lethal in 15-min exposures, they would be much less toxic in 3-min exposures. However, the margin of safety to treated fish is not high, and users
should be aware that overdosing could result in undue stress or mortality.
Benzocaine is uniformly toxic to different species of fish and at various water temperatures, pH's, and hard nesses. This consistency is an advantage because users need not be concerned about alterations in safety to the fish. The toxicity of many other fishery chemicals is influ enced by water characteristics, especially pH (Hunn and Alien 1974). Fasman (1976) reported the ionization con stant (pka) for benzocaine to be 2.38. At this pH, half the benzocaine would be in the ionized form and half in the un-ionized form (Dawson and Gilderhus 1979). At the pH's of 6.5-9.5, there would be little change in the concen tration of the un-ionized, lipid soluble form of benzocaine available for uptake; thus, little, if any, change in toxicity at these pH's would be expected.
The 96-h exposures were of little relevance for evaluat ing safety, but the values generated demonstrate that expo sure time beyond 1 h does not critically increase the tox icity. The concentrations of benzocaine in the water solutions remained nearly constant for 96 h, indicating that the chemical was not degraded and the fish did not remove a significant portion of the chemical at this loading rate. In some of the exposures for 96 h, dissolved oxygen fell below 50% saturation. As a result, toxicity increased in those tests. The ethanol solvent seemingly provided bacteria with a nutrient source that allowed them to prolif erate in some test vessels. Variability in the 96-h results is probably due to these bacterial growths.
Toxicity of benzocaine to fish compares closely to that of MS-222. Marking (1967) reported that for MS-222, the
Table 5. HPLC analysis of benzocaine concentrations of 7.5, 10, and 30 mg/L (calculated) in water at selectedtemperatures, hardness, andpH's at 0 and 96 h.
Benzocaine concentration (mg/L)
Temperature
7121712121212121212
Hardness
SoftSoftSoftVery softSoftHardVery hardSoftSoftSoft
pH
7.87.87.88.28.28.28.26.58.59.5
Calculated
1010107.57.57.57.57.57.57.5
Measured
0 ha, 96 ha
9.5, 9.49.4, 9.39.6, 9.0b7.1,6.47.3, 6.77.3, 6.97.3b, 6.86.8, 6.67.1 b, 6.86.9, 6.7
Calculated
30303030303030303030
Measured
0 ha, 96 ha
28.5, 29.428.4, 29.429.2, 29.328.5, 28.128.2, 29.128.4, 29.427.3, 27.627.0, 27.828.3,29.126.8, 26.9
"Duplicate samples. bAverage of triplicate analysis.
INVESTIGATIONS IN FISH CONTROL 102
24-h LCSO's ranged from 34 to 64 mg/L. Although the values for MS-222 are slightly higher than those for ben- zocaine, MS-222 was formulated as a methanesulfonate salt. The higher molecular weight of the formulation would be expected to yield an anesthetic less active than the benzocaine formulation. The toxicity of MS-222 also was affected little by water hardness or temperatures, and safety for rainbow trout in 15-min exposures to MS-222 was similar to that for benzocaine.
References
ASTM Committee E-35 on Pesticides. 1980. Standard practice for conducting acute toxicity tests with fishes, macroinverte- brates, and amphibians E729-80. Pages 1-25 in Annual book of ASTM standards. Part 46. End use and consumer products. American Society for Testing and Materials, Philadelphia, Pa.
Bell, G. R. 1967. A guide to the properties, characteristics, and uses of some general anesthetics for fish. 2nd ed. Bull. Fish. Res. Board Can. 148. 9 pp.
Committee on Methods for Acute Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. Environ. Prot. Agency, Ecol. Res. Serv., EPA 660/3-75-09. 61 pp.
Dawson, V. K., and P. A. Gilderhus. 1979. Ethyl-p-aminobenzo- ate (benzocaine): efficacy as an anesthetic for five species of freshwater fish. U.S. Fish Wildl. Serv., Invest. Fish Control 87. 5pp.
Fasman, G. D., editor. 1976. CRC handbook of biochemistry and molecular biology, section D Physical and chemical data. 3rd ed. Vol. 1. CRC Press, Cleveland, Ohio. 576 pp.
Gilderhus, P. A. 1989. Efficacy of benzocaine as an anesthetic for
salmonid fishes. N. Am. J. Fish. Manage. 9:150-153.Gilderhus, P. A., B. L. Berger, J. B. Sills, and P. D. Harmon. 1973.
The efficacy of quinaldine sulfate: MS-222 mixtures for the anesthetization of freshwater fish. U.S. Fish Wildl. Serv., In vest. Fish Control 54. 9 pp.
Gilderhus, P. A., and L. L. Marking. 1987. Comparative efficacy of 16 anesthetic chemicals on rainbow trout. N. Am. J. Fish. Manage. 7:288-292.
Hunn, J. B., and J. L. Alien. 1974. Movement of drugs across the gills of fishes. Pages 47-55 in H. W. Elliot, ed. Annual review of pharmacology. Vol. 14. Annual Reviews, Inc., Palo Alto, Calif.
Lennon, R. E., and C. R. Walker. 1964. Laboratories and methods for screening fish-control chemicals. U.S. Fish Wildl. Serv., Invest. Fish Control 1 (Circ. 185). 15 pp.
Litchfield, J. T, Jr., and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99-113.
Marking, L. L. 1967. Toxicity of MS-222 to selected fishes. U.S. Fish Wildl. Serv., Invest. Fish Control 12. 10 pp.
Marking, L. L. 1975. Toxicological protocol for the development of piscicides. Pages 26-31 in P. H. Eschmeyer, ed. Rehabilita tion of fish populations with toxicants: A symposium. Am. Fish. Soc. Spec. Publ. 4.
Marking, L. L., and F. P. Meyer. 1985. Are better anesthetics needed in fisheries? Fisheries (Bethesda) 10(6):2-5.
McErlean, A. J. 1967. Ethyl p-aminobenzoate: an anesthetic for cold-blooded vertebrates. Copeia 1967:239-240.
McFarland, W. H. 1959. A study of the effects of anesthetics on the behavior and physiology of fishes. Publ. Inst. Mar. Sci., Univ. Tex. 6:23-55.
U.S. Department of Agriculture. 1986. Interregional Research Project No. 4. Guidelines for IR-4 investigations. New Jersey Agricultural Experiment Station, Cook College, Rutgers Uni versity, New Brunswick. 6 pp.
Bill
s, T
erry
D.,
Geo
rge
E.
How
e, a
nd L
eif
L. M
arki
ng.
1990
. E
ffec
ts o
f W
ater
T
empe
ratu
re, H
ardn
ess,
and
pH
on
the
Tox
icit
y of
Ben
zoca
ine
to E
leve
n F
resh
wat
er F
ishe
s. U
.S.
Fish
. W
ildl.
Serv
., In
vest
. F
ish
Con
trol
102
. 6
pp.
The
toxi
city
of b
enzo
cain
e (e
thyl
4-a
min
oben
zoat
e; 9
8%)
to e
leve
n fr
eshw
ater
fi
shes
was
eva
luat
ed u
nder
var
ious
phy
sica
l an
d ch
emic
al c
ondi
tions
. U
nder
st
anda
rd te
st c
ondi
tions
(pH
7.8
,12°
C, s
oft w
ater
), th
e 24
-h L
C50
(mg/
L)
17.2
for
lake
stu
rgeo
n (A
cipe
nser
fulv
esce
ns),
was
22.
5 fo
r ra
inbo
w tr
out
(Onc
orhy
nchu
s m
ykis
s), 3
4.0
for
mus
kellu
nge
(Eso
x m
asqu
inon
gy),
24.
0 fo
r nor
ther
n pi
ke (
Eso
x lu
cius
),
19.0
for
com
mon
car
p (C
ypri
nus
carp
io),
25.
9 fo
r fa
thea
d m
inno
w
(Pim
epha
les
prom
elas
), 2
8.0
for
chan
nel
catf
ish
(Ict
alur
us p
unct
atus
), 2
8.1
for
stri
ped
bass
(M
oron
e sa
xati
lis)
, 21.
9 fo
r gr
een
sunf
ish
(Lep
omis
cya
nell
us),
21.
9 fo
r bl
uegi
ll (L
. m
acro
chir
us),
and
22.
0 fo
r w
alle
ye (
Stiz
oste
dion
vitr
eum
). T
ests
co
nduc
ted
at t
hree
wat
er t
empe
ratu
res,
fou
r ha
rdne
sses
, an
d th
ree
pH's
sho
wed
th
at n
one
of th
ese
vari
able
s in
flue
nced
toxi
city
. The
hig
her t
empe
ratu
res
test
ed (t
o 22
° C
) di
d no
t inc
reas
e th
e ra
te o
f to
xico
sis.
Rai
nbow
tro
ut s
urvi
ved
expo
sure
to
the
reco
mm
ende
d us
e co
ncen
trat
ion
of 2
5 m
g/L
for
15
min
but
did
not
sur
vive
ex
posu
re t
o 75
or
125
mg/
L.
Use
rs o
f be
nzoc
aine
sho
uld
be c
autio
ned
that
ov
erdo
ses
may
lea
d to
und
ue s
tres
s or
mor
talit
y in
exp
osed
fis
h. C
ompa
riso
n of
be
nzoc
aine
con
cent
ratio
ns b
y hi
gh p
erfo
rman
ce l
iqui
d ch
rom
atog
raph
y at
0 a
nd
96 h
sho
wed
no
degr
adat
ion
of th
e co
mpo
und
duri
ng t
he e
xpos
ure
peri
od.
Key
wor
ds:
Tox
icity
, ben
zoca
ine,
ane
sthe
tic,
envi
ronm
enta
l fac
tors
, fis
h._
Bill
s, T
erry
D.,
Geo
rge
E.
How
e, a
nd L
eif
L.
Mar
king
. 19
90.
Eff
ects
of
Wat
er
Tem
pera
ture
, Har
dnes
s, a
nd p
H o
n th
e T
oxic
ity
of B
enzo
cain
e to
Ele
ven
Fre
shw
ater
Fis
hes.
U.S
. Fi
sh.
Wild
l. Se
rv.,
Inve
st.
Fis
h C
ontr
ol 1
02.
6 pp
.
The
toxi
city
of b
enzo
cain
e (e
thyl
4-a
min
oben
zoat
e; 9
8%)
to e
leve
n fr
eshw
ater
fi
shes
was
eva
luat
ed u
nder
var
ious
phy
sica
l an
d ch
emic
al c
ondi
tions
. U
nder
st
anda
rd te
st c
ondi
tions
(pH
7.8
,12°
C, s
oft w
ater
), th
e 24
-h L
C50
(m
g/L
) 17
.2 fo
r la
ke s
turg
eon
(Aci
pens
erfu
lves
cens
), w
as 2
2.5
for
rain
bow
tro
ut (
Onc
orhy
nchu
s m
ykis
s),
34.0
for
mus
kellu
nge
(Eso
x m
asqu
inon
gy),
24.
0 fo
r nor
ther
n pi
ke (
Eso
x lu
cius
),
19.0
for
com
mon
car
p (C
ypri
nus
carp
io),
25.
9 fo
r fa
thea
d m
inno
w
(Pim
epha
les
prom
elas
), 2
8.0
for
chan
nel
catf
ish
(Ict
alur
us p
unct
atus
), 2
8.1
for
stri
ped
bass
(M
oron
e sa
xati
lis)
, 21.
9 fo
r gr
een
sunf
ish
(Lep
omis
cya
nell
us),
21.
9 fo
r bl
uegi
ll (L
. m
acro
chir
us),
and
22.
0 fo
r w
alle
ye (
Stiz
oste
dion
vitr
eum
). T
ests
co
nduc
ted
at t
hree
wat
er t
empe
ratu
res,
fou
r ha
rdne
sses
, an
d th
ree
pH's
sho
wed
th
at n
one
of th
ese
vari
able
s in
flue
nced
toxi
city
. The
hig
her t
empe
ratu
res
test
ed (
to
22°
C)
did
not
incr
ease
the
rat
e of
toxi
cosi
s. R
ainb
ow t
rout
sur
vive
d ex
posu
re t
o th
e re
com
men
ded
use
conc
entr
atio
n of
25
mg/
L f
or 1
5 m
in b
ut d
id n
ot s
urvi
ve
expo
sure
to
75 o
r 12
5 m
g/L
. U
sers
of
benz
ocai
ne s
houl
d be
cau
tione
d th
at
over
dose
s m
ay l
ead
to u
ndue
str
ess
or m
orta
lity
in e
xpos
ed f
ish.
Com
pari
son
of
benz
ocai
ne c
once
ntra
tions
by
high
per
form
ance
liq
uid
chro
mat
ogra
phy
at 0
and
96
h s
how
ed n
o de
grad
atio
n of
the
com
poun
d du
ring
the
expo
sure
per
iod.
Key
wor
ds:
Tox
icity
, ben
zoca
ine,
ane
sthe
tic, e
nvir
onm
enta
l fa
ctor
s, f
ish.
Bill
s, T
erry
D.,
Geo
rge
E. H
owe,
and
Lei
f L. M
arki
ng.
1990
. Eff
ects
of W
ater
Tem
pera
ture
, Har
dnes
s,
and
pH o
n th
e T
oxic
ity o
f Ben
zoca
ine
to E
leve
n Fr
eshw
ater
Fis
hes.
U.S
. Fis
h. W
ildl.
Serv
., In
vest
. F
ish
Con
trol
102
. 6 p
p.
The
toxi
city
of b
enzo
cain
e (e
thyl
4-a
min
oben
zoat
e; 9
8%)
to e
leve
n fr
eshw
ater
fis
hes
was
eva
luat
ed
unde
r va
rious
phy
sica
l an
d ch
emic
al c
ondi
tions
. U
nder
sta
ndar
d te
st c
ondi
tions
(pH
7.8
, 12
° C
, so
ft w
ater
), th
e 24
-h L
C50
(m
g/L)
17.
2 fo
r lak
e st
urge
on (
Aci
pens
erfu
lves
cens
), w
as 2
2.5
for r
ainb
ow tr
out
(Onc
orhy
nchu
s m
ykis
s), 3
4.0
for m
uske
llung
e (E
sox
mas
quin
ongy
), 24
.0 fo
r nor
ther
n pi
ke (
Eso
x lu
cius
), 19
.0 f
or c
omm
on c
arp
(Cyp
rinu
s ca
rpio
), 25
.9 f
or f
athe
ad m
inno
w (
Pim
epha
les
prom
elas
), 28
.0 f
or
chan
nel
catf
ish
(Ict
alur
us p
unct
atus
), 2
8.1
for
strip
ed b
ass
(Mor
one
saxa
tilis
), 2
1.9
for
gree
n su
nfis
h (L
epom
is c
yane
llus)
, 21.
9 fo
r blu
egill
(L. m
acro
chir
us),
and
22.0
for w
alle
ye (
Stiz
oste
dion
vitr
eum
). Te
sts
cond
ucte
d at
thr
ee w
ater
tem
pera
ture
s, f
our
hard
ness
es,
and
thre
e pH
's sh
owed
tha
t no
ne o
f th
ese
varia
bles
inf
luen
ced
toxi
city
. Th
e hi
gher
tem
pera
ture
s te
sted
(to
22°
C)
did
not
incr
ease
the
rat
e of
to
xico
sis.
Rai
nbow
trou
t sur
vive
d ex
posu
re to
the
reco
mm
ende
d us
e co
ncen
trat
ion
of 2
5 m
g/L
for
15 m
in
but d
id n
ot s
urvi
ve e
xpos
ure
to 7
5 or
125
mg/
L. U
sers
of b
enzo
cain
e sh
ould
be
caut
ione
d th
at o
verd
oses
m
ay le
ad to
und
ue s
tress
or m
orta
lity
in e
xpos
ed fi
sh. C
ompa
riso
n of
ben
zoca
ine
conc
entr
atio
ns b
y hi
gh
perf
orm
ance
liq
uid
chro
mat
ogra
phy
at 0
and
96
h sh
owed
no
degr
adat
ion
of th
e co
mpo
und
duri
ng t
he
expo
sure
per
iod.
Key
wor
ds:
Toxi
city
, ben
zoca
ine,
ane
sthe
tic, e
nvir
onm
enta
l fa
ctor
s, f
ish.
Bill
s, T
erry
D.,
Geo
rge
E. H
owe,
and
Lei
f L. M
arki
ng.
1990
. Eff
ects
of W
ater
Tem
pera
ture
, Har
dnes
s,
and
pH o
n th
e T
oxic
ity o
f Ben
zoca
ine
to E
leve
n Fr
eshw
ater
Fis
hes.
U.S
. Fis
h. W
ildl.
Serv
., In
vest
. F
ish
Con
trol
102
. 6 p
p.
The
toxi
city
of b
enzo
cain
e (e
thyl
4-a
min
oben
zoat
e; 9
8%)
to e
leve
n fr
eshw
ater
fis
hes
was
eva
luat
ed
unde
r va
riou
s ph
ysic
al a
nd c
hem
ical
con
ditio
ns.
Und
er s
tand
ard
test
con
ditio
ns (
pH 7
.8,
12°
C,
soft
wat
er),
the
24-h
LC
50 (
mg/
L)
17.2
for
lake
stu
rgeo
n (A
cipe
nser
fulv
esce
ns),
was
22.
5 fo
r rai
nbow
trou
t (O
ncor
hync
hus m
ykis
s), 3
4.0
for m
uske
llung
e (E
sox
mas
quin
ongy
), 24
.0 fo
r nor
ther
n pi
ke (
Eso
x lu
cius
), 19
.0 f
or c
omm
on c
arp
(Cyp
rinu
s ca
rpio
), 25
.9 f
or f
athe
ad m
inno
w (
Pim
epha
les
prom
elas
), 28
.0 f
or
chan
nel
catfi
sh (
Icta
luru
s pu
ncta
tus)
, 28
.1 f
or s
tripe
d ba
ss (
Mor
one
saxa
tilis
), 21
.9 f
or g
reen
sun
fish
(L
epom
is c
yane
llus)
, 21.
9 fo
r blu
egill
(L. m
acro
chir
us),
and
22.0
for w
alle
ye (S
tizos
tedi
on v
itreu
m).
Test
s co
nduc
ted
at t
hree
wat
er t
empe
ratu
res,
fou
r ha
rdne
sses
, an
d th
ree
pH's
show
ed t
hat
none
of
thes
e va
riab
les
infl
uenc
ed t
oxic
ity.
The
high
er t
empe
ratu
res
test
ed (
to 2
2° C
) di
d no
t in
crea
se t
he r
ate
of
toxi
cosi
s. R
ainb
ow tr
out s
urvi
ved
expo
sure
to th
e re
com
men
ded
use
conc
entr
atio
n of
25
mg/
L fo
r 15
min
bu
t did
not
sur
vive
exp
osur
e to
75
or 1
25 m
g/L.
Use
rs o
f ben
zoca
ine
shou
ld b
e ca
utio
ned
that
ove
rdos
es
may
lead
to u
ndue
stre
ss o
r mor
talit
y in
exp
osed
fish
. Com
pari
son
of b
enzo
cain
e co
ncen
trat
ions
by
high
pe
rfor
man
ce l
iqui
d ch
rom
atog
raph
y at
0 a
nd 9
6 h
show
ed n
o de
grad
atio
n of
the
com
poun
d du
ring
the
ex
posu
re p
erio
d.
Key
wor
ds:
Toxi
city
, ben
zoca
ine,
ane
sthe
tic, e
nvir
onm
enta
l fac
tors
, fis
h.
(Reports 87 through 89 are in one cover.)87. Ethyl-p-aminobenzoate (Benzocaine): Efficacy as an Anesthetic for Five Species of Freshwater Fish, by
V. K. Dawson and P. A. Gilderhus. 1979. 5 pp.88. Influences of Selected Environmental Factors on the Activity of a Prospective Fish Toxicant, 2-(Digeranyl-amino)-
ethanol, in Laboratory Tests, by C. A. Launer and T D. Bills. 1979. 4 pp.89. Toxicities of the Lampricides 3-Trifluoromethyl-4-nitrophenol (TFM) and the 2-Aminoethanol Salt of 2',5-Dichlo-
ro-4'-nitrosalicylanilide (Bayer 73) to Four Bird Species, by R. H. Hudson. 1979. 5 pp.
(Reports 90 and 91 are in one cover.)90. Accumulation and Loss of 2',5-Dichloro-4'-nitrosalicylanilide (Bayer 73) by Fish: Laboratory Studies, by
V. K. Dawson. J. B. Sills, and Charles W. Luhning. 1982. 5 pp.91. Effects of Synergized Rotenone on Nontarget Organisms in Ponds, by R. M. Burress. 1982. 7 pp.
(Reports 92 through 94 are in one cover.)92. Acute and Chronic Toxicity of Rotenone to Daphnia magna, by J. J. Rach, T. D. Bills, and L. L. Marking. 1988.5 pp.93. Toxicity of Rotenone to Developing Rainbow Trout, by T. D. Bills, J. J. Rach, and L. L. Marking. 1988. 3 pp.94. Oral Toxicity of Rotenone to Mammals, by L. L. Marking. 1988. 5 pp.
95. Deposition and Persistence of Rotenone in Shallow Ponds During Cold and Warm Seasons, by P. A. Gilderhus, V. K. Dawson, and J. L. Alien. 1988. 7 pp.
(Reports 96 and 97 are in one cover.)96. Effects of Environmental Factors on the Toxicity of Chloramine-T to Fish, by T. D. Bills, L. L. Marking,
V. K. Dawson, and J. J. Rach. 1988. 6 pp.97. Effects of Organic Matter and Loading Rates of Fish on the Toxicity of Chloramine-T, by T. D. Bills, L. L. Marking,
V. K. Dawson, and G. E. Howe. 1988. 4 pp.
98. History of Acute Toxicity Tests with Fish, 1963-1987, by J. B. Hunn. 1989. 16 pp.
99. Evaluation of 215 Candidate Fungicides for Use in Fish Culture, by T. A. Bailey and S. M. Jeffrey. 1989. 9 pp.
NOTE: Use of trade names does not imply U. S. Government endorsement of commercial products.
TAKE PRIDEin America
U.S. DEPARTMENT OF THE INTERIORFISH AND WILDLIFE SERVICE
As the Nation's principal conservation agency, the Department of the Interior has responsibility for most of our nationally owned public lands and natural resources. This includes fostering the wisest use of our land and water resources, protecting our fish and wildlife, preserving the environmental and cultural values of our national parks and historical places, and providing for the enjoyment of life through outdoor recreation. The Department assesses our energy and mineral resources and works to assure that their development is in the best interests of all our people. The Department also has a major responsibility for American Indian reservation communities and for people who live in island territories under U.S. administration.