INVESTIGATIONS IN FISH CONTROL - USGS · 2010-11-01 · Investigations in Fish Control, published by the Fish and Wildlife Service, include reports on the results of work at the National

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



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-nitrophenol (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



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.


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



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


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), muskellunge (Esox masquinongy), northern pike (Esox lucius\ common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), channel catfish (Ictalurus punctatus), 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 bluegill) 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 chromatography (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


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


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


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.


24-h LCSO's ranged from 34 to 64 mg/L. Although the values for MS-222 are slightly higher than those for benzocaine, 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, macroinvertebrates, 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-aminobenzoate (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.

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INVESTIGATIONS IN FISH CONTROL - USGS · 2010-11-01 · Investigations in Fish Control, published by the Fish and Wildlife Service, include reports on the results of work at the National