DOWNSTREAM MIGRATION (%)

70

50

---------- @--~--~~--y__~__@03 mg/liter |

trol 4
=S5Sng/liter

12 mg/liter
er | 18 mg/liter:

T I.
@----- Wf--- =-925 mg/liter

trol
—— ames Seu

Figure 8.

| _
3|

DAYS: POST RELEASE

Percent downstream mi
exposure to Tordon 101. A.

group following 360 h of Tordon 101 exposure. B
salmon released per group following 96 h of Tordon

gration of yearling coho salmon following
represents 209-213 coho salmon released per

+ Tepresents 107-115 coho
101 exposure.

50


9L

TABLE 3. ACUTE TOXICITY OF ACROLEIN TO VARIOUS FISH SPECIES.

Toxicity Le 5017
Species test (ug/t) pH Temperature. Alkalinity Hardness Reference
Ania calva (bowfin) static, lab 62 (24 h) 7.27.3 69-72°F 89-93 4O-41 Louder and McCoy (1962)
Carassius auratus (goldfish) field pond 2000(24 h) Jordan et al. (1962)
Carassius auratus static, lab 1000(3 h)2/ St. Amant et al. (1964)
field tests 2000(24 h)2/ 69-740F
Fundulus similis (killifish) flowing, lab 240 (48 h)3/ Butler (1965)
Gambusia affinis static, lab 149 (24 h) 7.5-7.3  69-720F 89-93 40-41 Louder and McCoy (1962)
(mosqui tof sh) 61 (48 h)
Lepom!'s macrochirus Flowing, Jab 77 (24 nya’ 60°F Burdick et al. (1964)
Lepomis macrochirus field pond 2000(24 h) : Jordan et al. (1962)
Lepomis macrochirus static, lab 140 (24 h) 7.2-7.3 69-72°F 89-93 4O-41 Louder and McCoy (1962)
{etuceiTts} 100 (96 h)
Micropterus salmoides static, lab 183 (24 h) 7.2-7.3  69-72°F 89-93 4o-41 Louder and McCoy (1962)
(largemouth bass) 160 (96 h)
Oncorhynchus tshawytscha static, lab 80 (24 h) 7-4-7.7 20°C 41-71 Bond et al. (1960)
chinook salmon 1350(2 h)2/
0. kisutch (coho salmon) static, lab 68 (96 h) 7-4-7.6 10°C 79-82 100-101 Lorz et al. (This study)
Pimephales promelas static, lab 150 (24 h) 7.2-7.3  69-720F 89-93 4o-41 Louder and McCoy (1962)
(fathead minnow) 115 (48 h)
Pimephales promelas Flowing, lab 84 (144 bh) 6.6-6.8 250°C 30 32 Macek et al. (1976a)
Rasbora heteromorpha flowing, lab 140 (24 h) 20°C 20 Alabaster (1969)
(harlequin fish) 60 (48 h)
Salmo gairdneri sti lab 5000(24)NES/ —7.5-8.2 55°F Applegate et al. (1957)
(rainbow trout) flowing, lab Tho (24) 8/ Folmar (1976)
static, lab 65 (2h) 74-727 20°C 41-71 Bond et al. (1960)
flowing, lab 46 (24) 60°F Burdick et al. (1964)

Salmo trutta fario
(brown trout)

Uconcentration of acrolein causing 59% mortality of exposed fish In tlme given (except as noted).

2/Total mortality in time given.

Vtest carried out in flowing seawater.

40% mortality.

5/No toxic effect of concentration tested; (no information as to percent active ingredient or why compound reacted so differently).
S/Unpublished data fish pesticide lab. Columbia Mo.


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.

The nine series are

Environmental Health Effects Research
Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)

interagency Energy-Environment Research and Development
“Special” Reports
Miscellaneous Reports

CONODNSWN =

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis

for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.


Lee)

90
_ 80
SZ 70
>
& 60
>
” 50
5
a 40
& 30
ry
a 20
OF
ob a
24 72 120 24 72 120 182 240
Freshwater Seawater

EXPOSURE TIME (hours) «

coho salmon during exposure to

Fi
gure 11. Percent survival of yearling
fval upon transfer to seawater.

di
iquat in freshwater and subsequent surv
TABLE 22. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

R AND THE SUBSEQUENT SURVIVAL

TO DIQUAT IN FRESHWATE!
FOLLOWING TRANSFER TO SEAWATER (APR. I9-MAY 5, 1977).

Concentration Percent Percent
Domi (mg/L) survival2/ Gill survival
ninal measured _(144-h exposure Fw) ATPase2/ (240-h_ SW)
Cc
ye! - 100 5.0 (4) 100
0. 5 0.23 100 NT 100
Vee 0.47-0.49 100 NT 100
5/0 1.05-1.06 100 4.9 (5) 100
10.0 4.94 100 NT 85
15.0 9.45 100 4.5 (6) 57
20.0 14.7 70 NT 36
. 19.4-19.7 42 NT 13
2,
Hiventy fish exposed per concentration.
th sample size in parentheses.

(Na,K)=-stimulated ATPase activity of gills mean wi

59


Hughes, J. S. and J. T. Davis. 1962a.

Toxicity of selected herbicides to
bluegill sunfish. Proc. La. Acad.

Sci. 25:86-93.

i icity to bluegill
Hughes, J. S. and J. T. Davis. 1962b. Comparative toxicity | Game
"3 sunfish and of granular and liquid herbicides. Conf. S. E. Assoc
Fish Comm. Proc. 16:319-323.

: sos il
Hughes, J. S. and J. T. Davis. 1963. Variations in toxicity to bluegil
sunfish of phenoxy herbicides.

Weeds 11(1) :50-53.

ttera-
Johnson, D. W. 1968. Pesticides and fishes - A review of selected liter:
ture. Trans. Amer. Fish. Soc. 97(4):398-424,

i jon

Johnson, S. L., R. D. Ewing and J. A, Lichatowich. 1977. Character izat in"
of gill (NatK)-activated adenosine triphosphatase from chinook sa
Oncorhynchus tshawytscha.

J. Exp. Zool. 199:345-354,

Jones, R. 0. 1962. Tolerance of the fry of common warm-water fishes te
some chemicals employed in fish culture. Proc. Ann. Conf. Southe
Assoc. Game Fish Comm, 16:436-445,

Jordan, L. S., B. E. Day and R. T. Hendrixon. 1962. Chemical contro! of
filamentous green algae.

Hilgardia. 32(9):441-443.

Phenols. pp. 665-707. In: P. C. Kearney and 0. oe
Herbicides:chemistry, degradation and mode of act
2. Marcel Dekker, Inc., New York, New York.

Kaufman, D.D. 1976,

Kaufman (eds.).
Vol.

Keenleyside, M. H. A. 1967. Effects of forest spraying with DDT in won
Brunswick on food of young Atlantic salmon. J. Fish. Res. Board Can.
24:808-822,

Kenaga, E. E. 1969. Tordon herbicides - evaluation of safety to fish and
birds. Down to Earth. 25(1) 35-9.

Kenaga, E. E. 1974. 2,4,5-T and derivatives:toxicity and stability in
the aquatic environment. Down to Earth. 30(3):19-25.

. id
Kennedy, M. U., B. J. Stojanovic, and F. L. Shuman, Jr. 1969. Chemica} o)
thermal methods for disposal of Pesticides. Residue Reviews. 29:89

Klingman, G. C. and F. M. Ashton.
practices.

1975.

Weed Science:principles and
John Wiley and Sons.

New York. 429 pp.

Koch, R. B., D. Desaiah, H. H,
biphenyls: effect of 1
Pimephales promelas. B

Yap and K. L. Cu 1
on cere exposure on ATPase activity in fish,
iu .

Contam. Toxicol. 7:87-92.


SECTION IV

METHODS
SELECTION OF TOXICANTS AND EXPERIMENTS

The Environmental Management Section (EMS) of the Oregon Department
of Fish and Wildlif,

e@ provided a list of approximately 30 herbicides that
have caused environmental concern in the past few years. This list was iment
reviewed with Dr. Logan Norris (Pacific Northwest Forest and Range Exper
Station) for herbicides that are currently used in forest management an 5
agriculture and could thus be Potential contaminants of aquatic ecosyster .
ce Laboratory agreed to undertake the analysis of water
or residues of herbicides chosen for study. Although in

The study was designed to determine the effects of freshwater exposure
to several herbicides on the acute toxicity of yearling coho salmon and ©}
the subsequent ability of the salmon to adapt to seawater. Following

acute exposure to a given herbicide, (Na,K)-stimulated ATPase activity of
the gill was monitored and survival

in seawater observed. If deaths P
occurred during the exposure to a herbicide, histological examination o
several tissues was conducted.

Based on acute ex
chosen for further tes
diquat on growth, seaw.
migratory disposition,
in yearling coho salmon
Study was limited to the

Posures, three of the thirteen herbicides were

ting. The effects of Tordon 101, dinoseb, and

ater tolerance, (Na,K)-stimulated ATPase activitys 5

histopathology, and tissue accumulation of herbicide:

following 12-15 days exposure were examined. Our

smolt life history stage in the salmon's develop~
an effect on year] ing coho salmon

may be considerably different from those which could affect alevins, fry,

or fingerling salmon.

EXPERIMENTAL FISH

Most experiments w.

lere conducted on 12-
1975 year class2/,

. to 17-month-old coho of the
Fish were hatched and reared from fertilized

Vvear class refers to year of spawning.

6


SECTION VII

REFERENCES

insecticides,

A
labaster, J. S. 1969. Survival of fish in 164 herbicides,
Int. Pest.

fungicides, wetting agents and miscellaneous substances.
Control 11(2):29-35.

Studies on the fate of 2,4-D and ester

Aly, 0. M. and S. D. Faust. 1964.
J. Agric. Food Chem. 12:541-

Serlvatives in natural surface waters.

A
Nderson, J. M. 1971. Assessment of the effects of pollutants on physio-~
177: 307-320.

logy and behaviour. Proc. Roy. Soc. Ser. B. Biol. Sci.

Anderson, J. M., and M. R. Peterson. 1969. DDT: Sublethal effects on

brook trout nervous system. Science? 164:440-441.

Applegate, V. C., J. H. Howell, A. E. Hall Jr., and M. M. Smith. 1957-
Toxicity of 4,346 chemicals to larval lampreys and fishes. U. S.
Fish and Wildlife Serv. Spec. Sci. Rep. Fish. 207. 157 pp-

Arthur, J. W. 1970. Chronic effects of linear alkylate sulfonate deter~
gent on Gammarus pseudolimnaeus, Campeloma decisum and Physa integra.
Water Res. 4:251-257.

7

Audus, L. J. 1950. Biological detoxication of 2,4-dichlophenoxyacetic acid

in soils: isolation of an effective organism. Nature 166: 356.

Axe, J. A., A. C. Mathers and A. F. Wiese. 1969. Disappearance of atrazine,
propazine and trifluraline from soil and water. Proc. South. Weed

Conf. 22:367. (Abstr.)-

Battelle-Northwest Laboratories. 1970. Degradation and depletion of herbi-
cides in drainage water and accumulation of residues in crops irrigated

with treated water. Unpublished Report. Richland, Washington.

Beasley, P. G., J. M. Lawrence, and H. | H. Funderburk, Jr. 1965. The
absorption and distribution of cl4-labeled diquat in goldfish,
Carassius auratus (Linnaeus). Proc. South. Weed Control Conf. 18:58).

Berry, ¢. R. uUr., C. B. Schreck, S. Van Horn and R. Strange. 1975-
Angler oriented objectives of an aquatic weed control program.
Annu. Conf. Southeast. Assoc. Game Fish Comm. 29:301-309.

nd M. L. Sharma. 1976.
xic concentrations of diquat.

Proc.

Bimber, D. L., R. W. Boening a Respiratory seress
in yellow perch induced by subto: to ¥-

Sci. 76(2) :87-90.
65


Analysis of surface, subsurface, and finished waters in lowa, where
atrazine is widely used in corn production, indicated that the herbicide
was present in small amounts (<50 ug/L). Atrazine levels in surface waters
correlated with discharge volume data for the river (Richard et al. 1975).

Laboratory and field tests have indicated that atrazine is moderately
toxic to fish in comparison to other herbicides. Macek et al. (1976b) in-
vestigated the effects of atrazine on survival, growth, and reproduction of
three species of fish. Utilizing soft water (hardness 33 to 40 mg/L) and a
éontinuous flow apparatus, Macek et al. were able to show in acute toxicity
tests that both the 96-h and incipient LC50 for fathead minnows were
15 mg/L atrazine (95% Cl 11-20). Their acute 96h LC50 for bluegills was
>8.0 mg/L and the incipient LC50 was 6.7 (5.4-8.4) mg/L atrazine, which
agrees with the 96-h LC50 of approximately 6 mg/L atrazine (wetable powder)
reported by Walker (1964) for this species. The 96-h LC50 for atrazine
toxicity to brook trout reported by Macek et al. (1976b) was 6.3 mg/L (4.1-
9.7) and the incipient LC50 was 4.9 mg/L (4.0-6.0). This is similar to the
48-h LC50 (12.6 mg/L) reported for rainbow trout in a static bioassay
(FWPCA 1968).

Bluegill and fathead minnow spawning, survival, and growth were not
affected by exposure to 0.095 and 0.213 mg/L atrazine, respectively (Macek
et al. 1976b). Hiltibran (1967) found that 10 mg/L granular atrazine did
not affect green sunfish embryo development, or bluegill] and green sunfish
survival over 8 days. Lake chub sucker fry (Ermyzon sucretta) survived
10 mg/L wetable powder atrazine. Similarly, brook trout parental survival,
egg production, and hatchability appeared to be unaffected by exposure to
<0.72 mg/L atrazine (Macek et al. 1976b). Survival and growth of brook
trout fry were, however, significantly reduced following 90 days of
exposure to 0.72, 0.45 and 0.24 mg/L atrazine. Analysis of muscle tissue
from bluegills, fathead minnows, and brook trout indicated that these fish
bloconcentrated detectable amounts of atrazine after prolonged exposure
(Macek et al. 1976b).

Walker (1964) observed no fish mortality after application of 2.0 to
6.0 mg/L atrazine to ponds infested by aquatic weeds. He suggested,
however, that atrazine had the potential to affect fish in ways other than
direct toxicity. A reduction in bottom fauna was observed immediately
following application. Among the most sensitive species were mayflies
(Ephemeroptera), caddisflies (Tricoptera), leeches (Hirudinea) and
gastropods (Musculium). Studies by Macek et al. (1976b) on the chronic
toxicity of atrazine to selected aquatic invertebrates ‘indicated that
morphological development of progeny is particularly sensitive. Exposure
of two successive generations of chironomids to 0.23 mg/L atrazine resulted
in reduced hatching success, larval mortality, developmental retardation,
and a reduction in the percentage of pupating larvae and emerging adults.
Continuous exposure to 0.25 mg/L atrazine significantly reduced production
of Daphnia. Development to the seventh instar of Fy gammarids exposed to
0.14 mg/L atrazine was reduced 25% below that of lower concentrations and
controls.

23


TABLE A-]. CONCENTRATIONS OF 2,4-D AND 2,4,5-T IN STEELHEAD TROUT
FRY FOLLOWING EXPOSURE TO ESTERON BRUSH KILLERS

Exposure concentration Residue found
(ug/L) Time Wet weight of (mg/kg)

nominal measured (h) tissue (g) 2,4-D 2,4,5-T

A. May 31-June 4, 1977

Control 32.9-34.0 96 15.27 <0.05 <0.06

75 35.2-37.2 96 10.30 0.90 1.92
450 77-4 96 11.48 41.0 ree
450 80.1 96 12.20 23.1 40.8
800 b/ 48 10.65 8.70 10.6
800 b/ 48 11.07 19.57 26.28
1200 b/ 48 10.75 8.28 7.81
1200 b/ 48 4.06 1.48 6.61
1200 (+ 48 h clean water)2/ 48 4.49 3.39 9.26
800 (mortalities)2/ 24 1.32 64.5 60.4
1200 (mortalities?2/ 24 16.78 16.47 100.5

B. June 12-16, 1977

Control 6.5-7.2 96 25.11 - -
800 202-269 96 19.6 5.12 13.94
1000 240-243 96 18.85 7.66 24.37
1200 226-246 96 19.98 11.35 25.13

2/whole body residues.
Samples lost during extraction.

95


O-Img/ L O-I mg/L
100 RR TO ie ke hen ake make make aie ake make ake HK

90 14 1Omg/L|
{\N
1 Fr ii \
+N L
s ror ‘ Xing)
xz 60 1, “o }
> |
WM SO- \ \ \
| el ‘
a 40r \ aa —o
O 30 \ &
ia \ 3
a 207 \500 \
loL mg/L % \
a sol A
oa 72 120 48 120 168 240
Freshwater

Seawater
EXPOSURE TIME (hours)

Figure 5. Percent survival of

yearling coho salmon during exposure to
Paraquat-CL in freshwater and s

ubsequent survival upon transfer to seawater-

TABLE 9. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO PARAQUAT-CL IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (MAR. 22-APR. 7, 1977).

Concentration Percent Percent
(mg/L) survivala/ Gill survival
nominal measured ()44-h exposure FW) ATPase2/ (240-h SW)
Control 0 100 3.04 100
0.25 0.14- 0.19 100 NTo/ 100
0.50 0.50- 0.52 100 4LG 100
1.0 1.08- 1.12 100 1.96 100
10.0 10.6 95 1.90 36
50.0 56.3 35 NT 0
100.0 112.7-113.7 0 NT
200.0 238.7-251.2 0 NT
a/ Twenty fish exposed per concentration.
P/ (wa ,X)-stimulated ATPase activity of the gills; mean of 5 fish.
°/not measured.

30


eens —
20ug/liter

(%)

MIGRATION

DOWNSTREAM

*

n n
23

° 1 : 3 5 7 9 iT] 3 16 I
DAY POST RELEASE

ation of yearling coho salmon following

Figure 10. Percent downstream migr:

Xposure to dinoseb (Dow Premerge). A. represents 203-219 coho salmon
360 h of dinoseb exposure. B. represents 98-115
b exposure except for

repeased per group following

th 0 Salmon released per group following 96 h of dinose

r 7 0 ug/L group exposed for 25 h. C. represents 107 and 197 coho salmon
eleased following 1) h exposure to 40 ‘and 60 ug/L dinoseb, respectively.

55


ATPase activity appeared unaffected by the herbicide. Histological exami-
nation of gill, liver, and kidney tissue indicated no apparent effect of
exposure to dicamba (Appendix 11). Following 144-h exposure to the toxicant,
the yearling coho salmon were challenged with seawater. Fish previously
exposed to the lowest concentration, 0.25 mg/L dicamba, showed a 32%
mortality during the 1] days of the seawater challenge (Table 7), however,
no deaths occurred at higher concentrations. The deaths began after 96 h

in seawater and concluded at about 200 h. No explanation is available for
this unusual pattern of mortality. The deaths followed the time pattern of

osmoregulatory failure noted in previous seawater challenge tests following
copper exposure (Lorz and McPherson 1977).

TABLE 7. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO DICAMBA IN FRESHWATER AND SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (JAN. 6-23, 1977).

Concentration Percent Percent

(mg/L) survival2/ Gill survival

nominal measured (144-h exposure FW) ATPaseb/ (268-h sw)
Control 0) 100 2.52 100.0
0.25 0.19- 0.22 100 NTo/ 68.4
0.50 0.40- 0.42 100 NT 100.0
1.0 0.54- 0.56 100 NT 100.0
5.0 3.15- 3.33 100 1.64 100.0
10.0 10.05-10.11 100 NT 100.0
50.0 50.5 -53.2 100 2.04 100.0
100.0 108.2-109.9 100 2.20 100.0

a Twenty fish exposed per concentration.

=< (Na,K)-stimulated ATPase activit:

y Of gills; mean of 5 fish.
&/Not measured.

KRENITE

Review of Literature: Toxicity to Fish and Behavior in the Environment

Krenite is a postemergence herbicide containing 41.5% (4 Ib a.i./gal)
ammonium ethy] carbamoyl phosphonate. It is registered for brush control on
Non-cropland areas such as railway and highway right-of-ways. drainage -ditch
banks, industrial plant sites, and other similar areas, including

use around domestic water supply reservoirs, lakes, and ponds. Krenite brush
control agent is absorbed and tr

t anstocated by plants after foliar appli-
cation, but little effect is evident until the following year when susceptible
Plants fail to leaf out.

a For this reason application is recommended 2 months
Prior to fall leaf colorati

ton at the rates of 3-5 lb/acre or 4 1bs/100
gal water for spot treatment.

Krenite has a high water solubility, is nonvolatile, and is rapidly
Inactivated in the soil (Oregon Weed Control Handbook 1977). Soil residue

26


chemical by mud particles . Woodiwiss and Fretwell (1974) estimated a 48-h
LC50 of 300 mg/L diquat for

brown trout in relatively hard water; however,
this is more than ten-fold higher than that given by Simonin and Skea
(1977) of 20.5 mg/L in water about one-half as hard. The majority of the
researchers (Table 21) noted that salmonids had a 96-h LC5O of 11-32 mg/L

diquat in moderately hard water. Bond et al. (1960) reported a 48-h Le50
of 28.5 mg/L diquat for chinook salmon whereas in our study, we estimated 7
the 96-h LC50 to be 30 mg/L for coho salmon. We did not test concentrations
>20 mg/L; and thus the shape of the toxicity curve may be considerably
steeper and would give a lower LC50 value. Surber and Pickering (1962)
indicated the importance of hardness, noting that the 96-h LC50 of diquat

in hard water was 2 to 10 times the level observed in soft water tests with
fathead minnows and bluegill sunfish.

Diquat has been shown to be toxic to fish fry. Bluegill, lake chub
sucker, and smallmouth bass fry survived for 3 days or less in 2.5 mg/L

diquat under static conditions (Hiltibran 1967). Folmar (1976) reported
that rainbow trout fry displayed no avoidance of 0.1, 1.0 or 10 mg/L
diquat.

Berry et al. (1975) found that treatment of Chickahominy Reservoir,
Virginia, with diquat resulted in diquat accumulation in the hydrosol.

Similar to other researchers, they found diquat was strongly adsorbed to
soil particles and desorption did not appear to occur.

Diquat has been found to be absorbed by fishes, but it does not :
appear to be cumulative as the herbicide is believed to be metabolized or
voided soon after it disappeared from the water (Beasley et al. 1965,
Calderbank 1972 and Newman 1970). Beasley et al. (1965) injected two .
goldfish intraperitoneally with cl4 labeled diquat and allowed them to swim
freely in Freshwater for 24 and 48h. Two additional fish were exposed to
4 mg/L diquat (C'4 labeled) for 24 and 72 h. The measured radioactivity
suggested that diquat moved primarily in the Plasma and accumulated
predominantly in the gastro-intestin

al tract. Placement of fish in fresh~
water decreased the labeled herbicide in variou
not in the gastro-intestinal tract,

freshwater.

S Organs and tissues, but ie
and increased the radioactivity of t

Experimental Results

Static exposure--

. quat to yearling coho salmon was f
estimated to be 30 mg/L and the 144-h LCO5O was 19 mg/L. When survivors °

Placed in seawater, deaths occurred ina
n all groups of yearling coho exposed to concen

trations greater than 1.0 mg/L (Fig. 11, Table 22). Although our sample
size was small, there was no apparent eff

ect of diquat on the (Na,K)-
stimulated ATPase activity of the gills.

tests were

58


TABLE A-2. SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING COHO SALMON

EXPOSED TO TORDON 101 IN FRESHWATER AND SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER.

Concentration Percent
(mg/L) 2/ Percent Gill survival2
nominal measured survival ATPase2/2/ (240-h SW)

A. 144 h exposure (Mar. 28, 1977-Apr. 13, 1977)

Control (220) 0.02 100 3.64 + 1.46 (19) 100 (20)
0.2 (220) 0.34-0.36 100 100 (22)
0.6 (220) 0.32-0.49 100 3.10 + 1.00 (19) 100 (22)
1.2 (220) -1.07-1,.24 100 loo (22)
1.8 (220) 0.94-1.25 100 3.93 + 1.55 (20) 100 (22)

B. 380 h exposure (Mar. 28, 1977-Apr. 25, 1977)

Control (180) 0.005 100 3.00 + 0.86 (19) 100 (30)
0.3 (180) 0.26-0.39 100 2.54 + 0.99 (19) 100 (30)
0.6 (180) 0.50-0.52 100 2.30 + 0.91 (20) 100 (30)
1.2 (180) —1.33-1.40 100 3.06 + 1.40 (20) 100 (30)
1.8 (180) 1.23+1.39 100 2.43 ¥ 0.80 (20) 96.7 (30)S/

2 Number of fish used in parenthesis.

(Na,K)-stimulated ATPase activity of gill; mean
sampled in parenthesis.

c/Exposed for 480 h to seawater as fish appeared to behave differently than
other groups. One death occurred after 400 h of exposure.

+ SE; number of fish

96


ESTERON BRUSH KILLER

Review of Literature: Toxicity to Fish and Behavior in the Environment

Esteron® Brush Killer contains equal concentrations of 2,4-D and
2,4,5-T [2 1b each/gal (a.e.)] as the Propyleneglycol buty] ether (PGBE)
ester. This ester of 2,4-D and 2,4,5-T is very soluble in oil and organic
solvents but has very low solubility in water. However, in the presence of

emulsifiers and with agitation, an emulsion is formed in water; Esteron
Brush Killer is applied in this manner.

The 2,4-D esters of low-molecular.
vapor pressure (Loos 1976).

are less volatile and present
animals.

-weight alcohols have an appreciable
The long-chain alcohols with an ester linkage
less of a hazard to non-target plants and

The distribution and absorption of these ester formulations is also
influenced by their rate of degradation to the parent compound. The speed

of hydrolysis of 2,4,5-T esters has been related to pH. Alkaline conditions
are more conducive to deesterification of these compounds than are acidic
conditions (Kenaga 1974).

Teasley and Williams (1969, in Kenaga. 1974)
Studied degradation rates of a number of 2,4,5-T esters including the PGBE
ester formulation. In wate

r of pH 6.5 all of the herbicides exhibited
similar rates of deesterification at a concentration of | mg/L (a.e.).

Hydrolysis rates for 2,4-D esters should be faster than those observed for
2,4,5-T esters of comparable structure,

Although most salts of 2,4-D and 2,4,5-T are low in toxicity to fish,
certain ester formulations of these herbicides are toxic to some species at

1 mg/L (Meehan et al. 1974, Kenaga 1974). Particularly toxic to fish are
PGBE esters, butoxyetha

nol (BE) esters, and butyl esters. The 48-h LC50 of
rainbow trout to the PGBE ester of 2,4-D and 2,4,5-T was 980 and 570 ug/L,
respectively (FWPCA 1968).

Much of the increased toxicity of ester formulations over that of
their parent compounds can be exp]

ained by differences in their physical
and chemical. properties. The 2,4,5-T esters have high partition coeffi-
cients, which are related to the greater absorption of the compounds into
fatty tissues such as skin and gills. The PGBE, BE, butyl, and isopropyl
esters of 2,4,5-T have relatively high Partition coefficients, which may
partially explain their high toxicity to fish (Kenaga 1974).
Sublethal effe D d for
fish (Cope 1966), Spawning of s ’ have been demonstrate

Imouth bass fry, 4
icid hey faile
to survive the 8-day duration of the test. cide, as they fa

36


Additional References Not Cited

Akhavein, A. and D. Linscott. 1968. The dipyridylium herbicides paraquat
and diquat. Residue review. 23:97-145.

Albaster, J. S. 1957. The toxicity of certain weedkillers to fruit.
Proc. 3rd. Brit. Weed Control Conf. p. 807-808.

Albaster, J. S. 1959. Toxicity of weedkillers, algicides and fungicides
to trout. Proc. 4th. Brit. Contr. Conf, 2 pp.

Coats, G., H. Funderburk, J. Lawrence and D. Davis. 1964.

Persistence of
diquat and paraquat in pools and Ponds. Proc. So. Weed Conf. 17:308-
320.

Cope, 0. B., E. M. Wood and G. H. Wallen. 1970. Some chronic effects of
2,4-D on the blue

gill (Lepomis macrochirus). Trans. Am. Fish. Soc.
99(1):1-12.

Davis, P. W., J. M. Friedhoff, and G. A. Wedemeyer. 1972. Organochlorine

insecticide, herbicide and Polychlorinated bipheny! (PCB) inhibition

of NaK-ATPase in rainbow trout. Bull. Environ. Contamin. and
Toxicol. 8(2):69-72.

Gangstad, E. 0., et al. 1974. Aquatic-use Patterns for 2,4-D dimethylamine
and integrated control.

U. S. Army Engineer, Vicksburg, Mississippi.
Tech. Rept. No. 7.

Harrison, J. W. E., and E,W. Rees. 1946.

2,4-D toxicity-]. Toxicity
toward certain species of fish.

Amer. J. Pharm. 118(2):422-425.

Hawkes, C. L. and L. A. Norris. 1977, Chronic oral toxicity of 2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD) to rainbow trout. Trans. Am. Fish.
Soc. 106 (6) :641-645.

Hiltibran, R. c. _1972. The hydrolysis of adenosine triphosphate by blue-
gill liver mitochondria in the presence of 2,4,5-T and silver derivatives:
Trans. Ilinois Acad. Sci, 65: 51-57.

Howe, D. J. T., and N. Wright. 1965.

The toxicity of paraquat and diquat.
Proc. 18th N.Z. Weed and Pe y Parad q

st Conf. p. 105-14.

Hughes, J. S. and J. T. Davis. 1962. Median tolerance limits reported in
parts per million of bluegill sunf

. ish to herbicides tested during
1961-1962. La. Wild. and Fish. Comm. Monroe, La. 3 pp.

78


APPENDIX TABLES

Number

CONCENTRATIONS OF 2,4-D AND 2,4,5-T IN STEELHEAD TROUT FRY
FOLLOWING EXPOSURE TO ESTERON BRUSH KILLER

SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING COHO SALMON

EXPOSED TO TORDON 101 IN FRESHWATER AND SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER.............ccccsess sees esse,

EFFECT OF TORDON 10] EXPOS

URE ON AVERAGE LENGTH, WEIGHT,
AND CONDITION FACTOR

PERCENT MIGRATION (TO JULY 6
SALMON RELEASED INTO A SMALL
AND CHRONIC EXPOSURE To ToRDO

» 1977) OF YEARLING COHO
COASTAL STREAM FOLLOWING ACUTE
N 101 (RELEASED APRIL 13, 1977).

PERCENT MIGRATION (TO JULY 6
RELEASED {NTO A SMALL COASTAI
CHRONIC EXPOSURE TO DINOSEB

» 1977) OF YEARLING COHO SALMON
L_ STREAM FOLLOWING ACUTE AND
(RELEASED MAY 5, 1977).......0000
CONCENTRATION OF DINOSEB IN VARIOUS TISSUES OF YEARLING

COHO SALMON FOLLOWING EXPOSURE TO SUBLETHAL AND LETHAL
CONCENTRATIONS OF DINOSEB

EFFECT OF DIQUAT EXPOSURE ON AVERAGE LENGTH, WEIGHT, AND
CONDITION FACTOR

PERCENT MIGRATION (TO JULY 6, 1977) OF YEARLING COHO SALMON
RELEASED INTO A SMALL COASTA\

L STREAM FOLLOWING ACUTE AND
CHRONIC EXPOSURE To DIQUAT (RELEASED MAY 26, 1977)


FOREWORD

Effective regulatory and enforcement actions by the Environmental
Protection Agency would be virtually impossible without sound scientific
data on pollutants and their impact on environmental stability and human
health. Responsibility for building this data base has been assigned to
EPA's Office of Research and Development and its 15 major field installa-
tions, one of which is in the Corvallis Environmental Research Laboratory
(CERL).

The primary mission of the Corvallis Laboratory is research on the
effects of environmental pollutants on terrestrial, freshwater, and marine
ecosystems; the behavior, effects and control of pollutants in lake systems;
and the development of predictive models on the movement of pollutants in
the biosphere.

This report describes a potentially adverse effect of pollutants on
fish such as salmon which must migrate from freshwater to seawater, and
demonstrates that under certain conditions exposure to sublethal levels of
pollutants can result in mortality when fish subsequently enter seawater.
Laboratory test methods are described which should detect this effect in
screening tests and the data obtained in this report should advance
knowledge on the effects of pollutants in aquatic ecosystems.

J. C. McCarty
Acting Director, CERL


and the dimethylamine formulations were the least toxic to bluegills of 11
formulations of 2,4-D tested (800-166 mg/L as 48-h LC50 depending on
batch). The isopropyl ester and butyl ester were the most toxic (0.8 and
1.3 mg/L as 48-h LC50 concentrations, respectively, for bluegills). Davis
and Hardcastle (1959) found differences in LC50 values for 2,4-D and other
herbicides when waters from two different sources were used in toxicity

tests.

Schultz and Harman (1974b) reviewed the literature concerning aquatic
use of 2,4-D compounds as a prerequisite for registration of 2,4-D for use
on irrigation canal banks and for use in moving water. They noted that
many formulations of 2,4-D are available, but the one most commonly used
in aquatic situations is the dimethylamine salt of 2,4-D (DMA-2,4-D). The
ester formulations have also been used, but are many times more toxic to
fish and other aquatic organisms than the dimethylamine salt. Meehan
et al. (1974) tested the toxicity of various formulations of 2,4-D to
salmonids and noted that <50 mg/L 2,4-D acid produced no mortality except
in pink salmon fry. The butyl ester, however, was very toxic causing
almost complete mortality in all species at concentrations >1 mg/L. The
isooctyl ester was the Jeast toxic of the ester formulations. Meehan
concluded that specific phenoxy herbicide ester formulations should be
chosen with regard to their impact on aquatic organisms if there isa
possibility that the chemical will enter the water.

Rodgers and Stal}ing (1972) exposed rainbow trout, channel catfish
and bluegills to a cl4 jabeled butoxyethanol ester of 2,4-D and studied
its uptake from the water by fed and fasted fish. Maximum residue concen-
trations were observed in most organs of fed fish within 1-2 h of exposure
and within 1-8 h exposure for fasted fish. The herbicide or its metabo-
lites were eliminated rapidly after maximum residue concentrations were
reached. Ina large scale application of 2,4-D (butoxyethanol ester) in
seven TVA reservoirs, Smith and Isom (1967) found little uptake of 2,4-D
by fish but some by mussels during 96 h of exposure. Significant concen-
trations of 2,4-D, however, were noted in isolated sediment samples up to

10 months after treatment.

Histological and biochemical changes were observed in bluegill sunfish

exposed to 2,4-D, Esteron 99 (propylene glycol butyl ether ester), in
ponds in Oklahoma (Cope et al. 1970). The pathology involved liver,

vascular system, and brain, with depletion of liver glycogen, globular
deposits in the blood vessels and stasis and engorgement of the brain

circulatory system.

Experimental Results

In our study, no mortalities were observed when yearling coho salmon
were challenged with up to 200 mg/L of 2,4-D (DMA) for 144 h (Table 10).
Similarly, no affect on the (Na,K)-stimulated ATPase activity of the gills
was noted. When coho salmon previously exposed to 2,4-D were challenged
with seawater little mortality occurred (Table 10). Therefore, comparison
of our test results to those of Meehan et al. (1974) indicates that it
would be safest to use the DMA formulation of 2,4-D tf the desired herbicidal

effects could be achieved.

33


re
Only atrazine caused deaths of coho salmon during the freshwater exposu
(25% mortality at 15 mg/L).

Dicamba (Banvel D),
+ 2,4-D) all produced une
salmon Previously exposed to onl

cide. No effect on the (Na,K)-stimulated ATPase activity of the gills

was noted although fish from concentrations causing mortality in sean
water were not always checked.

Acute (96-h) exposure to Tordon 101 had no apparent effect on
Migration of yearling coh

© salmon, whereas the two highest concentrate
did elicit a reduced migratory response of chronically exposed fish
days at 1.2 and 1.8 mg/L) as compared to control or low Tordon 101
€xposure groups. The conc i

Generally, except for the direct toxic effect of acrolein and .
dinoseb and effects of sublethal concentrations of diquat on migration
and seawater survival, the herbicide formulations tested did not appear
to affect smolting of yearling coho salmon Per sé as measured by sea~
water tolerance, (Na,K)-stimulated ATPase activity or migratory tendency:
!n our study, however, we tested only one life Stage of the salmonids
life history; whereas genetics, younger life stages, or food resources

may be considerably more affected as noted by Macek et al. (1976b) in a
study with atrazine,

The insecticides ODT
to modify salmonid Physiol
exposure to sublethal conc.
Peterson 1969, Elson et al
and Johansen 1972, Hatfiel

and sumithion (feni
ogy, behavior,
entrations (And,

trothion) have been shown
and learning ability following
€rson 1971, Anderson and .

+ 1972, Hatfield and Anderson 1972, Hatfield

d and Riche 1970, Keenleyside 1967, Saunders
1369, Symons 1973, 1977, Warner et al. 1966 and Wildish et al. 1971)-

Whether the herbicides tested would elicit Tesponses similar to these
insecticides is unknown. [t does suggest, however, that appropriate
Precautions be taken during the application of any herbicide to water-
sheds containing fish,

64


Mount, D. I. and C. E. Stephan. 1967. A method for establishing acceptable
toxican limits for fish-malathion and the butoxyethanol ester of
2,4-D. Trans. Amer. Fish. Soc. 98:185-193.

Mufrhead-Thompson. R. C. 1971. Pesticides and freshwater fauna. Academic
Press, London. 248 pp.

Mullison, W. R. 1970. Effects of herbicides on water and its inhabitants.
Weed Sci. 18(6):738-750.

National Research Council of Canada. NRC Associate Committee on Scientific
Criteria. 1974. Picloram: the effects of its use as a herbicide on
environmental quality. Publication No. NRCC 13684 of the Environmental

Secretariat. 128 pp.

Newman, J. 1970. Ecological implications of the use of paraquat and diquat
as aquatic herbicides. Chem. and Ind. No. 45:1424-1425.

Newman, J., and J. Way. 1966. Some ecological observations in the use of
paraquat and diquat as aquatic herbicides. Proc. Brit. Weed Contr.

Conf. 8:582-585.

Newman, A. S., J. R. Thomas, and R. L. Walker. 1952. Disappearance of 2,4-
dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid
from sot}. Proc. Soil Sci. Am. 16:21-24.

Newton, M. and J. A. Norgren. 1977. Silvicultural chemicals and
protection of water quality. U. S. Environmental Protection Agency.

EPA 910/9-77-036. Seattle, Wash. 224 pp.

Norris, L. A. 1967. Chemical Brush control and herbicide residues in the
Forest Environment pp. 103-123. In: M. Newton (ed.) Proc.
Symposium Herbicides and Vegetation Management, Corvallis, Oregon.

Norris, L. A. 1970. Degradation of herbicides in the forest floor.
pp. 397-411. In: C. T. Youngberg and C. B, Davey. (eds.). Tree Growth
and Forest Soils. Oregon State Univ. Press, Corvallis, Oregon.

Norris, L. A. 197]. Chemical brush control-assessing the hazard. J.
For. 69(10):715-720.

Norris, L. A. 1978. Toxic materials in forest streams. pp- 43-68.
In: Water Resources Research Institute Seminar. Toxic Materfal in
the Aquatic Environment. Oregon State University, Corvallis, Oregon.

Norris, L. A., M. Newton, and J. Zavitkovski. 1966. Stream contamination
with Amitrole following brush control operations with Amitrole-T.
pp. 20-23. Res. Progr. Rep., West. Weed Contr. Conf.

Norris, L. A. and M. L. Montgomery. 1975. Dicamba residues in streams
after forest spraying. Bull. Envir. Contamin and Toxic. 13(1):1-8.

73


United States Environmental Research EPA-600/3-79-071
Environmental Protection Laboratory June 1979
Agency Corvallis OR 97330

Research and Development

SEPA Effects of Selected
Herbicides on
Smolting of
Coho Salmon

>> SS»


001

TABLE A-6. CONCENTRATION OF DINOSEB IN VARIOUS TISSUES OF YEARLING COHO SALMON FOLLOWING
EXPOSURE TO SUBLETHAL AND LETHAL CONCENTRATIONS OF DINOSEB2/.

Concentration of dinoseb (ug/L)

Exposure concentration Sample skin muscle gill spleen gall bladder liver. kidney
Contro12/ a <0.01 <0.01 <0.09 <0.28 <0.11 <0.05 <0.04
b <0.03 <0.09 <0.15 <0.29 <0.09
20 ug/L (384 h) a <0.02 <0.03 0.09 1.4 <0.15 0.40 0.37
(0.06) (0.3) (0.06 (0.05)
b <0.03 <0.01 0.07 0.15 0.11
(0.03) (0.03) (0.03)
60 pg/L (144 h) a <0.02 <0.01 <0.08 0.61 0.77 <0.07 «0.04
(0. 38) (0.23)
b <0.03 <0.01 <0.09 <0.08 <0.11

a, . : + .
2/ On basis of wet weight of tissue examined;

were combined.

>/yalues given as less than (<) a number are below the detection limits;

indicate the detection limit.

to obtain an adequate tissue sample generally 3-5 fish

numbers in parentheses below a

number


DISCLAIMER

This report has been reviewed by the Corvallis Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify that the contents necessarily reflect
the views and policies of the Environmental Protection Agency, nor does

mention of trade names of commercial products constitute endorsement or
recommendation for use.


Lol

TABLE A-7. EFFECT OF DIQUAT EXPOSURE ON AVERAGE LENGTH, WEIGHT, AND CONDITION FACTOR.

Date Days Nominal con- Fork Condition

of of Tank centration length Weight factor

sample exposure No. (mg/L) cm + SE gtise KFL + SE

May 10, 1977 o2/ 1 Control 16.2 + 0.16 42.7 + 1.35 0.994 + 0.008
0 2 Control 16.8 + 0.27. 47.1 4 2.15 0,980 + 0.011
0 7 0.5 16.8 + 0.31 48.2 + 2.84 0.992 + 0.009
0 8 0.5 17.3 + 0.38 54.2 + 3.51 1.009 + 0.009
0 3 1.0 16.4 + 0.24 44.3 41.91 0.988 ¥ 0.008
0 4 1.0 16.9 0.27 48.74 2.18 0.985 ¥ 0.011
0 5 2.0 16.4 + 0.25 46.3 + 2.01 1.026 + 0.009
0 6 2.0 16.9 + 0.29 50.3 + 2.59 1.019 + 0.011
0 9 3.0 16.6 + 0.25 46.9 + 2.25 1.001 + 0.009
0 10 3.0 16.9 + 0,22 49.6 + 1.98 1.005 + 0.008

May 26, 1977 o/ 1 Control 16.8 + 0.20 47.3 +1.67 0.986 + 0.008
0 2 Control 16.5 + 0.20 44.8 + 1.54 0.984 + 0.009
12 7 0.5 17.5 + 0.28 53.6 + 2.90 0.977 + 0.007
12 8 0.5 16.9 + 0.26 49.2 + 2,28 0.990 ¥ 0.009
12 3 1.0 16.5 + 0.16 45.3 + 1.59 0.987 + 0.013
12 4 1.0 17.0 + 0.21 48.9 + 1.88 0.972 * 0.013
12 5 2.0 17.3 + 0.25 52.9 + 2.63 0.994 + 0.010
12 6 2.0 17.1 + 0.21 50.7 + 1.97 1.003 + 0.015
12 9 3.0 16.7 + 0.23 46.7 2.11 0.981 + 0.008
12 10 3.0 17.1 + 0.22 49.6 + 1.77 0.979 + 0.009

2 sample size 30
2/sample size 40


studies for ammonium thiocyanate (Demyanenko 1941). The reason for the
discrepancy is unknown although differences in water quality or species
tested are known to be important variables. Histological examination of
dying fish showed degenerative changes occurring in the liver, kidney and
gill of fish exposed to 200 mg/L (Appendix 11). The deaths observed in
seawater generally occurred within the first 24-48 h following transfer
from the toxicant and probably were not directly related to impaired
osmoregulatory ability.

TABLE 5. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO AMITROLE-T IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (JAN. 17-FEB. 6, 1977).

Concentration Percent Percent
(mg/L) survival? Gill survival
nominal measured (144-h exposure FW) ATPase?/ (336 h SW)
Control 0.03-0.06 100 2.1 100
0.25 0.20 100 2.1 100
0.50 0.37 100 NTo/ 100
1.0 0.89-0.97 100 2.7 100
25.0 24.8 100 2.9 56.3
50.0 48.6 55 2.4o/ 12.5
100.0 104.5 14.6 NT 0
200.0 200.7 0 NT NT

a/, Twenty to 21 fish exposed per concentration.

B/ (ata ,K) ~stimulated ATPase activity of the gill; mean of 5 fish.
~/Not measured.

~/Mean of 3 fish.

21


APPENDIX |. METHODOLOGY FOR ANALYSIS OF SPECIFIC HERBICIDES.

AMITROLE-T

1.

3.

Summary of method: The method is an adaptation of Southerland
(1964). Aminotriazole is removed from water by adsorption on an
ion exchange resin. The eluate from the resin is decolorized with
activated charcoal. N-(I-Naphthyl) ethylenediamine dihydrochloride
is used to form a chromophoric moiety which is measured in a
colorimeter at 460 nm.

Reagents: Dowex 50W-X8
3-amino-s-triazole
Sulfuric Acid, reagent grade (concentrated, 75% and 0.75%)

Sodium nitrite, reagent grade (0.5% aqueous solution,
prepare fresh daily)

Sulfamic acid, reagent grade (5.0% aqueous solution,
prepare fresh daily)

N-(1-Naphthyl) ethylenediamine dihydrochloride,
reagent grade (1.0% aqueous solution, prepare
fresh daily)

Charcoal, activiated ("Darco'! G-60)
Ammonium hydroxide, reagent grade, 2 Normal solution
Apparatus: Widemouth plastic bottles, 500 mL (sample containers)

Magnetic stirrers and stir bars
Chromatographic columns

Hot plates

Colorimeter

Timer

Suction flask (500 mL)
Sintered glass funnels (medium porosity)
Beakers (600, 400 & 100 mL)
Erlenmeyer flasks (50 mL)
Volumetric flasks (25 mL)

Pipet

Glass beads (3 mm)

81


Schwartz, H. J. Jr. 1967. Microbial degradation of pesticies in aqueous
solution. J. Water Poll. Contr. Fed. 39:1701-1704.

Sergeant, M., D, Blazek, J. H. Elder, C. A. Lembi and D. J. Morre. 1970.
The toxicity of 2,4-D and picloram herbicides to fish. Proc. Indiana

Acad. Sci. 80:114-123.

Simonin, H. A. and J. C. Skea. 1977. Toxicity of diquat and cutrine to
fingerling brown trout. N. Y. Fish and Game Jour. 24(1):37-45.

Smith, G. E. and B. Isom. 1967. Investigations of effects of large-scale
applications of 2,4-D on aquatic fauna and water quality. Pestic.

Monit. J. 1:16-21.

Southerland, G. L. 1964. 3-Amino-s-Triazole. pp. 639-642. In: Gunter
Zweig (ed.) Analytical Methods for Pesticides, Plant Growth Regulators,
and Food Additives. Volume IV, Herbicides. Academic Press. New York.

Sprague, J. B. 1969. Measurement of pollutant toxicity to fish - |.
Bioassay methods for acute toxicity. Water Res. 3:793-821.

St. Amant, J. A., W. C. Johnson, and M. J. Whalls. 1964. Aqualin as a
fish toxicant. Prog. Fish-Cult. 26(2):84-88.

Sund, K. A. 1956. Residual activity of 3-amino-1,2,4-triazole in soils.
J. Agr. Food Chem. 4:57-60.

Surber, E. W. and G. H. Pickering. 1962. Acute toxicity of endothal,
diquat, hyamine, dalapon, and silvex to fish. Prog. Fish Cult.
24:164-171.

Symons, P. E. K. 1973- Behavior of young Atlantic salmon (Salmo salar)
exposed to or force-fed fenitrothion, an organophosphate insecticide.
J. Fish. Res. Bd. Can. 30(5) 2651-655.

Symons, P. E. K. 1977. Dispersal and toxicology of the insecticide feni-
trothion; predicting hazards of forest spraying. Residue Res. 68:
1-6.

Tarrant, R. F., and L. A. Norris. 1967. Residues of herbicides and diesel
cil carriers in forest water. pp- 81-88. In: Proc. Symp. Herb. in
Veg. Manage. Corvallis, Oregon.

Teasley, J. |. and P. W. Williams. 1969. The degradation of four esters
of chlorophenoxyalky! acids used in aquatic weed control. Unpublished
manuscript. Southeast Water Control Laboratory USDI, Athens, Georgia.
Cited In: E. E. Kenaga. 1974. Down to Earth. 30(3) 219-25.

Templeman, W. G. 1955. The use of plant growth substances. Ann. Appl. Biol.
42:162-173.

75


SECTION !11

INTRODUCTION

Herbicides have become a widely used and nearly indispensable tool in
reducing the competition of brushy hardwoods in intensive silviculture of
Douglas-fir in the Pacific Northwest. As Norris (1971) pointed out,
however, it is necessary to know the behavior of these chemicals to allow
their safe use, particularly with respect to water contamination.

Herbicides can enter streams through direct application to stream
surfaces, in overland flow during periods of intense precipitation or by
leaching through the soil profile. The probability of overland flow or
leaching of an herbicide to streams depends largely on the persistence and
Movement characteristics of the chemical, properties of the soil, and the
degree to which precipitation infiltrates the soil surface. Leaching is a
slow process capable of moving only small quantities of chemical short
distances. Overland flow of water (and herbicide) on forest land seldom
occurs because the infiltration characteristics of the forest floor and
soil greatly exceed rates of precipitation (Norris and Moore 1971).

Norris (1967) reported that direct application of chemicals to stream
surfaces is the principal mechanism of chemical entry to aquatic systems.
This type of contamination can be prevented or minimized through the use
of buffer strips and attention to the details of application.

The opportunities for entry of herbicides from agricultural lands to
Streams are similar to those from the forest except where herbicides are
used to contro] aquatic or streambank vegetation. Many forest and agri-
cultural herbicides are applied in the early spring and summer months when
anadromous salmonids are normally migrating downstream. A careful evalua-
tion of the possible toxic hazards of herbicides to aquatic life is
therefore necessary to insure that use of these valuable tools does not
Cause toxic impacts on aquatic organisms.

Evaluation of the probability of occurrence of toxic impacts on
aquatic organisms requires a consideration of two factors. One is the
inherent toxic properties of the chemical involved, and the second is the
Probability that the organisms will be exposed to toxic amounts or concen-
trations of this chemical. The probability of exposure is related to both
temporal and spatial relationships between aquatic organisms, treated
areas, application of herbicides and the behavior of the chemical in the
environment. Behavior of chemicals in the environment includes their
Movement, persistence, and fate within the aerial, terrestrial, and aquatic
Portions of the environment. Of primary importance is the mechanism of
chemical entry to streams.


TABLE 16. SURVIVAL OF YEARLING COHO SALMON AT THREE FISH DENSITIES
EXPOSED TO TORDON 22K OR TORDON 22K PLUS AMMONIUM CHLORIDE
IN FRESHWATER, AND THE SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (FEB. 11-28, 1977).

Fish Concentration (mg/L) Percent survival

density Nominal Measured — NH4CT  NH3-NB/ THI~h

No./tank __Tordon (added) exposure FW 260-h SW
10 Control - 0 0.34 100 100
20 Control - 0 0.70 100 100
5 Control - 0.80 1.05 100 100
10 0.05 0.058 0 0.25 100 100
20 0.05 0.048 0 0.64 100 100
10 0.10 0.11 0 0.38 100 100
20 0.10 0.11 0) 0.63 100 100
10 0.25 0.50 0 0.23 100 100
20 0.25 0.42 0) 0.65 100 100
5 0.25 0.32 0.80 1.01 100 100
10 0.50 0.59 0 0.22 100 100
20 0.50 0.71 0 0.61 100 100
> 0.50 0.67 0.80 1.04 100 100
10 0.80 0.89 0 0.27 100 100

a/ : .
— Measured ammonia level, average of six daily values.

TORDON 101

Review of Literature: Toxicity to Fish and Behavior in the Environment

The Dow Chemical Company's Tordon® 101 formulation contains 5.7% a.e.-

(0.54 1b/gal) triisopropanolamine salt of picloram plus 21.2% a.e.

(2.0 1b/gal) 2,4-D as the triisopropanolamine salt. The interaction of
Picloram with 2,4-D enhances the translocation of 2,4-D in plants (Foy
1976). Other reported interactions are either adaptive or competitive.
Tordon 101 is used in the control of annual and perennial weeds, woody
Plants, and vines on non-crop lands including right-of-ways. Label speci-
fications suggest Tordon 101 application at the rate of 1/2 to 3 gal/acre
for the control of broadleaf weeds, and 1 to 4 gal/acre for woody plants

and vines.
ish has been reviewed by Kenaga (1969).

Salmonids were exposed to varying levels of the toxicant in Lake Huron
water at 10°C. Lynn (1965 in Kenaga 1969). reported the 96-h LC50's for
brook, brown, and rainbow trout were 50.9, 48.8, and 31.8 mg/L (a.e.) 2,4-
D, and 13.7, 13.1 and 8.6 mg/L (a.e.) picloram, respectively. Fathead
minnows were considerably more sensitive to Tordon 101. The 96-h LC50 for
this spectes (10°C) was 13.7 mg/L 2,4-(a.e.) and 3.7 mg/L (a.e.) picloram.

The effect of Tordon 101 on f

45


Reagents: Diethyl ether (distilled over sodium)
Benzene (Burdick & Johnson, Distilled in Glass TM)
Potassium hydroxide (reagent)
Sulfuric acid (concentrated, reagent)
Alumina, activated (Fisher A-540)
Diazomethane
n-Hexane (distilled over sodium)
Acetone (Burdick & Jackson, Distilled in Glass TM)

Apparatus: Separatory funnels fitted with teflon stopcocks
Beakers

Volumetric flasks

Graduated cylinders

Pipet, Pasteur, disposable

Heating mantle, 250 mL

Round-bottom boiling flask (250 mL)

Condenser (West, 24/40 $)

Centrifuge (International Model U or equivalent)

Centrifuge bottles, 250 mL

Tissue grinder (Tek Mar Model SDT or equivalent)

Gas chromatograph (Varian Model 1200 or equivalent
with scandium E.C. detector)

Flash evaporator (CaLab Model 5101 or equivalent)

Procedure:

Sample extraction: Weigh sample, cut in small pieces and place in
250 mL round-bottom boiling flask. Add 50 mL of 95% ethanol, grind
with tissue grinder. Add 100 ml 50% potassium hydroxide in water
solution, place in a heating mantle and attach a reflux condenser.
Reflux for 1 hour. Cool, transfer contents to centrifuge bottle and
centrifuge at 2000 rpm for 20 min. Decant into separatory funnel,
acidify with concentrated sulfuric acid, and extract with three 75 mL

aliquots of benzene. Evaporate the extract to dryness with the aid of
a flash evaporator at 40°C.

Derivation and cleanup: Add the ethanol solution of diazomethane
(Hopps 1970) drop by drop until the yellow color persists. Swirl the
flask gently, allow to stand 10 min, and evaporate just to dryness.
Prepare the alumina column by tamptng a plug of glass wool

into a disposable Pasteur pipet and pour in a I-in layer of activated
alumina. Transfer the methylated residue to the column with two 3 mL

Portions of hexane. Discard the hexane eluate. Elute the DNBP
methyl ester with 4 mL ethyl ether. Collect the ether in a 5 mb
volumetric flask.

Gas chromatography and

quantification are identical with the method for
water analysis.

DIQUAT AND PARAQUAT

This method is taken from Pack (1967). The control water samples were clear
and contained no absorbing species at 31

0 nm or 256 nm, therefore, we were

88


. ‘as
Blackburn, R. D. and L. W. Weldon. 1962. Control of southern maid (Naja
quadalupensis) and othe

r submerged weeds in south Florida trrigaryon
and drainage channels. Proc. South. Weed Control Conf. 15:254-255-
Bohmont, B. L. 1967.

Toxicity of herbicides to livestock, fish, honey
bees and wildlife.

West. Weed Cont. Conf. 21:25-27.

Bond, C. E., R. He Lewis, and J. L. Fryer. 1960.

d.)
herbicidal materials to fishes. Pp. 96-101. In: C. M. Tarzwell, (e

Biological problems in water pollution. Trans. 1959. Semin., Robert
A. Taft Sanit. Eng. Cent. Tech. Rept. W60-3,

Toxicity of various

i r
Bond, C. E., J. D. Fortune, Jr. and F, Young. 1965. Results of preliminary
bioassays with kurosol SL a

nd dicamba. Prog. Fish. Cult. 27(1):49-51-
Burchfield, H. P., D. E. Johnson. 1965.

residues. U.S, Dept. of Health, Edu

Service, Bur. of State Sery. (Env. He:
Washington D.C,

+ icide
Guide to the analysis of pestictt
cation, and Welfare, Public ie
alth). Office of Pesticides,

Burdick, G. E., H. J, Dean, and E. J. Harris. 1964, Toxicity of aqua i.
to fingerling brown trout and bluegills.° N.Y. Fish Game J. 11:10

Burnet, A. M. R. 1972, Effect of paraquat on invertebrates in a Canterbury
Stream, New Zealand. N. Z. J

+ Mar. Freshwater Res. 6(4) :448-455.
Burnside, 0. ¢,

> &. A. Wicks,
dicamba,

: and C. R. Fenster. 1971, Dissipation of 25.

Picloram and 2,3,6-TBA across Nebraska. Weed Sci. 19:323-3

Butler, P. A. 1965, Effects of herbicides on estuarine fauna. Proc. South
Weed Conf, 18:576-580.

ations in the development of
Outlook on Agriculture

Calderbank, A. and P. Slade,

1976. Diquat and Paraquat. pp. 501-540. Ins
P. C. Kearney and p. D. Kaufman (eds.), Herbicides:chemistry, degra~
nylons and mode of action. Vol. 2. Marcel Dekker, Inc., New York,
lew York,

Campbell, R. D., T. P. Leadem gnd0. W. Johnson. 1974. the "In Vivol!
effect of Pp, p!

DDT on Nat-Kt-Activated ATPase activity in rainbow
heen gairdneri). Bull. Environ, Contam. & Toxicol. 11(5):

Carter, L., 1971. Marine Research Station, Br
results, In: Cal]

ixham, England, unpublished
dns derbank, A, and P. Slade. Diquag and
Pp.501-540. In: P

1976. Diquat and paraquat.
ae C. Kearney and D. D. Kaufman (eds.) Herbicides:cheTM
Near gdat on and mode of action. Vol. 2. Marcel Dekker, Inc., New Yorks
et ork.

66

isthY


studies have indicated that, in 2 weeks, half of the herbicide is converted
to carbamoyl phosphonic acid which is subsequently converted to CO) and
humic acid fractions within 8 weeks. Bottom sediments lose Krenite in 3
months or less (Dr. James Harrod, duPont Co., unpublished report).

There is little published data on the toxicity of Krenite to fish.
Unpublished findings of E. |. duPont de Nemours and Company, Inc. (technical
pamphlet) indicate that rainbow trout and fathead minnow have a 96-h LC50
of 1,000 mg/L (product), while bluegill sunfish exhibit a 96-h LC50 of 670
mg/L (product). Laboratory tests have demonstrated that Krenite is not
bioaccumulated. Residues in fish tissues were comparable to the concen-
tration of the herbicide in the water (Newton and Norgren 1977).

Experimental Results

No mortalities were observed in yearling coho salmon exposed to
Krenite concentrations up to 200 mg/L. When survivors were transferred to
seawater only minimal mortality occurred (Table 8). Krenite is not very
toxic to coho salmon, as neither freshwater survival nor subsequent seawater
survival were affected. There was no apparent effect of Krenite on the
(Na,K)-stimulated ATPase activity of the gill (Table 8).

TABLE 8. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO KRENITE IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (FEB. 2-17, 1977).

Concentration Percent Percent
(mg/L) survival 2/ Gill survival
nominal (144-h exposure FW) ATPase®/ (216-h SW)
Control 100 2.2 100
0.25 100 1.8, 93
0.50 100 NT= 100
1.0 100 1.7. - 100
10.0 100 NT 100
50.0 100 NT 100
100.0 100 NT 100
200.0 100 2.7 100

¥, Twenty fish exposed per concentration.
P/ (va ,K) -stimulated ATPase activity of gills; mean of 5 fish.

°/Not measured.

PARAQUAT

Review of Literature: Toxicity to Fish and Behavior in the Environment

Paraquat is the common name for 1,1'-dimethy1-4,4'-bipyridinium ion.
It is formulated under the trade names Ortho Paraquat and GramozoneR.

27


In the first exposure test approximately 50% of the fry exposed to 1200 ug/L
(nominal concentration) for 48 h died (Table 13 A), whereas in a test 2
weeks later, only a single death was recorded at 1200 pg/L even though the
test was run for 96 h (Table 13 B). Unfortunately the water samples from
the first 800 and 1200 ug/L groups were lost during extraction. Thus we do
not know if total concentration of the herbicide were similar between the
two tests. As was noted in the test with yearling coho salmon, the measured
concentration of Brush Killer was only a fraction of the expected. Reasons
for the discrepancy in both the nominal and measured concentrations of the
herbicide and deaths observed between the two tests are unknown.

TABLE 13. SURVIVAL OF BIG CREEK WINTER STEELHEAD TROUT FRY
EXPOSED TO ESTERON BRUSH KILLER, FAIRPLAY LABORATORY,

Osu (MAY 31-JUNE 4, 1977).

Concentration Percent
(ug/L) Exposure survival
nominal measured time-h?/ (range)
A. May 31-June 4, 1977
Control 32.9-34.0 96 (60) 100
1 12.1-12.8 96 (60) 100
7 13.2-13.5 96 (60) 100
25 17.7-17.9 96 (60) 98.3 (100-96.7)
75 35.2-37.2 | 96 (60) 100
150 43.9-44.3 96 (60) 100
450 77.4-80.1 96 (60) / 100
800 b/ 48 (60)2 88.3 (100-76.7)
1200 b/ 48 (63)&/ 50.8 ( 80-24.2)
B. June 12-16, 1977
Control 6.5- 7.2 96 (60) 100
800 202.0-269.0 96 (60) 98.4 (100-96.7)
1000 2h0.0-243.0 96 (60) 98.4 (100-96.7)
1200 226.0-246.0 96 (60) . 98.4 (100-96.7)

a, Total number of fish exposed in replicated tanks under static conditions.

$2) +
BY, Samples lost during extraction.
Tanks stocked June 2, 1977 in the morning;
afternoon of same day.

toxicant exposure initiated in

Wildlife Service, Fish-Pesticide
Research Unit (personal communication) found 96-h LC50 values of 600-1000 pg/L
with the PGBE ester of 2,4-D for cutthroat trout fry. He did not find
toxicity to be affected by pH or water hardness. [na recent publication
Woodward and Mayer (1978) noted that survival of cutthroat trout alevins

was significantly reduced when they were continuously exposed to concen-
trations of 60 and 124 pg/L 2,4-D PGBE ester. The authors suggest the no-
effect concentration for cutthroat trout to be 31 ug/L 2,4-D PGBE ester.

Dr. D. Woodward, U. S. Fish and

39


0-5 mg/L

a ee
100 ‘a en rs ee Cae

90 OL

——~oL, ~ 25a

a Fl _
2 70 HI ~“—O-
z oll
Bll

ail
=
all
© 30} 115m
a& 2otld

10 seis

a a0 48° "96~=—=«44~—~S A288

Freshwater Seawater

EXPOSURE TIME (hours)

cent survival of yearling coho salmon during exposure to
cloram) in freshwater and Subsequent survival upon transfer

Tordon 22K

Figure 6. Per
(pi
to seawater.

ammonia groups (Table 16). Seawater deaths,
therefore, did not appear to be involved with fish density, or ammonia
levels during toxtcant exposure,

Ay


Following a simulated field application Woodward and Mayer (1978) recommended
that water residues of the ester should not exceed 100 ug/L after a single
application nor 31 ug/L following multiple applications.

Histological examination of the steelhead trout fry showed liver and
kidney tissues to be normal, but gill tissue (including controls) showed
evidence of hypertrophy of the chloride cells (Appendix 11).

Examination of steelhead trout fry showed evidence of 2,4-D and
2,4,5-T residues following exposure to Brush Killer (Table A-1). The whole
body residues generally increased with increasing concentration and

exposure time. Fish that were moribund or dead exhibited the highest body

residues (Table A-1). Fish placed in clean water for 48 h following 48 h of
toxicant exposure showed slightly reduced residue levels.

TORDON 22 K (PICLORAM)

Review of Literature: Toxicity to Fish and Behavior in the Environment

Tordon® (4-amino-3,5,6-trichloropicolinic acid) or picloram is one of
the most potent and effective Plant growth regulators currently employed
(Foy 1976). There are three basic formulations: potassium salt,
triisopropanolamine salt, and isooctyl ester. Tordon 22K contains 21.5%
picloram (a.e.) as the potassium salt.

Picloram is effective against most perennial, broadleaf, herbacious
weeds and many woody species (Klingman and Ashton 1975). It ts routinely
used along right-of-ways (utility lines, Pipelines, highways, railways) and
on pastureland, rangeland, and forests, where it is applied at rates
ranging from 1/8 to 2 Ib/acre (a.j.).

The effectiveness of the picloram herbicides has been directly
correlated with their inherent potency, translocatability and comparative
resistence to degradation (Foy 1976). Goring et al. (1965) reported that
losses of picloram in the soil ranged from 58 to 96% within 1 year after
application, and 78-100% within 2 years. Insignificant amounts of picloram
are lost through volatiliz

ation. Picloram has been shown to undergo
extensive photodegradation by ultr.

s aviolet light and sunlight in aqueous
solutions or soil surfaces (Hall e

t al. 1968).

lation of picloram is slow in temperate climates

Ouncil Canada 1974, NRCC), decomposition in the soil

logical processes (Hance 1969). Adsorption to soil
in neutral or alkaline sandy loam soil, but increases
'ncreasing organic content, and increasing concen-
iron and aluminium oxides (Foy 1976).

Tordon 22K is highly soluble in water (40% w/w). Movement of the
herbicide in the soil profile is governed by the net water flow, with
maximum losses occurring under warm, humid conditions, following heavy
rainfall, and in light soils low in organic content. The removal of
picloram through rainfall and leachin

9 Is one of the major factors governing

Although degrad
(National Research C
does occur by nonbio
colloids is minimal
with decreasing pH,
trations of hydrated

4o


Hall, R. C., C. S. Giam, and M. G. Merkle. 1968, The photolytic degra~
dation of picloram. Weed Res. 8:292-297.

Losses of atrazine in run-

Hall, J. K., M. Pawlus and E. R. Higgins. 1972.
1:172-176.

off water and soil sediment. J. Environ. Qual.

Hance, R. J. 1969. Further observations on the decomposition of herbicides
in soil. J. Sci. Food Agric. 20:144-145.

Hardy, J. L. 1966. Effects of Tordon herbicides on aquatic chain organisms.
Down to Earth. 22(2):11-13.

Harris, C. I. and T. J. Sheets. 1965. Persistence of several herbicides

in the field. Proc. N. E. Weed Cont. Conf. 19:359.

Hatfield, C. T., and J. M. Anderson. 1972. Effects of two insecticides on
the vulnerability of Atlantic salmon (Salmo salar) parr to brook trout
(Salvelinus fontinalis) predation. J. Fish. Res. Board Can. 29:27-29.

Hatfield, C. T., and P. H. Johansen. 1972. Effects of four insecticides on
the ability of Atlantic salmon parr (Salmo salar) to learn and retain
a simple conditioned response. J. Fish. Res. Board Can. 29:315-321.
Hatfield, C. T., and L. G. Riche. 1970. Effects of aerial Sumithion®
spraying on juvenile Atlantic salmon (Salmo salar L.) and brook
trout (Salvelinus fontinalis Mitchill) in Newfoundland. Bull.
Contam. Toxicol. 5:440-442.

Environ.

Hiltibran, R. C. 1967. Effects of some herbicides on fertilized fish eggs
and fry. Trans. Am. Fish. Soc. 96 (4) :414-416.

Hiltibran, R. C., D. Underwood, and J. Fickle. 1972. Fate of diquat in
the aquatic environment. Univ. of Illinois, Water Res. Center, Res.

Rep. No. 52. 45 pp-

Hoar, W. §. 1939. The weight-length relationship of the Atlanti
J. Fish. Res. Board Can. 4:441-460.

c salmon.

Hopps, H. B. 1970. Preparations and reactions of Diazomethane. Acta.

3(4) 39,

House, W. B., L. He Goodson, H. M. Gadberry, and K. W. Dockter. | 1967.
Assessment of ecological effects of extensive or repeated use of
herbicides. Advanced Research Projects Agency. Department of Defense.
ARPA No, 1086, Kansas City, Missouri. U. S. Dep. Com. AD #824314.

369 pp.
o striped bass

Hughes, J toxicity of 30 chemicals t
» J. 8. 1973. Acute toxicity of 3 Fish Comm.

(Morone saxatilis). Proc. Annu. Conf. West. Assoc.

53:399-418,

69


Juntunen, E. T., and L. A. Norris. 1972. Field application of herbicides-
avoiding danger to fish. Oreg. Agric. Exp. Stat. Spec. Rept. 354.
26 pp.

Lawrence, J. M. 1962. Aquatic herbicide data. U. S. Dept. of Agric.
Agriculture Handbook No. 231. 133 pp-

Loeb, H. A., and W. H. Kelly. 1963. Acute or oral toxicity of 1,496
chemicals force-fed to carp. U. S. Dept. Interior. Fish and Wild. Ser.
Spec. Scient. Rept. Fish No. 471. 124 pp.

May, B. E., R. S. Hestand and J. M. VanDyke. 1973. Comparative effects of
diquat plus copper sulfate on aquatic organisms. Weed Sci. 21(3):
249-252.

Meyer, F. A. 1966. Aquatic plant control. p. 70-95. In: A. Calhoun, (ed.).
Inland Fisheries management. Calif. Dept. Fish and Game.

Norris, L. A. and R. A. Miller. 1974. The toxicity of 2,3,7-8-Tetra
chlorodibenzo-p-dioxin (TCDD) in guppies Poecilia reticulatus Peters).
Bull. Envir. Contam. and Toxicol. 12(1):76-80.

Pimentel, D. 1971. Ecological effects of pesticides on non-target species.
Exec. Office of the President. Office of Science and Technology.

Wash. D.C. 220 pp-

Plumb, T. R., L. A. Norris, and M. L. Montgomery. 1977. Persistence of
2,4-D and 2,4,5-T in chaparral soil and vegetation. Bull. Environ.

Contam. and Toxicol. 17(1):1-18.

Rawls, C. K. 1965. Field tests of herbicide toxicity to certain estuarine
animals. Chesapeake Sci. 6(3):150-161.

Schultz, D. P., and E. W. Whitney. 1974. Monitoring 2,4-D residues at
Loxahatchee National Wildlife Refuge. Pestic. Monit. J. 7(3/4):

146-152.

Snow, J. R. 1948. A preliminary study of the toxicity of 2,4-D to pond
fishes and its effectiveness in the control of emergent species of pond
weeds. MSc thesis Auburn Univ. 87 pp., Auburn, Ala.

Walker, C. R. 1971]. The toxicological effects of herbicides and weed
control on fish and other organisms in the aquatic ecosystems. Proc.
Eur. Weed. Res. Counc. Int. Symp. Aquatic Weed. 3:119-126.

Way, J., J. Newman, N. Moore and F. Knoggs. 1971. Some ecological effects
of the use of paraquat for the control of weeds in small lakes. J.

App]. Ecol. 8:509-532.

Yokote, M., S. Kimura, H. Kumada and Y. Matida. 1976. Effects of some
herbicides applied in the forest to the freshwater fishes and other
aquatic organisms. IV. Experiments on the assessment of acute and
subacute toxicities of 2,4,5-T to the rainbow trout. Bull. Freshwater

Fish. Res. Lab. (Toyko) 26(2):85-93.

79


TABLE 10. SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING COHO SALMON

EXPOSED TO 2,4-D IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (DEC. 29, 1976-JAN. 14, 1977)-

Concentration Percent Percent
(mg/L) survival2/ Gill survival
nominal measured (144-h exposure FW) ATPase?/ (240-h_ SW)

Control 0 100 1.4 100.0
0.25 0.25- 0.18 100 nTe/ 94.8
0.50 0.52- 0.59 100 NT 100.0
1.0 1.07 100 1.6 100.0
10.0 10.4 100 NT 100.0
50.0 59.6- 64.0 100 NT 100.0
100.0 106.8-115.4 100 1.0 100.0
200.0 170.0-237.0 100 1.3 100.0

2/ twenty fish exposed per concentration.

— (Na,K)~stimulated ATPase activity of the gills; mean of 5 fish.
£/Not measured.

2,4,5-T

Review of Literature: Toxicity to Fish and Behavior in the Environment

The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) is identical
to 2,4-D with the exception of an additional chlorine atom on the number 5
carbon in the ring. Many of the roses, legumes, and broadleaved plants
that are resistent to 2,4-D are controlled with 2,4,5-T. The herbicide has
been cleared for use on pastures, rangelands, right-of-way, and forests.

It is not registered for use on food crops nor for use in water, on ditch
banks, around homes or recreational areas, or near populated areas. The
herbicide 2,4,5-T is normally applied at 1

to 2 lb/acre (a.i.) for area
treatment or at 2 to 4 Ib/gal (a.i.) Per 100 gallons of solution for spot
treatment.

The water solubility of 2,4,5-T (238 mg/L) is less than that of 2,4-D
[(600 mg/L) (Montgomery et al. 1976)], and this influences its pattern of
use. The sodium salt of 2,4,5-T, for example, unlike that of 2,4-D, has a
low solubility and is difficult to get into a herbicidally active solution.
Amine salts have been shown to form complexes with sodium, iron, calcium,
and magnesium.

Under conditions favorable for microbial degradation, 2,4,5-T has a
longer residual life in soil than 2,4-D. Normal persistence of 2,4,5-T in
soils is from 45 to 270 days (Newman et al. 1952). Norris et al. (1977)
discussed the persistence of 2,4,5-T in a Pacific Northwest forest. The
2,4,5-T level (in the forest floor) declined 90% during the first 6 months
following application, and there was less than 0.02 kg/ha remaining after
1 year or 0.76% of the originally applied 2,4

r »5-T. Other properties of the
ester and amine formulations are comparable to those of 2,4-D.

34


EPA-600/3-79-071
dune 1979

EFFECTS OF SELECTED HERBICIDES ON SMOLTING OF COHO SALMON

by

Harold W. Lorz
Susan W. Glenn
Ronald H. Williams
Clair M. Kunkel
Research and Development Section
Oregon Department of Fish and Wildlife

and

Logan A. Norris
Bobby R. Loper
Pacific Northwest Forest and Range Experiment Station
Corvallis, Oregon 97331

Grant #R-804283

Project Officer

Gary A. Chapman
Western Fish Toxicology Station
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330

CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330


TABLE A-4.

PERCENT MIGRATION (TO JULY 6, 1977) OF YEARLING COHO SALMON

RELEASED INTO A SMALL COASTAL STREAM FOLLOWING ACUTE AND
CHRONIC EXPOSURE TO TORDON 101 (RELEASED APRIL 13, 1977).

Nominal concentration

(

mg/L)

Percent migration
Days post release

1-5 6-10 11-20 21-30 3i+
1. Acute exposure - 96 h
Control 60.9 63.6 63.6 63.6 63.6
0.3 59.8 64.5 65.4 67.3 67.3
0.6 50.4 51.3 52.2 53.9 53.9
1.2 55.0 56.0 56.0 56.9 56.9
1.8 61.5 62.4 62.4 62.4 62.4
2. Chronic exposure - 360 h
Control 46.0 47.9 48.3 48.8 48.8
0.3 48.4 52.6 54.0 54.0 54.0
0.6 42.7 45.5 47.4 48.3 48.3
1.2 31.1 37.8 39.2 39.2 39.2
1.8 30.2 34.9 39.2 39.6 39.6

98


LS

Te 507 ‘Alkalinity Hardness
Species Toxicity test (mg/t) _pH Temperature (mg/t_as CaC03) Reference
Oncorhynchus kisutch static, lab. 30 (96 h) 7-4-7.6 10% 79-82 100-101 Lorz et al. (this
coho salmon-yeartings) study)
0. tshawytscha static, lab. 29.5 (24 h) TeM-7.7 20°C 41-71 Bond et al. (1960)
(chinook salmon) 28.5 (48 h)
0. tshawytscha static, lab. 29.0 (48 h) fui chead- Thompson
1971)
Pimephales promelas static, lab. 10.0 NTE (96 h)2/ 75°F Lawrence et al. (1965)
(fathead minnow)
prome las static, 1h (96 h) Teh 25sec 22 22 Surber and Pickering
~ 130 (96 h) 8.2 25°C 299 379 (1962)
Rasbora hetermorpha flowing, lab. 73-93 (48 h) 20°C 20 Alabaster (1969)
{harlequin Fish)
: . evs 0,
Salmo gairdneri static, lab. 5-0 LD, (96 h)=! 65 F Lawrence et al. (1965)
~“{ratnbow trout), 10
S. gairdneri flowing, lab 70 (48 h) 20° 250 Alabaster (1969)
$. gairdneri static, lab 1o nTe2/ 55°F U.S. Fish & Wild.
Service (1963)
S$. gairdnert static, field 11.2 (96 h) 8.2 48-89°F 233 280 Gi'tderhus (1967)
concrete pond
Sz gairdnert static, lab. 20.0 (48 h) 13°C Cope (1966)
$. gairdnert flowing, lab. 10.0 ne’ 8.0 89.5 _ Fotmar (1976)
S. trutta static, lab. 32.6 (24 h) o.,
(Brown trout-fingerlings) static, lab. 20.4 (96 h) 15 18.3°C too Simonin & Skea (1977)
S. trutta static, lab. 300 (48 n)S/ 7.6-8.0 10 % 210-290 Woodiwiss & Fretwell
—— 570 (48 n)&/ (1974)
Suizorredion vitreum static, lab. 2.1 (96 h) 8.2 48-89°F 233 280 Gilderhus (1967)
zy Modified from Folmar 1977.
3 Concentration of diquat causin:

4 NIE = No toxic effect.

5y Test carried out in seawater.
gy *PProximate threshold toxicity 1D,
Concentration of aquacide and Reg:

9 for 96 h contact period.
lone given as 48 H 1050 values, respectively.

1g 50% mortality of exposed fish in time given.


Residue analyses of aquatic organisms exposed tp picloram indicate
that this herbicide is not bioconcentrated in invertebrates or along food
chains (NRCC 1974). Daphnia exposed to 1 mg/L of the potassium salt of
picloram had whole body residues of the herbicides equal to that present in
the water (Hardy 1966). Bioconcentration of picloram (acid) was not
evident in mosquito fish exposed to | mg/L (a.e.) for 18 days (Youngson and
Meikle 1972). The concentration factor for these fish on a wet weight,
whole body basis was only 0.02. The 18 days of exposure to picloram was
qacauate to achieve a steady state level of accumulation in the mosquito-

ish.

Experimental Results

In our study, the 24h LC50 of Tordon 22K was estimated to be
17.5 mg/L. There was no apparent effect of picloram on the (Na, K)-stimulated
ATPase activity of the gills (Table 15). Histological examination of fish
exposed to 5 mg/L Tordon 22K for 144 h revealed abnormal liver and gill
tissues (Appendix I1). When the survivors of the Tordon (22K exposure were
Placed in sea water, a 25% mortality occurred in the group previously
exposed to 0.25 mg/L (Fig. 6). We do not know the reason for these deaths
in seawater after exposure to this low Tordon 22K concentration. Similar
Seawater mortalities at low concentrations occurred following the Tordon
101 and Dicamba exposures, and they also were not explainable--different
tanks were used for seawater challenge tests for the three chemicals and
oxygen concentrations were @9 mg/L when the mortalities occurred.

TABLE 15. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO TORDON 22K IN FRESHWATER AND SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (DEC. 9-27, 1976).

Concentration Percent Percent

(mg/L) survival 2/ Gill survival

Rominal measured _(144-h exposure Fw) ATPase2/ (288-h SW)
Control 0-0.12 100 2.5 100
0.25 1.67 100 nTe/ 6
0.50 1.89 100 NT 100
0.75 2.76 100 NT 100
1.0 3.89-4.23 100 2.2 100
5-0 10.54-11.84 100, 1.8 100
15.0 31.81-41.23 oY : NT
30.0 37.95-45.10 oe/ NT

a .
¥, Twenty fish exposed per concentration.
of Nak) -stimulated ATPase activitys.mean of 4 fish.
afNet measured.

ell dead in 48 h.

~fAll dead in less than 8 h.

43


40 ug/L dinoseb, 336 h (3 fish) - There was some hydropic change in one of
the livers and occasional scattered necrotic cells. The other two livers
Were normal. The second segment of the proximal tubule had occasional cells
with pyknotic nuclei and first proximal segment cells contained eosinophilic
droplets. Gills were normal, tissue changes from this treatment were minor.

60 ug/L dinoseb, 168 h (6 fish) - Livers, kidneys and gills from all fish
were normal. There appeared to be no effect on these tissues.

100 ug/L _dinoseb, 114 h (4 fish) - All the livers had peripheral bile necrosis
and extensive diffuse necrosis of the remaining parenchymal cells. All
regions of the kidney tubules as well as most of the hematopoietic tissue
were necrotic. The gill epithelium was totally necrotic and sloughed from
the gill lamellae. This treatment was severe and extremely toxic to the

tissues examined.

DIQUAT

Control, 360 h (5 fish) - Livers, kidneys and gills of these fish were
normal.

us) mg/L _diquat, 144 h (3 fish) - Livers showed hydropic degenerative changes
in centrolobular regions and some necrosis in one liver. Kidneys of all
three fish were normal but gills showed both limited hypertrophy and hyper-

Plasia of epithelial cells.

20 mg/L diquat, 144 h (1 fish) - This fish had extensive hydropic degeneration
and necrosis of the liver. The kidney was normal but the gills had both
hypertrophy and hyperplasia of epithelial cells.

3.mg/L digquat 360 h (5 fish) - Several foci of degenerate and occasionally
necrotic cells were present in the liver. The kidneys also had numerous
degenerate and some necrotic tubule cells, particularly in the collecting
ducts. Both hypertrophy and hyperplasia of lamellar and interlamellar
epithelium were present in the gills.

93


Fish exhibited a narrow range of susceptibility to acrolein. Louder
and McCoy (1962) noted the 24-h and 96-h LC50 values for largemouth bass
(Micropterus salmoides), bluegills, bowfins (Amia calva), mosquito fish
(Gambusia affinis) and fathead minnows (Pimephales promelas Rafinesque)
ranged from 62 to 183 ug/L. Burdick et al. (1964) found reduced toxicity
corresponding to increasing size for bluegills but not for brown trout

fingerlings.

Macek et al. (1976a) investigated the effect of chronic exposure to
acrolein on fathead minnows. These authors noted that adult fathead
minnows exposed to acrolein concentrations of 0 to 41.7 ug/L for 30 to 60
days showed similar rates of spawning, growth, and survival. However,
they observed only 2% survival of larval fish exposed to 41.7 ng/L acrolein.
The 6-day incipient LC50 for fathead minnows exposed to acrolein was 8h
ug/L, and the estimated maximum acceptable toxicant concentration (MATC)

was >11.4 <41.7 wg/L.

Under field conditions, Green (1960) found that acrolein applied at
1.0 to 2.0 mg/L could kill carp, Cyprinus carpio, and threadfin shad,
Dorosoma petenense. Largemouth bass and bluegill appeared not to be
harmed by 5.0 mg/L Aqualin, but Moen (1961) found Aqualin to be toxic to
fish at 0.5 mg/L when applied to a pond. Meyer (1961) found Aqualin was
toxic to fish when applied to ponds at rates as low as 0.2 mg/L. Catfish,
Ictalurus sp. and sunfish, Lepomis sp. succumbed at } to 2 mg/L Aqualin,
while buffalo fish, Ictiobus sp. were killed at 0.2 mg/L. Chemical control
of filamentous green algae was investigated by Jordan et al. (1962), but
they found acrolein to be erratic in its performance and the only herbicide
tested that injured fish. Furthermore, they noted 50% mortality of
bluegill and goldfish in 24 h following the third and fourth applications
of acrolein at 2 mg/L. Applications of 4 mg/L produced 98% mortality of
the bluegill and 95% mortality of the goldfish within 24 h. Placement of
fish in ponds 2 weeks after application of 4 mg/L resulted in death of all
of the goldfish and only 3% survival of the bluegills. These findings are
contrary to those of Moen (1961), but probable differences in water quality
and an eightfold greater Aqualin concentration may account for the extended

toxic condition.

During the 1960's there was interest in acrolein as a fish repellent.
Louder and McCoy (1962) reported that acrolein had been used successfully
by the lowa State Conservation Commission to drive fish downstream into a
weir in the Raccoon River. They concluded, however, that the herbicide
was not suitable for collecting fishes in lotic waters since it required a
lethal dose to repel fish. Testing of overt avoidance reaction of rainbow
trout fry to acrolein revealed that the fry probably would not avold a

lethal concentration (Folmar 1976).

Experimental Results

The 96-h LC50 for yearling coho salmon was estimated at 68 ug/L
acrolein (Fig. 3, Table 4). It was the most toxic water soluble herbicide
tested. There was no apparent effect of acrolein on (Na,K)-stimulated

17


Salinity of the water was maintained at 30 + 0.5 °/oo with a temperature
of 10 + 1°, The seawater exposure period was a minimum of 10 days; if
mortality was still occurring, longer observation periods were used.

ASSESSMENT OF COEFFICIENT OF CONDITION

Each month, after a 24-h starvation period, about 40 fish were selected
randomly from the stock and exposure tanks, anesthetized, weighed, and

Measured. Individual fish were weighed to 0.1 g and fork length was
determined to 0.1 cm. The coefficient of condition (K) was determined for

each fish in the sample using the formula K = 100 wW/L3, where W denotes
weight in grams and L denotes fork length in centimeters (Hoar 1939). Any
change in condition factor compared to controls probably reflected an
effect of the herbicide on metabolic processes because the feeding behavior

Appeared to be unaffected except in those fish exposed to dinoseb.

HISTOLOGICAL EXAMINATION

Gill, liver and kidney tissue were usually excised from freshly
killed fish and preserved in Bouins solution for histological examination.
Three to five fish were collected from herbicide concentrations that
Caused death during the static exposure. Five chronically exposed fish
were collected from each Tordon 101, dinoseb and diquat concentration.

Or. J. D. Hendricks, Dept. of Food Science and Technology (Oregon State
Dr. Hendrick's

University) made the histological examination of the fish.
analyses are in Appendix II, but will be referred to in the results and

discussion whenever histopathological damage was noted for a particular

herbicide.

DOWNSTREAM MIGRATION

The effect of Tordon 101, dinoseb and diquat on migratory disposition
was assessed by releasing marked control and herbicide-exposed yearling
coho salmon into a tributary of the North Fork Alsea River and trapping
them 6.4 km downstream. The trap was checked daily for the first 10 days
following release, then every second or third day thereafter. On the day
Prior to release, about 100 fish from each 1.54-m exposure tank (long
exposure) and 50 fish from each 0.61-m tank (short exposure) were anes~
thetized in MS-222, weighed and marked by freeze branding and a fin clip.
No fish smaller than I! cm were released as the "parr-smolt transformation"!
is markedly size dependent. Releases were made between April 13 and May

f seaward migration of wild juveniles. Trapping

26, 1977, the norma] time o
was terminated July 6, 1977, more than a month after the last release.

43


L6

TABLE A-3- EFFECT OF TORDON 101 EXPOSURE ON AVERAGE LENGTH, WEIGHT, AND CONDITION FACTOR.

Date Days Nominal con- Fork Condition
of of Tank centration length Weight factor
sample exposure No. (mg/L) cm + SE g & SE KFL + SE
Mar. 9, 1977 02/ 1 Control 15.5 40.32 42,942.42 1.118 + 0.012
0 2 Control 15.1 ¥ 0.17 39.94 1.29 1.163 ¥ 0.013
0 7 0.3 14.8 40.29 37.9 * 2.16 1.134 ¥ 0.009
0 8 0.3 15.2 40.26 40.5 ¥ 1.86 1.145 ¥ 0.010
0 3 0.6 14.8 40.28 37.6 £2.10 1.125 ¥ 0.009
i} 4 0.6 15.2 + 0.22 40.4 + 1.63 1.130 + 0.012
0 5 1.2 15.1 + 0.25 39.2 + 1.83 1.118 + 0.011
0 6 1.2 15.40.20 Al. £1.49 1.111 ¥ 0.012
(0) 3 1.8 14.9 + 0.23 39.0 + 1.85 1.156 + 0.014
0 10 1.8 14.9 + 0.17 37.4 + 1.23. 1.125 + 0.009
Apr. 12, 1977 o2/ 1 Control 15.7 + 0.21 39.1 + 1.58 0.998 + 0.007
) 2 Control 15.4% 0.17 37.94 1.14 1.022 ¥ 0.008
15 7 0.3 15.6 + 0.19 39.9 + 1.64 1.035 + 0.011
15 8 0.3 15.5 * 0.18 39.2 41.34 1.043 ¥ 0.009
15 3 0.6 15.5 + 0.25 39.6 + 1.88 1.024 + 0.008
15 4 0.6 15.6 + 0.24 40.1 41.96 1.036 ¥ 0.011
15 5 1.2 14.7 £0.16 33.5 + 1.18 1,038 + 0.018
15 6 1.2 15.6 ¥ 0.23 41.7 41.98 1.067 ¥ 0.021
15 9 1.8 15.34 0.20 36.941.47 1.016 0.011
15 10 1.8 15.4 + 0.21 38.5 + 1.55 1.038 + 0.009

2/ sample size 30

sample size 40


DICAMBA

Review of Literature: Toxicity to Fish and Behavior in the Environment

Dicamba (3,6-dichloro-o-anisic acid) is one of the most extensively
used benzoic acid herbicides in the United States. Production of dicamba
in 1971 was estimated to be 6 million Ibs of active ingredient (Lawless et
al. 1972). The herbicide was introduced in the early 1960's for the
control of preemergence and postemergence broadleaf weeds in cereal grains
(Frear 1976 and Velsicol Chem. Co. 1967). Phenoxy-tolerant broadleaf weeds
and brush species are also controlled by foliar and area applications of
dicamba. Current registration for this herbicide is for use on barley,
corn, oats, wheat, pasture and rangeland, where it is applied at rates
ranging from 1/4 to 8 lb/acre. The dimethylamine salt of dicamba, formu-
lated as a liquid, is sold under the trade name of Banvel, but is also
available in granular form as the acid or amine salt.

Dicamba has been shown to exhibit intermediate persistence in many
soil types when compared to other herbicides (Burnside et al. 1971),
remaining phytotoxic for several months (Klingman and Ashton 1975). It has
a relatively high water solubility (7900 mg/L) and has been demonstrated to
move within the soil profile with water flux. Studies by Trichell et al.
(1968) have shown that runoff losses of dicamba are limited. The vapor
pressure of dicamba is quite low, so minimal amounts are lost through
volatility (Montgomery et al. 1976). Photodecomposition of dicamba is
similarly limited. Indirect evidence from a number of studies suggests
that microbial degradation may be instrumental in reducing dicamba per-
sistence (Frear 1976 and Hahan et al. 1969). Norris and Montgomery (1975)
noted that following spraying of a brushy area in coastal Oregon with 1.12
kg dicamba/ha, water residue levels rose sharply to 37 ppb about 5 h after
spraying and then declined slowly to background levels by 37.5 h.

The effects of dicamba on fish have not been well investigated.
Toxicity tests with dicamba indicate a low toxicity to salmonids and warm
water fish species. In static tests conducted by Bond et al. (1965) the 24
and 48-h LC50 values for juvenile coho salmon were 151 and 120 mg/L active
ingredient, respectively (methyl orange alkalinity approximately 55 mg/L,
pH about 7.7). Rainbow trout were killed by a concentration of 320 mg/L
dicamba in 72 h (Bond et al. 1965). Bohmont (1967), in his literature
review of Cope's 1962 and 1963 work, however, reported an estimated 48-h
LC50 for rainbow trout and bluegill of 35.0 mg/L and 130 mg/L dicamba
(Banvel D), respectively. Hughes and Davis (1962b) reported 24 and 48-h
LC50 values of 600 and 410 mg/L for the liquid formulation of dicamba for
the bluegill; however, when Banvel D acid was adsorbed onto vermiculite
they found a 24-h LC50 of 20 mg/L.

Experimental Results

No mortalities were observed in yearling coho salmon exposed to dicamba
(Banvel) concentrations up to 100 mg/L (Table 7). Gill (Na,K)-stimulated

25


Coho salmon that received 96 h exposure to Tordon 101 (0.3-1.8 mg/L) prior.
to release did not migrate as well as the control group except for the

0.3 mg/L group which again showed slight migratory stimulation. In the
other 96-h exposure groups (0.6, 1.2, and 1.8 mg/L) the migratory response
was less than observed for the control but no relationship to concentration
of Tordon 101 was apparent (Fig. 8b). The majority of downstream movement
of all groups of coho salmon occurred within the first 5 days of release

(Table A-4).

Histological examination of gill, liver, and kidney tissue of coho
salmon from the various chronic (15-day) Tordon 101 exposures showed only
peribiliary necrosis in the liver that was not present in the controls
(Appendix II). The consequences of this type of tissue degeneration or the
effect on the fish in terms of ultimate survival is unknown.

Coho salmon that had been exposed for 15 days to 1.8 mg/L Tordon 101
and a comparable group of control fish were killed and several tissue and
Organ systems excised and analyzed for bioconcentration of Tordon 101. No
apparent accumulation of 2,4-D or picloram was noted. Youngson and Meikle
(1972), however, did find residues of 0.21 mg/kg in mosquitofish exposed to

1 mg/L picloram for 18 days.

DINOSEB (PREMERGE)

Review of Literature: Toxicity to Fish and Behavior in the Environment

Dinitrophenols are the oldest organic chemicals patented for selective
weed control (Kaufman 1976). Two of these compounds, dinoseb (2-sec-buty!-
4, 6-dinitrophenol) and dinosam have become widely used herbicides. The
Phenol form of dinoseb is oil soluble and formulated as an emulsifiable

Concentrate. The amine or ammonium salts of dinoseb are water soluble and
are formulated under a variety of trade names. Dow Premerge (alkanol amine
al use of dinitroamines and

salt) was utilized in this study. The gener
ammonium salts has been for postemergent control of most seedling weeds and
Grasses in cereal, tree, and vegetable crops. Application rate of the
amine salt of dinoseb is normally from 1.5 to 6 1b/acre (a.i.) depending
Upon the crop. Dinoseb is also applied in some areas to control ditch bank
weeds (Woodward 1976).

lly dependent upon their
ke and absorption

id solutions, but

The fate and behavior of herbicides is genera

tate of decomposition, volatilization, movement, uptal

(Kaufman 1976). Dinitrophenols appear to be stable in ac i
Undergo photolysis at alkaline pH. Volatilization or codistillation of

dinoseb has been shown to occur under conditions of soll acidity, high |
temperature, and surface soil moisture. Heavy rains following application
May cause leaching, particularly in alkaline soils; although in mineral
Clays, adsorption may slow the rate of leaching (Montgomery et al. 1976,
Kaufman 1976). Certain soil microorganisms readily degrade dinitrophenols.
he residual life of dinoseb under warm, motst soit conditions is 3-5
Weeks; residual carry-over is not expected to occur from one season to the

Next (Klingman and Ashton 1975).

4g


Conte, F. P., H. H. Wagner, J. Fessler and C. Gnose. 1966. Development of
osmotic and ionic regulation in juvenile coho salmon Oncorhynchus

kisutch. Comp. Biochem. Physiol. 18:1-15.

ope, 0. Bee 1966. Contamination of the freshwater ecosystem by pesticides.
esticides in the environment and their effects on wildlife. J. Applied

Ecol. (Suppl.) 3:33-44.

Cope, 0. B., E. M. Wood and G. H. Wallen. 1970.
sot the bluegill (Lepomis macrochirus) .

Some chronic effects of
Trans. Am. Fish. Soc.

Crosby, D. G. and H. 0. Tutass. 1966. Photodecomposi tion of 2,4-dichloro-
Phenoxyacetic acid. J. Agric. Food Chem. 14:596-599.

Cc
utkomp, L. K., H. H. Yap, E. Y. Cheng and R. B. Koch. 1971. ATPase
activity in fish tissue homogenates and inhibitory effects of DDT and

related compounds. Chem-Biol. Interactions 3:439-447.
i}
avis, J. T. and W. §. Hardcastle. 1959. Biological assay of herbicides
or fish toxicity. Weeds 7:397-404.

Further observations on the toxicity

Davi
avis, J. T. and J. S. Hughes. 1963. ne weed f
roc. South. Weed Conf.

of commercial herbicides to bluegill sunfish.
16:337-340.

D,
ays 8. EL, L. S. Jordan, and R. T. Hendrixon. 1961. The decomposition of
amitrole in California soils. Weeds. 9:443-456.

1957. Effects of herbicides and

D
emyanenko. 1941. In; P. F. Springer.
North Carolina Pesticide Manual.

fungicides on wildlife. pp- 87-106.

E
arnest, R. D. 1971. The effect of paraquat on fish in a Colorado farm

Pond. Prog. Fish Cult. 33(1)327-31-
E .
ton, J. G. 1970. Chronic malathion toxicity to the bluegill (Lepomis
macrochirus, Rafinesque). Water Res. 4:673-684.
gical effects of metals in

E}
Sler, R. 1973. Annotated bibliography on biolo
repatic environments. U.S. Environ. Prot. Agency. Ecol. Res. Ser.
PA-R3-73-607. Corvallis, Oregon. 287 pp-

tated bibliography on biolo-

Eis
ler, R. and M. Wapner. 1975. Second anno
U.S. Environ.

gical effects of metals in aquatic environments.
rot. Agency Ecol. Res. Ser. EPA-R-3-75-023.

E

Ison, P, F., J. W. Saunders,and V. Zitko. 1972. Impact of forest-based
urces In New Brunswick.

industries on freshwater-dependent fish reso
ish. Res. Board. Can. Tech. Rep. 325:26 pp. -

67


0-30y9/L (5-30yg/U)
a a a ie i Caine

50 g/L e. Control

50 pg/L

PERCENT SURVIVAL

L L
24 72 120 24 96 168 240 280
Freshwater Seawater

EXPOSURE TIME (hours)

Figure 3. Percent survival of yearling coho salmon during exposure to
acrolein in freshwater and subsequent survival upon transfer to seawater.

AMI TROLE-T

Review of Literature: Toxicity to Fish and Behavior in the Environment

Amitrole (3-amino-s-triazole) with an equal molar amount of ammonium
thiocyanate is formulated under the trade name Amitrole-T. It is registered
for non-crop land clearing for right-of-ways, industrial premises, lawn
renovation, hardwood nurseries, and ditch banks. Amitrole-T has also been
found to be very effective in the control of aquatic vegetation such as
water-hyacinths. For spot treatment, Amitrole-T is normally applied at
1/2 lb active ingredient (a.i.) per 12 gal of water. As an area spray it
is applied at rates up to 4 lb per acre.

The literature contains many publications describing the fate of
applied amitrole. In a study of 55 different California soils, Day et al.
(1961) reported that amitrole disappeared rapidly within 2 weeks after
application. Rapid decomposition of amitrole was also observed ina
similar study in Oregon soils (Freed and Furtick 1961). Sund (1956) noted
that amitrole adsorbs rapidly and tightly to soil particles having a high

base exchange capacity and a high organic matter content. Amitrole complexed
readily with metals in soil.

19


Graduated cylinder, 100 mL

Heating mantle, 250 mL

Centrifuge (International Model U or equivalent)

Centrifuge bottles, 250 mL

Beakers

Flash evaporator (Calab Model 5101 or equivalent)

Tissue grinder (Tek Mar Model SDT or equivalent)

Gas chromatograph - MicroTek 2000 MF, or equivalent,
with Dohrmann model €200B microcoulometric
detector with halogen titration cell.

Procedure:

Sample extraction: Weigh sample, cut in small pieces and place in
250 mL round-bottom boiling flask. Add 50 mL of 95% ethanol, grind
with tissue grinder. Add 100 mL 50% potassium hydroxide in water
solution, place in a heating mantle and attach a reflux condenser.
Reflux for one hour. Cool, transfer contents to centrifuge bottle
and centrifuge at 2000 rpm for 20 min. Decant into separatory
funnel, acidify to <pH 2.0 with concentrated sulfuric acid, and
extract with three aliquots (100, 75, 60 mL) of ether. Evaporate to
2 mL with a stream of dry nitrogen and transfer with small aliquots
of ether to a 10 mL volumetric flask. Evaporate to dryness using a

flash evaporator. The derivation, gas chromatography and quantification
are identical with the method for water analysis.

DINOSEB

1.

Residues in water

Summary of method: The method is based on Zweig and Sherma (1972b).
Samples are collected and stabilized with sodium hydroxide. The
samples are adjusted to pH < 2, extracted with ether and alkylated

to form the methyl ester. Detection and quantification is accomplished
using electron-capture gas chromatography (ECGC).

Reagents:

Diethyl ether (distilled over sodium)

Sulfuric acid (concentrated, reagent)
Sodium hydroxide (reagent)
Diazomethane

Apparatus:

Glass jars with teflon lined caps

Separatory funnels fitted with te
Beakers

Volumetric flasks

Graduated cylinders

Gas chromatograph - Varian Model 1200 or equivalent with
scandium E.C. detector.

Flash evaporator (CaLab Model 5101 or equivalent)

flon stopcocks

86


ACKNOWLEDGMENTS

This investigation was supported in part by the U. S. Environmental
Protection Agency, Research Grant R-804283, and was funded through the
Corvallis Environmental Research Laboratory, Corvallis, Oregon. The U.
S. Department of Agriculture, Pacific Northwest Forest and Range Experi-
ment Station, accepted responsibility for chemical analyses of the herbicides
tested and provided the necessary personnel and equipment. Many con-
tributed to this study and their assistance is gratefully acknowledged:
Dr. G. A. Chapman, Project officer, Western Fish Toxicology Station
(WETS), EPA, provided technical assistance and guidance. Dr. N. A.
Hartmann devised the statistical test to determine if downstream movement
of the treatment groups was statistically different from the control.

Dr. M. Newton, Oregon State University School of Forestry, provided one
of the chemicals, Krenite, used in the study. Ms. Carroll Burkett,
Forest Science Laboratory, assisted in the chemical analyses.

The authors gratefully acknowledge Drs. G. A. Chapman, A. V. Nebeker
and H. H. Wagner who provided constructive criticism on the manuscript.

xi


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activated adenosine triphosphatase of gills: role in adaptation o
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Ernster, L. R., R. Svetterstrom and 0. Lindberg. 1950. A method for the
determination of tracer phosphate in biological material. Acta. Chem.
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Federal Water Pollution Control Administration.

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Secretary of the Interior. Fed. Wat. Poll. Cont. Admin. U. S.
Dept. Int. Washington, D.C. 234 pp.

1968. Water quality

Folmar, L. C. 1976.

Overt avoidance reactions
herbicides,

of rainbow trout fry to nine
Bull. Environ.

Contam. Toxicol. 15(5):509-514.

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Foy, C. L. 1976. Picloram and related compounds.

pp. 777-813. In;
and D. D. Kaufman (eds.) Herbicides: chemistry, degra~
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Frear, D. S$. 1976. The benzoic acid herbicides. pp. 542-608. In; P. c.
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Freed, V. H. and W. R, Furtick. 1961.

The persistence of amitrol in soil-
Hormolog. 3:3-4,
Gilderhus, P. A, 1967. Effect

of diquat on bluegills and their food
Organisms. Prog. Fish Cult. 29(2) 367-74.

Goring, C. A., C. R, Youngson, and J. W. Hamaker. 1965,
disappearance from Soils. Down to Earth. 20(4) 23-5,

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Persistence of four
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J. Am. Water Works Assoc. 58: 325-332.
Haas, R. C., C. J. Scifres

191) Occue s R. R. Hahn, and G. 0. Hoffman.
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68

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Norris, L. A., M. L. Montgomery, and E. R. Johnson. 1977. The persis-

tence of 2,4,5-T in a Pacific Northwest forest. Weed Sci. 25(5):
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Rodgers, C. A. and D. L. Stalling. 1972.

Dynamics of an ester of 2,4-D
in organs of three fish species.

Weed Sci. 20:101-105.

Sanders, H. 0. 1970. Toxicities of some herbici

des to six species of
fresh water crustaceans. J, Water. Poll.

Control Fed. 42(8) 31544-1550.

Saunders, J. W. 1969. Mass mortalities and behavior of brook trout and
juvenile Atlantic salmon in a stream Polluted by agricyltural pesticides.
J. Fish. Res. Board Can. 26:695-699.

: Uptake and distribution of 2,4-D dimethylamine salt
in channel catfish, bluegills, and largemouth bas

+ lth Weed Sci.
Soc. of Amer., Dallas, Texas. . ee

Schultz, D. Pa 1973. Dynamics of a salt of (2,4-dichlorophenoxy) acetic
acid in fish, water and hydrosol. J. Agric. Food Chem. 21(2):186-192.
Schultz, D. P. and P. D, Harman. 197ha.

Resid -'
mud and fish 1971. S!dues of 2,4-D in pond waters,

Pestic. Monit. J. 8(3):173-179,
Schultz, D. P. and P. D. Harman. 1974b.
use of 2,4-D in fisheries. U. S. Fis

A review of the literature on the
18. NTIS PB~235457, 90 pp.

h & Wild. Ser. Rep. No. FWS-LR-74~

74


2

FIGURES

Number Page
1 Diagram of flow-through diluter......eseeeeeseee eevee cere 9

Exposure tanks with diluter in background...........-.e.5+- 10

Percent survival of yearling coho salmon during exposure to
acrolein in freshwater and subsequent survival upon transfer
tO SCAWATEL. cere cece reer ee rere tere reese eteresecsecsecesene 1G

Percent survival of yearling coho salmon during exposure
to Amitrole-T in’ freshwater and subsequent survival upon
transfer to Seawater......seeeee sees eee neee see eeeeeeeeeeee 22

Percent survival of yearling coho salmon during exposure
to paraquat-CL in freshwater and subsequent survival upon

transfer to SCAWAteressrsececccceecrersertseseteressesseces 30

Percent survival of yearling coho salmon during exposure
to Tordon 22K (picloram) in freshwater and subsequent
survival upon transfer to seawater....-..seeeeee eee eeeeee es hh

Percent survival of yearling coho salmon during exposure
to Tordon 101 (2,4-D + picloram) in freshwater and
subsequent survival upon transfer to Seawater......ssseeee. 47

Percent downstream migration of yearling coho salmon
following exposure to Tordon 101..........-- see eee ceeeeeee 50

Percent survival of yearling coho salmon during
exposure to dinoseb (Dow Premerge) in freshwater and
subsequent survival upon transfer to Seawater.....ssereeee. 52

Percent downstream migration of yearling coho salmon
following exposure to dinoseb (Dow Premerge).........ee0065, 55

Percent survival of yearling coho salmon during exposure
to diquat in freshwater and subsequent survival upon
transfer to seawater.....eeeeeseeeeeeeee dee eeeeeeeeseeeeee 59

Percent downstream migration of yearling coho salmon
following exposure to diquat...scceseeeeseceeseceseveceeees 61


TECHNICAL REPORT DATA .
(Please read Instructions on the reverse before completing)

1, REPORT NO. 2. 3. RECIPIENT’S ACCESSION NO.
EPA-600/3-79-071
4. TITLE AND SUBTITLE 5, REPORT DATE
Effects of Selected Herbicides on Smolting of Coho June 1979 issuing date
Salmon 6, PERFORMING ORGANIZATION CODE
7. AUTHOR(S) lg. PERFORMING ORGANIZATION REPORT NO.

Harold W. Lorz, Susan W. Glenn, Ronald H. Williams,
Clair M. Kunkel, Logan A. Norris and Bobby R. Loper

9. PERFORMING ORGANIZATION NAME AND AODRESS 10. PROGRAM ELEMENT NO.
Oregon Department of Fish and Wildlife 1BA608
Research and Development Section, 28655 Highway 34, Ti CONTRACT/GRANT NO.
Corvallis, Oregon R-804283
12. SPONSORING AGENCY NAME AND ADORESS 13. TYPE OF REPORT AND PERIOD COVERED
Corvallis Environmental Research Laboratory Final 1-5-77 to 1-4-78
Office of Research and Development 14, SPONSORING AGENCY CODE
U.S. Environmental Protection Agency
Corvallis, OR 97330 EPA/600/02

15. SUPPLEMENTARY NOTES
With the technical assistance of the Pacific Northwest Forest and Range Experiment
Station, Corvallis, Oregon.
16, ABSTRACT
Static 96-h LC50 values for 12 water-soluble herbicides with yearling coho salmon in
freshwater were: acrolein 68 ug/1; dinoseb 100 ug/1; picloram 5,0-17.5 mg/1; 2,4,5-T
(triethylamine) > 10 mg/1; atrazine> 15 mg/l; diquat 30 mg/1; amitrole-T 70 mg/1;
paraquat 76 mg/1; dicamba> 100 mg/1; and Krenite and 2,4-D (dimethylamine) > 200 mg/1.
Amitrole-T, diquat, and paraquat exposure in freshwater reduced the survival of
salmon smolts placed in seawater. Diquat also inhibited downstream migration of
smolts. Under normal field use, acrolein and dinoseb could produce mortality of all
life stages of salmonids if treated irrigation waters were released into streams
prior to herbicide inactivation. The use of diquat at recommended treatment
levels could reduce downstream migration of smolts and decrease survival in seawater.
All other herbicide formulations tested appeared to have no effect on smolting of
coho salmon.

17. KEY WORDS AND DOCUMENT ANALYSIS
a DESCRIPTORS. [b-lDeNTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Herbicides, Salmon, Animal Behavior, Animal
Migrations, Water Balance, Histology, 06/A,F,T

Pollution, Toxicology.

18, OISTRIBUTION STATEMENT. 79. SECURITY CLASS (This Report) 21, NO. OF PAGES
unclassified x 116 _
20. SECURITY CLASS (This page) .
Release to Public unclassified

EPA Form 2220-1 (Rev. 4=77) ° 103



Marking, L. L. and R. G. Piper. 1976. Carbon filter for removing
therapeutants from hatchery water. Prog. Fish-Cult. 38(2):69-72.

Marston, R. B., D. W. Schults, T. Shiroyama, and L. V. Snyder. 1968.
Amitrole concentrations in creek waters downstream from an aerially
sprayed watershed sub-basin. Pestic. Monit. J. 2(3):123-128.

Matida, Y., Y. Furuta, H. Kumada, H. Tanaka, M. Yokote, and S. Kimura.
1975. Effects of some herbicides applied in the forest to the fresh-
water fishes and other aquatic organisms. 1. Survey on the effects
of aerially applied sodium chlorate and a mixture of 2,4-D and 2,4,5-T

on the stream community. Bull. Freshwater Fish. Res. Lab. (Tokoyo)
25(1) :41-53.

Matida, Y., S. Kimura, H. Tanaka, and M. Yokote. 1976. Effects of some
herbicides applied in the forest to the freshwater fishes and other
aquatic organisms. III. Experiments on the assessment of acute

toxicity of herbicides to aquatic organisms. Bull. Freshwater Fish.
Res. Lab. (Tokyo) 26(2):79-84.

McBride, R. K. and B. D. Richards. 1975. The effects of some herbicides
and pesticides on sodium uptake by isolated perfused gills from the
carp Cyprinus carpio. Comp. Biochem. Physiol. 51(C):105-109.

McKim, J. W. and D. A. Benoit. 1971.
copper and survival growth and rep
(Salvelinus fontinalis).

Effects of long-term exposures to
_Teproduction of brook trout
J. Fish. Res. Board Can. 28(5) 1655-662.

Meehan, W. R., L. A. Norris, and H. S, Sears. 1974. Toxicity of various
reopens of 2,4-D to salmonids. J. Fish. Res. Board Can. 31 (4):
485.

Meyer, F. P. 1961. Notes from June 6, 1961 report. United States Depart-
ment of the Interior. p, 240. In: Louder, D. E. and E. G. McCoy. 1962.
Preliminary investigations of the use of aqualin for collecting fishes-
Proc. Annu. Conf. Southeast Assoc.

Game Fish Comm. 16:240-242.

Miles, H. M. and L. S. Smith. 1968. tonic regulation in migration juvenile
coho salmon Oncorhynchus kisutch. Comp. Biochem. Physiol. 26:381-398-

Moen, T. 1961. Notes from panel discussion on herbicides taken from the

Weed Control Soc. Conf. Chicago, 111. Feb. t
D. E. Louder and E. G. HcCoy. 1962. Tear ee bs 20. Jn

Preliminary Investigations of
the use of aqualin for collection fishes. Proc. léth Ann. Conf.
S.E. Assoc. Game Fish. Comm. Pp. 240-242. :
Montgomery, M. L., H. V. Freed, and J. M. Witt. 1976. Chemical and °

Properties. pp. 11-33. In: Oregon Weed Control handbook. Extension
Service, Oregon State University, Corvallis, Oregon.
Mount, D. I. 1968. Chronic toxicit
D Y Of copper to f i
(Pimephales promelas, Rafinesque). Water Research 2-218.009""

72


TABLE A-5. PERCENT MIGRATION (TO JULY 6, 1977) OF YEARLING COHO SALMON
RELEASED INTO A SMALL COASTAL STREAM FOLLOWING ACUTE AND
CHRONIC EXPOSURE TO DINOSEB (RELEASED MAY 5, 1977).

Percent migration
Nominal concentration Days post release
ug/L 1-5 6-10 11-20 21-30 31+

1. Acute exposures - 96h

Control (115)2/ 60.0 63.5 66.1 66.1 66.1
10 (113) 53.1 55.7 55.7 56.6 57.5
20 ( 98) 62.2 66.3 67.3 68.4 68.4
40 (110) 47.3 51.8 54.5 54.5 54.5
60 (105) 2/ 43.6 52.5 54.5 56.4 56.4
2. Chronic exposures - 285 h
Control (103) 47.6 52.4 54.5 54. 5a
Control (100) 59.0 65.0 65.0 65.0 65.0
10 (104) 59.6 65.4 68.2 69.2 69.2
10 (105) 60.9 68.6 Th 72.4 72.4
20 (100) 59.0 64.0 66.0 66.0 66.0
20 (103) 43.7 49.5 50.5 51.5 51.5
40 (109) 39.4 53.2 55.0 56.0 56.0
60 ( 88)2/79/ 26.4 35.2 37.5 37.5 38.6
60 (109) 2/ 35.8 49.5 56.9 56.9 56.9

a/; Number of fish released in parenthesis
2/24 h exposure
48 h exposure

Y some mortality occurred in the exposure tanks prior to marking and release.

99


Reagents: Diethyl ether (distilled over sodium)
Sulfuric acid (concentrated, reagent)
Sodium hydroxide (reagent)

Di azomethane
BF 3-methanol
Apparatus: Widemouth plastic bottles (500 mL)
Separatory funnels fitted with Teflon stopcocks
Beakers

Volumetric flasks
Graduated cylinder

Gas chromatograph - MicroTek 2000 MF, or equivalent,

with Dohrman model C200B microcoulometric detector
with halogen titration cell.

Procedure:

Sample collection: water samples are c
Plastic bottles whic!

€ complete hydrolysis of any of the herbicide
ester which might be Present. The samples are transported to the
laboratory and stored at 2°,

Sample extraction: Transfer 200 mL of water sample to a 500 mL
separatory funnel. Adjust to pH < 2

with concentrated sulfuric acid.
Extract with three successive aliquots of ether (100, 80, 60 mL). The
ether extracts are collected in a 250 mL beaker, and evaporated to
less than 20 mL. The ether layer is carefully decanted away from any
water and into a 30 ml beaker. The residual extract is rinsed with
three small aliquots of ether adding the rinsings to the beaker. The
extract is evaporated to 2 mL wi
concentrated extract is transfer
a 10 ml volumetric flask
evaporated to dryness on a flash evaporator.
Derivation: The Methyl es » and picloram are formed
using an ethereal diazomethane reagent. The methyl esters of 2,4-D
and 2,4,5-T may be formed using the diazoy

methane reagent (Hopps, 1970)
or a BF3-methanol reagent (Burchfield and Johnson, 1965).

ters of dicamba

Gas ch romatography:

Gas chromatog raphy parameters:

Coulometer settings: 300 2, 250 mv bias, low gain mode.
Column: 4 mm 1.D. x 1.2 m glass packed with
5% OV-1 on 80/100 Gas Chrom Q.

84


Dinoseb has a high toxicity to man, animals, and fish, The acute and
chronic effects of dinoseb on cutthroat and lake trout was investigated by
Woodward (1976), who found that toxicity was largely dependent upon water
quality. Decreasing the water pH resulted in a corresponding Increase in
dinoseb toxicity to fish. Similar findings were reported by Lipschuetz and
Cooper (1961) for technical grade dinoseb. Decreasing the pH from 8.0 to

+3 increased the toxicity of dinoseb to rainbow trout by a factor of 5.
High water temperature and high hardness also tend to maximize the toxicity
of dinoseb to fish, but to a lesser extent than pH (Webb 1951, Woodward

1976).

The 96-h LC50's, under varying conditions of pH, temperature, and

hardness, ranged from 0.41 to 1.35 mg/L dinoseb for cutthroat trout and
from 0.032 to 1.40 mg/L for lake trout (Woodward 1976). Lipschuetz and
Cooper (1961) observed similar dinoseb toxicity values for rainbow trout.
Their 24-h LC50 at pH 8.0 was 0.30 mg/L dinoseb (tech.), and at pH 6.9 was
0.073 mg/L (18°C). Western blacknose dace (Rhinichthys atratulus) in water
of pH 8.0 and a temperature of 21°C had a 24-h LC50 of 0.24 mg/L dinoseb
(tech.). Using a dinoseb formulation composed o
and secondary amylbuty! dinitrophenol, Webb (1951) reported a 2h-h LC50 for
redside shiners (Richardsonius balteatus hydroflux) of 0.16 mg/L in soft
water (18 mg/L hardness, pH 7-6) and of 0.24 mg/L in hard water (105 mg/L
hardness, pH 8.2).

Woodward (1976) did not observe cumulative mortality of lake and
cutthroat trout with chronic exposures 8 to 12 days) to dinoseb. However
Prolonged exposures to 0.005 to 0.010 mg/L dinoseb affected yolk absorption
time and fry growth. Yolk absorption time increased by 6 to 9 days over
that of the controls, and fry growth was reduced at all concentrations of

dinoseb tested.

Experimental Results

Static exposure--

In our study, Dow Premerge was highly toxic to yearling

during the stati dd d sharp rise in
tatic freshwater exposures. A sudden and sharp ,
Tr tan ty occurred between 75 ug/L (no deaths) and 100 ug/L (90% mortality

tn Thd i ahah LC5O was calcula
as noted in P19 i i Nominal concentrations

While the 144-h L + 88 ug/L

=h LC50 was estimated to be ug/L. t
veh used to calculate the LC50 values since the measured concentrations
Combined aliquots from daily samples) were extremely varial

only a fraction of Coho salmon é:
the expected (Table 19). Co
500 ug/L dinoseb were all dead within 24 h, with 40-45% dying during the

1ayst 30 i tying fish (control to
minutes of exposure (Fig- 9). When the surv ving

ro ug/L dinoseb) vere transferred to seawater, all fish survived, ever

Ose that appeared weakened from the prior toxicant exposure (Table 19)-.

0 apparent dinoseb effect was noted on the (Na,k)-stimulated ATPase

activity of the gills (Table 19)-

51

f secondary buty! dinitrophenol


O-I5mg/L

PERCENT SURVIVAL

100 yc nro = mH BO IQ ESI AS IES FES FET ITA
30 Va
AN
80 .
9 AL osmg/t
70 4 A
60 r \ Niersccccceenencndh
50 \
| Q
40 .
| 30 mg/L \,.0.25 mg/L
30 + \ _
20 IL QO
10 cong/t
olk | Lt —E—E——— Ll
24 72 120 48 96 144 192 240 360
Freshwater Seawater

EXPOSURE TIME (hours)

yearling coho salmon during exposure to

Figure 7. Percent survival of
in freshwater and subsequent survival upon

Tordon 101 (2,4-D + picloram)
transfer to seawater.

Another test with Tordon 101 was initiated in mid-March. Half of the
test tanks were dosed with toxicant stock that was mixed fresh daily while
the other tanks were dosed with toxicant stock that was prepared when the
experiment was initiated. There appeared to be no difference in toxicant
ptfect regardless of when the toxicant stock was mixed (Table 18). The

ighest toxicant exposure concentration (22 mg/L) used in the March test
was close to the estimated 24-h LC50 of December (20 mg/L), however, 95% of
the yearling coho salmon exposed to 22 mg/L Tordon 101 (nominal concen-
pration) died. The reason for the poor recovery of Tordon 101 is unknown,
ut if the measured values are real, then yearling coho salmon are more
Sensitive as smolts than as non-smolts. There was no mortality noted
during the seawater challenge of fish from groups representing either of
the methods used in toxicant prepa t the sole survivor of 22

ration, excep
mg/L Tordon 101 died in the first sure (Table 18).

2h h of the seawater expo

47


Woter inlets with . .
U U spray heads (well, Float (connected via microswitch to toxicant manifold)
heated or chilled water) { / /

ee, \ \ /f Toxicant
\ T 7
Head 4 bon ween eee we. +
Box 7] Ji forty
eefobe ene age ------ ~~~
& NENT ge
pe a
Air stone + i; f
Waste Air stone “15m
water oS ° ed eee =a=t —
Toxicant ie H zai \
Manifold H
H Microswitch and
y valve (allows
= dumping toxicant
a a \4 water or
v electricity fails}
H waste
Mixing ” : water
Boxes
EA
Distribution pipes To Control Tanks Toxicant
to exposure. Yonks* Pump
TM~ 2.6m >|
Covered
Toxicant
Reservoir

Figure 1. Diagram of flow-through diluter.


Experimental Results

No deaths occurred in yearling coho salmon exposed to Esteron Brush )
Killer for 96 h at <800 ug/L (nominal concentration under static conditions
nor in the flow-through exposure tanks (210 ug/L maximum nominal concen-
tration). No deaths were observed in the subsequent seawater challenge
tests (Table 12). In both the static and flow-through systems the measured
amount of Brush Killer was very low even though extra care was taken in the

mixing of the toxicant solutions. The reason for the low recovery is
unknown.

TABLE 12. SURVIVAL OF YEARLING COHO SALMON EXPOSED TO ESTERON

BRUSH KILLER AND SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (MAY 31-JUNE 16, 1977).

Concentration Percent Percent
(ug/L) survival2/ survival
nominal Measured (96-h_ exposure FW) (290-h_ SW)

A. Static exposures

Control 3.6- 4.0 100 100 (15)2/
1 1.9- 2.6 100 too (20)
7 2.4- 2.8 100 100 (15)
25 7.0- 7.2 100 100 (20)
75 30.7- 35.4 100 100 (15)
150 45.0- 51.0 100 100 (20)
300 72.6- 73.6 100 100 (15)
450 104.1-111.6 100 100 (10)
800 147.7-159.0 100 100 (10)
B. Flow-through exposures
Control 0 100 100 (20)
35 23.7 100 100 (20)
70 WL .4- 45.7 100 100 (20)
Tho 59.6- 74.0 100 100 (20)
210 84.3- 88.3 100 100 (20)
@/Twenty fish ber concentration

exposed in static test and 40 fish per con-
centration in flow through.

Histological examination of coho salmon exposed to 800 ug/L Esteron
(nominal concentration) for 96 h indicated no apparent affect on either liver
or whaney tissue. The gints however, showed the following abnormalities?
curved gi ‘laments, epithelium fre uently h i ms
In some lamellae (Appendix It). 4 YNypertrophied, and aneuris

Steelhead trout fr

y were also exposed to Esteron Brush Killer to
determine if younger age groups were more susceptible than yearling fish.

38


TABLE 14. MORTALITY DATA FOR SEVERAL FISH SPECIES EXPOSED
TO TORDON 22K FOR 96 HoURS2/,

Water temperature Concentration Percent

Fish species (OF) (mg/L) a.e. mortality
Black bul lhead 50 91 2/ 50

69o/ 0
Bluegill 65 5.4 50
Brook trout 50 91 50

69 oO
Brown trout 50 52 50

22 0
Fathead minnows 50 29 50

22 0
Green sunfish 50 91 50

39 0
Lake emerald shiner 69-78 30 50
Rainbow trout 50 58 50

22 0
o/Moditiea from Kenaga (1969).

Calculated or derived 96~h LC50 values.
Highest concentration Producing no mortality.

he 99% analytical grade picloram (1.2 ppm,
were not affected whereas the technical grade or the 22% commercial
formulation of picloram (for up to 1 hour) caused Immobilization but not
death (Sergeant et al. 1970). Recovery of normal swimming response followed
transfer to clean pond water. Two subsequent exposures to the herbicide
shortened the recovery times; however, after a fourth exposure, many of the
fish failed to recover. Analytical grade picloram did not affect green
sunfish swimming behavior

2 + Sergeant and coworkers suggested that technical
grade and commercial formulations of picloram might contain a toxic impurity

Electron Micrographs of hepatocytes from
technical picloram (1

green sunfish exposed to
+2 mg/L) for 1 d.

ay revealed abnormalities in these
cells (Sergeant et al. 1970). These ultrastructural changes involved the

disappearance of rough-surfaced endoplasmic reticulum and an increase in
tubular or smooth endoplasmic reticulum. These changes were also accom-

panied by liver enlargement. Increasing the exposure time of the herbicide
from 1 to 5 days did not alter the ultrastructural Pattern.

42


Lane, J. R, 1967. DNBP. pp. 385-396. In: Gunter Zweig (ed.) Analytical
Methods for Pesticides, Plant Growth Regulators and Food Additives.
Volume V, Additional Principles and Methods of Analysis. Academic

Press, New York, N.Y.

Lawless, E. W., R. von Rumber, and T. L. Ferguson. 1972. The pollution
potential in pesticide manufacturing. U. S. Environ. Prot. Agency,
Washington, D. C. Pesticide Study Series No. 5, pp. 213 782/3BA. 276 pp.

Lawrence, J.oM., H. H. Funderburk, Jr., R. D. Blockburn, and P. G. Beasley.
1965. The status of diquat and paraquat as aquatic herbicides. Proc.
Ann. Conf. Southeast Assoc. Game Fish Comm. 18:247-257.

leadem, T. P., R. D. Campbell and D. W. Johnson. 1974. Osmoregulatory
responses to DDT and varying salinities in Salmo gairdneri - |. Gill Na-
K-ATPase. Comp. Biochem. Physiol. 49A:197-205.

Lipschuetz, M. and A. L. Cooper. 1961. Toxicity of 2-secondary-buty1-4,6-
dinitophenol to blacknose dace and rainbow trout. N. Y. Fish Game J.
8(2):110-121.

Loos, M. A, 1976. Phenoxyalkanoic acids. pp. 1-128. In: P. C. Kearney
and D. D. Kaufman (eds.). Herbicides:chemistry, degradation, and mode

of action. Vol. 1. Marcel Dekker, Inc., New York, New York.

lorz, H, W. and B. P. McPherson. 1977. Effects of copper and zinc on smoltifi-
cation of coho salmon. U. $. Environ. Prot. Agency, Ecol. Res. Ser.
EPA-600/3-77-032. Corvallis, Oregon. 62 pp.

torz, H. We, R. H. Williams and C. A. Fustish. 1978. Effect of several
Metals on smolting in coho salmon. U. $. Environ. Prot. Agency, Ecol.
Res. Ser, EPA-600/3-78-090. Corvallis, Oregon. 85 pp.

Mouder, D. &, and E. G. McCoy. 1962. Preliminary investigations of the use
Of aqualin for collecting fishes. Proc. Ann. 16th. Conf. Southeast.
Assoc. Game Fish Comm. pp. 240-242.

1951.

bo
wry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. them
em.

Protein measurement with the Folin phenol reagent. J. Biol.
193:265-275,

L
Yn, E. 1965. A review of toxicological information on Tordon
erbicides. Down to Earty. 20(4):6-8.

K. S. Buxton and P. A. Costa. 1976a.

fleas and fathead minnows.
Ser. EPA-600/3-76-099, Duluth,

Mac.
sk K. J., M.A, Lindberg, S. Sauter,
Oxicity of four pesticides to water
+ Environ. Prot. Agency, Ecol. Res.

Minn. 58 pp.
Ma
Sek, Ky J.) K. S, Buxton, S. Sauter, S. Gnilka and J. W. Dean. 1976b.
Chronic toxicity of atrazine to selected aquatic invertebrates and
Fishes. uU. §. Environ. Prot. Agency, Ecol. Res. Ser. EPA~600/3-76-

7, Duluth, Minn. 50 pp.

71


Concern regarding the protection of surface waters and aquatic life
has prompted numerous evaluations of the effects of chemicals on aquatic
invertebrates and fishes. Much of the toxicological research with aquatl
biota has, however, been limited to the development of acute toxicity
values to measure the effects of the chemicals on these organisms. More
recently, chronic exposure of fish and aquatic organisms has received .
attention because numerous parameters can be evaluated as indices of toxic
effects (Arthur 1970, Eaton 1970, Macek et al. 1976a, b, McKim and Benoit
1971, and Mount 1968). The “laboratory fish production index'' as defined
by Mount and Stephen (1967) reflects toxic effects on reproduction, growt ’
Spawning behavior, egg hatchability and fry survival. A parameter which
has received little attention is the effect of chemicals on seaward
migration and saltwater adaptation of anadromous Species.

The seaward migration of juvenile coho salmon (Oncorhynchus kisutch)
normally occurs during the spr

ing of their second year of life. They are
fully euryhaline several months earlier (Conte et al. 1966, Otto 1971)
provided they have achieved a threshold size of 9 cm. The experimental
transfer of juvenile salmonids from freshwater to seawater is followed by
a transient but marked disturba

nce of plasma water-electrolyte balance
(Conte et al. 1966, Miles and Smith 1968). This osmotic disturbance is

caused by the physiological changes necessary to adapt from freshwater

osmoregulation (salt retention, water excretion) to seawater osmoregulation

(salt excretion, water retention). These disturbances are minimized at
the time of normal seaward migration or “parr-smolt transformation'' (Wagner
1974a, b).

Zaugg and Wagner (1973) presented data showing that one of the
physiological factors correlated with migratory behavior in steelhead
trout (Salmo gairdneri) was an elevation of (Na,Kk)-stimulated adenosine
triphosphatase (ATPase) activity in the microsomes of gills. This enzyme
activity doubled during the Parr-smolt transformation of coho salmon and
steelhead trout (Zaugg and McLain 1970, 1972, and Zaugg and Wagner 1973).
ATPase activity in salmonids

increases rapidly during seawater exposure,

reaching a maximum after about 30 days, and is thought to be an important
factor in maintaining salt (os

motic and ionic) balance in fish (Epstein et
al. 1967, Zaugg and McLain 1970). Lorz and McPherson (1977) showed that
sublethal levels of copper inhibited the (Na,K)-stimulated ATPase activity:
Several workers have reported "in vitro!’ inhibition of ATPase by chlorinated
hydrocarbon insecti

cides and polychlorinated biphenyls (Campbell et al.
1974, Koch et al. 1972, and Leadem et al. 1974)

Widespread interest in the occurrence
hydrocarbon insecticides,

has resulted in several

aquatic ecosystems (Cope
1968, and Mullison 1970).
mixtures of cadmium or zine
coho salmon (Lorz and McPhe

and persistence of chlorinated

herbicides and heavy metals in surface waters

reviews of the effects of these chemicals on

1966, Eisler 1973, Eisler and Wapner 1975, Johnson
Our earlier research indicated that copper and
with copper were detrimental to smolting in

son 1977, Lorz et al. 1978). Therefore in the


Experimental Results

Static exposure --

In our study, a static toxicity test in December indicated a 2h-h neve
of Tordon 101 to be 20 mg/L (concentration of 2,4-D and picloram combined) 5
and the toxicant appears to have a steep mortality curve (Fig. 7). Tordon
101 appeared to cause a slight reduction in the (Na,K)-stimulated ATPase
activity of the gills, but this lowered activity did not appear to affect
seawater survival (Table 17). The reduced survival during the seawater
challenge of yearling coho salmon Previously exposed to 0.25 and 0.50 mg/L
is an interesting but unexplained Phenomenon (Fig. 7). A number of the

dead and dying fish were examined for disease or Parasites to determine if

this could possibly account for the deaths, but no abnormal conditions were
noted.

TABLE 17. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON

EXPOSED TO TORDON 101 IN FRESHWATER AND THE

SUBSEQUENT SURVIVAL FOLLOWING TRANSFER TO SEAWATER
(NOV. 30-DEC. 21, 1976).

Perceni
Concentration survival 2/ Percent
(mg/L) (144-h Gill survival Gill c/
nominal Measured exposure FW) ATPase2/ (360-h SW) ATPase’
Control - 100 1.9 100 4.0
0.25 0.29 100 nTa/ 25 4.9
0.50 0.50- 0.62 100 NT 65
1.0 1.32- 1.59 100 1.4 100 5-0
5.0 6.42- 7.35 100 1.5 100 4.0
15.0 18.1- 19.8 100 0.9 100
30.0 31.5 ogy -
60.0 67.8 of/

a/, Twenty fish exposed per concentration.
(Na ,K)-stimulated ATPase activity; mean of 4 fish (freshwater) .
&/Mean of 5 fish (seawater),
=/Not measured.
£/all fish dead within 60 h.
all fish dead within 2.5 h.

46


ABSTRACT

The 96-h LC50 values of several herbicides
Oncorhynchus kisutch, were determined. All 96
under static conditions at 10°C in freshwater
ranging from 70-83 mg/L and 85-93 mg/L (as Cai
herbicides acrolein and dinoseb were the most
soluble herbicides tested, having 96
respectively. Atrazine,
freshwater with 96

to yearling coho salmon,
-h tests were conducted
of alkalinity and hardness
C03), respectively. The
toxic of the 12 water

-h LC50 values of 68 and 100 ug/L,
diquat and picloram were moderately toxic in
-h LC50 values ranging from 10-30 mg/L.

Fish exposed to Amitrole-T, diquat and Paraquat in freshwater all

exhibited dose-dependent effects in subsequent seawater entry tests. The

other herbicides tested Produced little or no dose-related mortality when
fish were challenged with seawater. No apparent affects on the (Na,K)-
stimulated ATPase activit:

y of the gills were observed with any of the
herbicides tested.

The effect of sublethal concentrations of Tordon 101, dinoseb and
diquat on migratory disposi

tion was tested by releasing herbicide-exposed
salmon into a natural stream; only diquat produced a significant reduction
in downstream migration.

Under normal (field) application acrolein and dinoseb could affect
survival of all life stages of salmonids if water from treated irrigation
ditches were released into t!

he stream or river without sufficient holding
or detoxifying time. The use of diquat at recommended treatment levels
could reduce downstream migration of smolts and possibly affect their
survival in seawater. A11 other herbicide formulations tested appeared
to have no effect on smoltin,

g of yearling coho salmon; however, atrazine
has been shown to affect growth of young salmonids and survival of inverte-

brates at very low concentrations. Effects of the herbicides on other

life stages of coho salmon or different formulations of the herbicides
might produce considerably different results,

This report was submitted in fulfillment of grant R-804283 by the
Oregon Department of Fish and Wildlife under the partial sponsorship of
the U. S. Environmental Protection Agency. This report covers the period
January 5, 1977 to June 30, 1978,


~ SECTION VI

GENERAL DISCUSSION

herbinegeremPted to determine the 96 h-LC50 value of 12 water soluble
tested les to yearling coho salmon. The toxicity of the compounds
most t varied from <0.1 to >200 mg/L. Acrolein and dinoseb were the
amnenton tes with 96-h LC50 values of 68 and 100 ug/L, respectively;
were an ethyl carbamy] phosphonate (Krenite) and 2,4-D (dimethyl! amine)
snbenad taal aNd non-toxic at concentrations of 200 mg/L. Several
1976) f ers (Hughes and Davis 1963, Meehan et al. 1974 and Woodward

ture ave discussed the importance of alkalinity, hardness, pH, tempera~
para; and especially chemical formulation of the herbicide, as these
meters related to observed toxicity to fishes.

but particularly salmonids, would succumb to

c ;
oncentrations of acrolein or dinoseb if exposed at the approved
Control Handbook 1977). When coho salmon

yeblication rates (Oregon Weed
rlings were removed from either herbicide and placed in seawater, few
(Na, K)-stimulated ATPase activity of

afditional deaths occurred. The
gills appeared unaffected by either herbicide.

Most fish species,

cumulative over periods of

compounds may be
hasized the impor-

cl
hronic exposure. Sprague (1969) and Eaton (1970) emp!
tal exposures long enough to adequately

aon. of maintaining experimen

increas lethal threshold concentrations. The persistence of dinoseb

In oun ae the possibility of continuous aquatic habitat contamination.
result dinoseb flow-through exposures, concentrations of 4O and 60 ug/L
wher ted in almost total mortality during the 11 days of exposure;
mortalte in an earlier static experiment, 75 ug/k for 6 days caused no
cutth ity. Woodward (1976) did not observe cumulative mortalities in

In roat trout or lake trout exposed to dinoseb for 8 days. Similarly,
Our study concentrations of 10 and 20 ug/L for 15 days did not cause

any deaths.

The toxicity of some c

par. Acute exposure of yearling coho salmon to Amitrole-T, diquat and

aquat produced moderate mortality (30-75 mg/L); and the subsequent
esponse, even at

No statistically

cpa t lenge with seawater elicited a dose-dependent r
si entrations that were sublethal in freshwater. _
of ene icant effect of the herbicides on (Na,k)-stimulated ATPase activity
di e gill was observed. Coho salmon acutely and chronically exposed to
quat exhibited migratory inhibition upon release into a smal] coastal

Stream.
minimal or no effects

o mortality in the
ATPase activity.

and 2,4,5°T exhibited
j.e. little or a

Atrazine, Krenite, 2,4-D,
y-stimulated

1 .
sean iting of yearling coho salmon,
water challenge test, and no effect on (Na,K

63


ATPase activity of the gills and little effect on seawater tolerance
following toxicant exposure (Table 4). Our 96-h LC50 value is similar to
values given by Bond et al. (1960) and Burdick et al. (1964) for salmonids.
Histological examination of fish tissues (gill, kidney and liver) indicated
that acrolein had detrimental effects which appeared to be concentration
dependent (Appendix I1).

TABLE 4. SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING
COHO SALMON EXPOSED TO ACROLEIN IN FRESHWATER
AND THE SUBSEQUENT SURVIVAL FOLLOWING TRANSFER
TO SEAWATER (MAY 10-27, 1977).

Concent rat ion?/ Percent Percent
(ug/L) survival2/ Gill survival
nominal (144-h exposure FW) ATPase?/ (280-h Sw)
Control 100 4g 93.8
5 100 nta/ 100.0
10 100 4.7 100.0
20 100 NT 100.0
30 100 NT 100.0
50 95 5.2 86.7
75 0 NT NT
100 C) NT nTa/
y ictual concentration not measured.

Twenty fish exposed per concentration.

°/na,K-activated ATPase activity of the gill; umoles ATP hydrolyzed/mg
protein/h, mean of 4 fish. .

@/Not measured, no survivors in the higher concentrations.

The toxicity of acrolein was recently demonstrated on the Rogue River
above Grants Pass, Oregon, where Magnicide H, a gaseous form of acrolein,
was Introduced into an irrigation canal for the control of algae and
submerged vegetation (Oregon Department of Fish and Wildlife, notes in
Environmental Management Section file August 1977). The release of the
treated irrigation water within 24 h of treatment rather than after the
recommended holding time of 6 days (manufacturer recommendation on toxicant
contatner) appeared to be the cause of total mortality of fish in a 10-mile
section of the Rogue River below the spill. The Oregon Department of Fish
and Wildlife estimated that 238,000 fish were killed, including 42,000
salmonids, with an estimated value of $284,000.


DOWNSTREAM MIGRATION (%)

Control’

exposure to diquat.

0.5 mg/liter “|
o—O—

LO mg/liter _]
3.0 mg/liter 7]

gt L L

19 23 27

Control.
1,0 mg/liter 7

sof ee 0.5 mg/liter,

—_ 3.0 mg/liter

SOF 4
we ep SE mgiter_ 5
40 4
30 4
20 4
10 4

° [as ee ee Oe ee ee De 1 L i

ul 3 5 7 9 1 13 (5 a i9 23 27

DAYS: POST RELEASE
Figure 12. Percent downstream migration of yearling coho salmon following
A. represents 178-183 coho salmon released per group
B. represents 98-105 coho salmon

following 285 h of diquat exposure.
released per group following 96 h of diquat exposure.

61


A release of dinoseb-exposed coho salmon yearlings was made into
Crooked Creek May 5, 1977, and the downstream migration of the various ed
groups monitored. Coho salmon exposed to 10 ug/L dinoseb for 14 days ever
slightly better migration than the controls or the 20 ug/L group, however,
no significant difference in migration times were noted for the three
groups (Fig. 10A). Of the yearling coho salmon exposed for 96 h, the hough
control and 20-ug/L dinoseb treated fish showed the best migration, alt
the 10-and 40-yg/L groups had only 10% fewer migrants (Fig. 10B). The
group exposed to 60 ug/L for 24 h also exhibited a 10% lesser migratory
tendency than the controls (Fig. 10B). Fish exposed to 40 or 60 ug/L
dinoseb for 48 h showed a dose-dependent inhibition in average percent tes
downstream migration (Fig. 10C). Considerable variation in migration ra
occurred, however, between replicated exposure tanks [controls, 20 and i-
60 ug/L (Table A-5)]; and only one of the two 60 ug/L groups (which exper

enced mortality during the toxicant exposure) showed statistically less
migration than the controls (P = 0.05).

Yearling coho salmon exposed to 100 ug/L dinoseb for 114 h showed ex
tensive necrosis of the liver, kidney, and gil} lamellae (Appendix 11).
Histological examination of tissues of coho salmon exposed to 60 and
40 ug/L dinoseb for 168 and 336 h, respectively, showed only minor de-
generative changes (Appendix WI). This lack of histological damage is

rf Ss
S Of exposure to dinoseb. Tissue sample’

. S
b showed some present in the eight tissi
examined (Table A-6). The spleen, gall bladder, liver, and kidney appea
to be the sites of major accumulation.

DIQUAT

Review of Literature: Toxicity to Fish and Behavior in the Environment

Diquat (6,7-dihydrodipyrido(1, 2-a: 2!,
the trade names of OrthoR Diquat and RegoneR,
an aquatic herbicide in canals, lakes, and
crops such as alfalfa, clover, and vetch,
diquat's Properties and i

I-c) pyrazinediium ion) has
It is registered for use aS

Ponds, and on non-food seed

For additional information on

Diquat application rates of 1.5 to 2.5 mg/L or | gal per surface acre
have been shown to be effective in the control of aquatic vegetation in
small, stillwater Ponds in western Oregon. There appears to be a wide
margin of safety between this rate of application and lethal levels of
diquat to fish in hard water. Under laboratory conditions, Bimber et al-
(1976) found that yellow perch experienced a Significant level of
respiratory stress at diquat concentrations comparable to those normally
applied for aquatic weed control (no water quality given). Diquat is
moderately toxic as shown by its acute toxicity to various fish species
(Table 21). The toxicity has been Shown to va
upon hardness and pH (Surber and Pickering 1962), Wilson and Bond (1969)
found that the addition of pond mud to the test aquaria changed the 96-h
LCSO of diquat for the amphipod (dyalella azteca) from 0.046 to 6.8 mg/L.
The reduction in toxicity was Probably due to the adsorption of the

Sh


pH @7.03, ammonia <0.10 mg/L and hardness @100 mg/L as CaC03. Water
ed at 10 + 1°C in all static tests. In the flow-

temperature was maintain
through test the temperature pattern was similar to the average of the semi-
tures of the North Fork of the Alsea River

monthly maximal and minimal tempera
(9.0°C in March increasing to 12.3°C in June). Water temperatures were

monitored in the static and flow-through systems with continuous recording

thermometers.

CHEMICAL ANALYSIS

The concentration of each herbicide was analyzed from composite water
samples for each exposure tank. However, no analyses, were conducted for
acrolein or krenite. In the static toxicity tests a composite water
sample was obtained by combining daily samples of 80 ml of test water ina
container with an appropriate fixative. The water samples were generally
taken several hours after the daily exchange. In the flow-through system
a weekly composite water sample was obtained by combining datly samples of
50 ml of test water. At least two weeks usually elapsed between sampling
and subsequent extraction and analysis. During this period the samples

were stored in a cool area.

for each herbicide tested is described in

f tests the measured concentrations varied

d or nominal; however, no explanations are

The discrepancies (between measured and nominal
d because of problems in extraction or

the physical-chemical behavior of the

The analytical method
Appendix 1. In a number o
considerably from the desire
known for the deviations.
concentration) possibly resulte
analysis or may have been due to
herbicide solution in the exposure tank.

GILL ATPASE ACTIVITY
activity was measured on individual fish by

the whole gill homogenate method (Johnson et al. 1977). Previous charac-
S conducted on coho salmon (Lorz et al.

terization of this enzyme assay wa
1978). Enzyme activity was measured at 37°C. The released inorganic
phosphate was measured by the method of Ernster et al. (1950). (Na,K)-
Stimulated ATPase activity was calculated as the difference between rates
ence or absence of 0.5 mM

of inorganic phosphate liberated in the pres
Ouabain. Protein was measured by a modification of the method of Lowry et
al. (1951). Sample sizes ranged from 5-6 fish in static tests and 10-20

fish in the flow-through system.

(Na,k)-stimulated ATPase

TOLERANCE TO SEAWATER

Generally groups of 10-20 fish from each exposure tank (fol lowing
14h-h static, 144-h flow-through, or 15-day flow-through toxicant expo-
sures) were tested for tolerance to seawater. The percentage of these
yearling coho salmon surviving the seawater challenge thus provided a
Presumptive measure of the fish's osmoregulatory ability. Exposed: fish
and controls were transferred directly from the exposure tanks into 0.61-m
fiberglass tanks containing 120 L of natural seawater. The seawater was
continuously aerated and 85% of the 120 L was exchanged once daily.

1]


Paraquat formulations are presently available as chloride salts (Lawrence
et al. 1965). The pure chloride salt is a white solid that forms a dark
red aqueous solution. Paraquat is a nonselective, quick-acting herbicide
and desiccant, and is used extensively due to its effectiveness on grasses
and most broadleaf weed species (Calderbank and Slade 1976). Paraquat is
registered for use as a directed spray on preplant treatment in many tree
crops and several field crops including alfalfa, corn, soybeans, sugar
beets, and tomatoes. It is also employed as a preharvest desiccant on
cotton, potatoes, and soybeans, and in aquatic weed control. Normal
application rates for paraquat are 1/4 to 1 1b (a.i.)/acre, and 0.1 to
1.5 mg/L in the aquatic environment (Calderbank and Slade 1976).

Paraquat dichloride is a quarternary ammonium salt, highly soluble in
water, insoluble in most organic solvents, and nonvolatile. The herbicide
has little or no residual activity due to its rapid inactivation by irreversible
adsorption to soil. The persistence of Paraquat in water is highly variable
and dependent upon water movement, weed density, and the Presence of mud,
suspended silt, and sunlight. Herbicide residues are normally reduced to
0.01 mg/L or less in 6 to 14 days. Herbicide adsorbed to the hydrosol
persists for long periods but is Probably not biologically active (Calderbank
and Slade 1976). Grzenda et al. (1966) found that paraquat applied to
ponds at rates of 2.1] and 2.5 mg/L persisted tn the water for 6 and 23

days, respectively. No desorption of paraquat from the bottom clays was
observed 3 months after initial application.

Burnet (1972) found that am,
levels in drift fauna following
2 mg/L paraquat.

phipods were reduced to 5% of pretreatment
treatment of a New Zealand stream with

Paraquat and diquat do not appear to be bioconcentrated in fish.

Calderbank and Slade (1976) reported on Some unpublished findings of Carter
(1971). Rainbow trout exposed to these herbicides (1.0 mg/L) for 16 days
accumulated 0.5 to 0.6 mg/L whole body re.

sidues of either herbicide.
Residues were detected in the skin,

gills, gut, and organs, but not in the
muscle. Upon transfer of the fish t

o freshwater the residues steadily
declined. Similar findings for whole-

body residues of diquat and paraquat
Were reported by Cope (1966). When several ponds were treated with | mg/L
paraquat (emulsion) or diquat, Paraquat bioaccumulation was detected in
bluegill sunfish (1.21 mg/L whole-body residues) but little diquat bio-
accumulation was detected (0.09 mg/L). Residues of Paraquat in rainbow
trout, green sunfish, and channel catfish (Ictalurus punctatus) were
0.37 mg/L or less.

Earnest (1971) found that Paraquat levels ranged from 0.58 kg i
green sunfish to 1.86 mg/kg i i $ aeena/kg In
Colorado pond. Residue levels in bluegills reached a maximum of 1.58 mg/kg
8 days after treatment and then declined.

A comparison of normal Paraquat usage levels with fish toxicity suggests
that the direct poisoning of fish by fie 5 yoeee

t Id application is a remote possibility.
Bluegitls, largemouth bass, fathead minnows, channel catfish, and rainbow
trout exhibit approximate threshold (LC10) toxicity levels of 5 mg/L

28


TABLE 18. SURVIVAL OF YEARLING COHO SALMON EXPOSED TO
TORDON 101 IN FRESHWATER, AND THE SUBSEQUENT
SURVIVAL FOLLOWING TRANSFER TO SEAWATER
(MAR. 13-29, 1977).

Concentration Percent Percent
(mg/L) survival2/ survival
nominal Measured (44-exposure FW) (240-h_ SW)

New stock daily

Contro} 0 100 100
0.25 0.13-0.16 100 100
0.50 0.12-0.13 100 100
1.0 - 100 100

22.0 8.41-8.83 5 0
Stock mixed Mar. 13, 1977

0.25 0.19-0.38 100 100

0.50 0.41-0.50 100 100

22.0 9.21-9.7 0 -

2/ twenty fish exposed per concentration.

Flow-through exposure--

Following marking (fin excision) and time for acclimatization to new
surroundings, yearling coho salmon were exposed to Tordon 101 in the flow~
through system beginning on Mar. 28, 1977. Groups of yearling coho salmon
were placed in seawater following 144-h and 380-h exposure to the four

Tordon 101 concentrations. No deaths occurred in either the fish exposed
to Tordon 101 or the control fish

during the 240 h of seawater exposure
(Table A-2). The (Na,K)-stimulated ATPase activity was not affected by any
of the Tordon 101 concentrations tested (Table A-2). Exposure to Tordon
101 for 360 h produced no apparent effect on the condition factor of
yearling coho salmon (Table A-3). The fish all fed well during the
exposure, and the general decline in condition factor was probably the
normal change in the length-weight

e relationship that occurs as the fish
undergo transformation to smolts (Wagner 197h4a).

A release of 10 groups of coho salmon exposed to Tordon 101 for 360 h
and 5 groups exposed for 96 h was made into Crooked Creek on April 13,
1977, and downstream movement was monitored. The coho salmon exposed to
0.3 mg/L for 15 days showed slightly stimulated migration compared to the
controls, whereas the other concentrations (0.6, 1.2, and 1.8 mg/L) showed
a dose-dependent inhibitory response. Approximately a 10% difference in
movement was noted between controls and the group receiving 1.8 mg/L
Tordon 101 over the 84 days that migratt

on was monitored (Fig. 8a). The
differences observed, however, were not Statistically significant (P = 0.05) «

4G


Probability estimates for downstream migration occurring from each
release were tested for significance between the control group and each
treatment group. Significance (P=0.05) was determined by the z test where
the null hypothesis tested was:

Hg: Py = Pe
Ha: Pr # Pc
Po - Pr
z=
Po. (i-Pc) Pe SIP)
+
nc aT

and where: P = a/n

a
n

number of migrants
number of fish released.

A 7-km section of the stream was electrofished from above the release
sites to the weir in an attempt to collect non-migrants. No coho that had
been released earlier were found, although native cutthroat and rainbow of
comparable size to the released fish were caught. There were only two
small areas in the stream where our electrofishing gear appeared inadequate
because of the depth of the water. Thus, fish that had not migrated by
early July apparently died as a result of predators, stress, natural
causes or latent effects of the toxicant exposure.


Procedure:

Resin adsorption and desorption of aminotriazole:
water sample into a 400 mL beaker.
stirrer and stir for 30 min.

Measure 200 mL of
Add 10 g Dowex 50W-X8, place on
Set up a chromatographic column with
1g of resin in the bottom. Rinse the sample and resin from the

beaker into the column with distilled water, allow the water to drain
through the column and discard the water. Elute the aminotriazole

from the resin with 200 mL of 2N ammonium hydroxide into a 600 mL
beaker.

Acid digestion and cleanup:

Add 2 or 3 glass beads and boil on a hot
Plate until volume is 25-50

mL, cool and add 3 mL of 75% sulfuric acid
solution. Boil for 10 min, maintaining volume with water rinsing.
Carefully add 1 g charcoal, boil for another 10 min, maintaining
volume at 20 to 25 mL with water rinse.

Filter hot solution through sintered
rinse with 4x25 mL portions of hot 0.
followed by 100 mL of hot water.

glass funnel with suction and
75% sulfuric acid solution

bring to volume.

Color development:

Transfer a 5 mL aliquot of sample into a 50 mL
erlenmeyer, add 3 mL

concentrated sulfuric acid and cool in an ice

f 0.5% sodium nitrite, and swirl; after 10 min,
sulfamic acid, and swirl. After 10 min add 10
drops 1.0% N-(1-Naphthy!) ethylenediamine dihydrochloride and swirl.
Read against a reagent blank at 460 nm within 30 min. Using a
standard curve for aminotriazole, determine the amount of 3-amino-s-
triazole in the 5 mL aliquot.

ATRAZINE

1.

Summary of method: This method fs based on Zweig and Sherma (1972a).
Atrazine is extracted from water with methylene chloride. Analysis is

ic microcoulometric gas chromato-

Reagents: Methylene chloride, reagent grade

Methanol, reagent grade
Apparatus: Glass jars with teflon-lined caps
Separatory funnels (500 mL)

Graduated cylinders (500 € 100 mL)
Beakers (250 mL)

Volumetric flasks
Gas chromatograph, MicroTek 20
with Dohrmann model C200B

with halogen titration cel]

OOMF, or equivalent,
microcoulometric detector

82


Data on toxicity of triethylamine salts of 2,4,5-T to aquatic organisms
are scarce. Kenaga (1974) reviewed the literature concerning the toxicity
to fish ef 2,4,5-T and its derivatives. Exposure to the commercial formulation
DED-WEED® at concentrations >72 mg/L (a.e.) for 24 and 96-h resulted in at
least 50% mortality of bluegill sunfish, channel catfish, and fathead
minnows. Exposure of rainbow trout to the same formulation [>72 mg/L DED-
WEEDR (a.e.)] resulted in 50% mortality in 24 h whereas at concentrations
of 0.07-0.72 mg/L (a.e.) for 96 h exposure Kenaga also reported mortality
rates of 50%. Spot (Leiostomus xanthurus) exposed to 0.4 mg/L (a.e.) for
24 h showed no mortalities. The fathead minnow exposed to 40.2 mg/L for
72 h had no mortality, but at concentrations >72 mg/L for 24 or 96 h at
least 50% mortality was observed (Kenaga 1974). Davis and Hughes (1963),
utilizing the triethylamine salt of 2,4,5-T (Crop Rider®) found the 24 and
48-h LCSO for bluegill sunfish to be approximately 53 mg/L (a.e.).

Experimental Results

No deaths were observed in yearling coho salmon exposed to concen
trations of 6-7 mg/L of 2,4,5-T (triethylamine salt, Weedar) for 144 h
(Table 11). Similarly, there was no apparent effect on (Na,K)-stimulated
ATPase activity of the gills. When the fish were placed in seawater only
minimal mortality occurred (Table 11). The lack of mortality during the
toxicant exposure was surprising considering that concentrations of 0.07-
0.72 mg/L (a.e.) were reported to cause 50% mortality in 96 h to rainbow
trout (Kenaga 1974); however, no water quality data were presented.
Generally, LCSO values for herbicides obtained at 24 h have been about 10
times greater than the LC50 concentration noted for 96-h exposure.

TABLE 11. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED TO
2,4,5-T (WEEDAR) IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (MAR. 31-APR. 17, 1977).

Concentration Percent / Percent

(mg/L) survival? Gill survival

nominal measured (144-h exposure FW) ATPase?/ (240-h_ SW)
Control 0 100 4.06 100
0.05 0.016-0.021 100 nTe/ 95
0.10 0.041-0.043 100 NT 100
0.25 0.13 -0.15 100 NT 100
0.50 0.27 100 NT 100
1.0 0.57 -0.74 100 3.52 100
10.0 6.64 -7.0 100 4.66 100

2/ twenty fish exposed per concentration.
c (Na ,K)-stimulated ATPase activity of the gills;
Not measured.

mean of 5 fish.

35


Trichell, D. W., H. L. Morton and M. G. Merkle. 1968. Loss of herbicides
in runoff water. Weed Sci. 16(4):447-449.

Tweedy, B. G. and L. D. Houseworth. 1976. Miscellaneous herbicides. pp.
815-833. In: P. C. Kearney and D. D. Kaufman (eds.). Herbicides:

chemistry, degradation and mode of action. Vol. 2. Marcel Dekker, Inc.,
New York, New York.

United States Fish and Wildlife Service. 1963. Pesticide-wildlife studies,

a review of Fish and Wildlife Service investigations during 1961 and
1962. Fish Circ. 167.

Velsicol] Chemical Corp. 1967. Banvel D. General Bulletin No. 521-2.

Wagner, H. H. 1974a. Photoperiod and temperature regulation of smolting
in steelhead trout (Salmo gairdneri). Can. J. Zool. 52(2):219-234.

Wagner, H. H. 1974b. Seawater adaptation independent of photoperiod in
steelhead trout (Salmo gairdneri). Can. J. Zool. 52(7):805-812.

Walker, C. R. 1964. Simazine and other s-triazine compounds as aquatic
herbicides in fish habitats. Weeds 12(2):134-139.

Warner, R. E., K. K. Peterson, and L. Borgman. 1966. Behavioural

pathology in fish: A quantitative study of sublethal pesticide toxi-
cation. J. Appl. Ecol. 3:223-247.

Webb, W. E. 1951. Toxicity of certain pesticides in fish. Idaho Fish and

Game Department. p. 118. In: M. Lipschuetz and A. L. Cooper. 1961.
Toxicity of 2-secondary-buty1-4,6-dinitrophenol to blacknose dac:
and rainbow trout. N. Y. Fish Game J. 8(2):110-121.

Wellborn, T. L. Jr. 1969. The toxicity of nine therapeutic and herbicidal
compounds to striped bass. Prog. Fish-Cult. 31(1):27-32.

Wildish, D. J., W. G. Carson, T. Cunningham, and N. J. Lister.
Toxicological effects of some or
Atlantic salmon.

1971.
t ganophosphate insecticides to
Fish. Res. Board Can. MS Rep. 1157:22 pp.

Wilson, D. C. and C. E. Bond. 1969.

The effects of the herbicides Diquat
and Dichlobenil (CasoronR) on pon 4

Di n d invertebrates. Part 1. Acute
toxicity. Trans. Am. Fish. Soc. 98 (3) : 438-443,

R

Wojtalik, T. F., T. F. Hall, and L. L. Hill. 1971. Monitoring ecological
conditions associated with wide-scale applications of DMA 2,4-D to
aquatic environments. Pestic. Monit. J. 4(4) 2184-203.

Woodiwiss, F. Ss. and G. Fretwell. 1974. The toxicities of sewage effluents,
industrial discharges and some chemical substances to brown trout
(Salmo trutta) in the Trent River Authority area. Water P Control
(G.B.) 73:396-401. . er Poll. Con

76


Flow-through exposure-~

As dinoseb was found to be extremely toxic to coho salmon and had the
potential for interacting with anadromous salmon because of its use in weed
contro] along ditches and irrigation canals, it was chosen for study in the
flow-through system. Following marking (fin excision) and a 5-day accli-
matization period, yearling coho salmon were exposed to dinoseb (Dow-
Premerge) beginning April 19, 1977. Two days after initiation of the
exposure, the fish in the highest concentration (60 ug/L) began to turn
dark, eat poorly, and die. The deaths occurred at concentrations con-
siderably lower than had previously caused death under static conditions in
late February. Whether the smolting of the fish, the use of a flow-through
system or a combination of both was responsible for the greater toxicity is
unknown. During the first 6 days of exposure to dinoseb, 93% mortality
occurred in the groups receiving 60 ug/L and 8% mortality was noted in the
4Q-ug/L group. Two deaths occurred during the sea water challenge in the
group originally exposed to 60 yg/L dinoseb for 144 h (Table 20). Con-
sidering the poor physical appearance of the 40 and 60 ug/L exposed groups
prior to placing in salt water, it was surprising that there were so few
deaths (Table 20). The majority of dark colored fish regained their
silvery coloration during the 10-day seawater challenge. There were no
mortalities during either toxicant exposure or seawater challenge of the
coho salmon exposed to 10 or 20 ug/L dinoseb for 16 days. However, all
the fish exposed to 60 ug/L and almost all exposed to 40 ug/L died during
the 16 days of exposure (Table 20). The gill (Na,K)-stimulated ATPase
activity was unaffected after 6 or 16 day exposures to dinoseb (Table 20).

TABLE 20. SURVIVAL OF YEARLING COHO SALMON EXPOSED TO DINOSEB
IN FRESHWATER AND SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (APR. 19-MAY 16, 1977).

Concentration Percent
(ug/L) Percent Gill survivala/
nominal measured survival 2/ ATPase®/ (240-h SW) +
A. thh-h exposure
Control 0.5 100 (270) 6.93 (10) 100.0 (20)
10 Te4- 7.9 100 (250) nT~ 100.0 (20)
20 15.3-17.3 100 (250) 6.54 (10) 100.0 (20)
4O 38.6 92 (300) 5.36 (10) 100.0 (20)
60 54.5-58.3 7 (339) 90.0 (20)
B. 384-h exposure
Control 0.5 100 (270) 6.81 (20) 100.0 (30)
10 6.2- 8.8 100 (250) NT 100.0 (30)
20 18.3-17.3 100 (250) 6.43 (20) 100.0 (39)
mt) 30.7-38.6 6 (300)9/ NT NT
60 52.9-60.0 0 (339)2

3/Number of fish exposed in parentheses.

—/ (Na,K)-stimulated ATPase activity;mean with sample size in parentheses.
qfot measured.

Terminated on day 11 to restock tanks.

£/all dead within 11 d.

53


Woodward, D. F. 1976. Toxicity of the herbicides dinoseb and picloram to
cutthroat trout (Salmo clarki) and lake trout (Salvelinus namaycush) .
J. Fish. Res. Board Can. 33:1671-1676.

Woodward, F. D. and F. L. Mayer Jr., 1978. Toxicity of three herbicides
(Butyl, Isoocty] and Propylene glycol butyl ether esters of 2,4-D) to
cutthroat trout and lake trout. U. S. Fish and Wild. Serv. Tech.
Papers. 97:1-6.

Yeo, R. R. 1967. Dissipation of diquat and paraquat, and effects on
aquatic weeds and fish. Weeds. 15(1):42-46.

Youngson, C. R. and R. W. Meikle. 1972. Residues of picloram acquired by
a mosquito fish, Gambusia sp. from treated water. The Dow Chemical
Company, Walnut Creek, California. Unpublished Report GH-1210. p. 65.
In: National Research Council of Canada. NRC Associate Committee on
Scientific criteria. (1974). Picloram: the effects of its use as a
herbicide on environmental quality. Publication No. NRCC 13684 of

the Environmental Secretariat.

Zaugg, W. S. and L. R. McLain. 1970. Adenosine triphosphatase activity in
gills of salmonids: seasonal variations and salt water influence on
coho salmon, Oncorhynchus kisutch. Comp. Biochem. Physiol. 35:587-596.

Zaugg, W. S. and L. R. McLain. 1972. Changes in gill adenosine-triphos~
phatase activity associated with parr-smolt transformation in steelhead
trout, coho, and spring chinook salmon. J. Fish. Res. Bd. Canada

29:167-171.

Zaugg, W. S. and H. H. Wagner. 1973. Gill ATPase activity related to parr-
smolt transformation and migration in steelhead trout (Salmo gairdneri):
Influence of photoperiod and temperature. Comp. Bicohem. Physiol.
45B:955-965.

Zweig, G. and J. Sherma. 1972a- Miscellaneous Pesticides - Triazines. pp.
240-245. In: Gunter Zweig (ed.) Analytical Methods for Pesticides,
Plant Growth Regulators, and Food Additivies. Volume VI, Gas Chromato-
graphic Analysis. Academic Press. New York.

Zweig, G. and J. Sherma. 1972b. DNBP (Dinoseb). pp- 639-642. In: Gunter
Zweig (ed.). Analytical Methods for Pesticides, Plant Growth Regulators,
and Food Additives. Volume VI, Gas Chromatographic Analysis. Academic

Press. New York.

77


O-Img/L

RR KR RK KO le he Kern ake max ee a=
4 ~
$ \
z A ee se
2 25mg/L
a Na 259:
| oad
2
rs}
ry
50mg/L
"es
pOmyt ss yg tg
120 192 240 "536

Freshwater Seawater

EXPOSURE TIME (hours)

Figure 4. Percent survival of yearling coho salmon during exposure to
Amitrole-T in freshwater and subsequent survival upon transfer to seawater.

ATRAZINE

Review of Literature: Toxicity to Fish and Behavior in the Environment

Atrazine (2 chloro-4 ethylamino-6~isopropylamino-s-triazine) is a wide
spectrum symmetrical triazine herbicide. It is widely used to control many
broad leaf and grass weeds in the production of corn, macadamia nuts,
pineapples, perennial ryegrass, sorghum and winter wheat. Atrazine is also
employed in some areas for selective weed control in Christmas tree farming,
grass-seed production, highway right-of-way clearance, and conifer refores-
tation. Hall et al. (1972) noted that more than 100 million pounds of
atrazine is applied annually to agricultural lands in the United States.

Atrazine is not as strongly bound to soil particles as is the triazine
herbicide simazine. Its lower solubility and reduced adsorption add to its
mobility in the soil. Axe et al. (1969) studied the residual life of
atrazine in the soil and found that only 33% of the herbicide remained
after 5 days. Under most climatic and edaphic conditions, atrazine (2-4
lb/acre) has residual phytotoxicity for 4-7 months (Harris and Sheets
1965). Residual carryover after repeated application is minimal.

22


Number

12

13

16

20

21
22

23

SURVIVAL OF YEARLING COHO SALMON EXPOSED TO ESTERON
BRUSH KILLER AND SUBSEQUENT SURVIVAL FOLLOWING TRANSFER

TO SEAWATER (MAY 31-JUNE 16, 1977)..esececeeeceerereeseuees

SURVIVAL OF BIG CREEK WINTER STEELHEAD TROUT FRY EXPOSED
TO ESTERON BRUSH KILLER, FAIRPLAY LABORATORY, OSU

(May 3t-June 4, Si) ee

MORTALITY DATA FOR SEVERAL FISH SPECIES EXPOSED TO
TORDON 22K FOR 96 HOURS...... cee e eee eee enn c eee necesscnnns

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO TORDON 22K IN FRESHWATER AND SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (DEC. 9-27, 1976)...........

SURVIVAL OF YEARLING COHO SALMON AT THREE FISH DENSITIES
EXPOSED TO TORDON 22K OR TORDON 22K PLUS AMMONIUM

CHLORIDE IN FRESHWATER, AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (FEB. 11-28, 1977).......00

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO TORDON 101 IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (NOV. 30-DEC. 21, 1976).....

SURVIVAL OF YEARLING COHO SALMON EXPOSED TO TORDON 101
IN FRESHWATER, AND THE SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (MAR. 13-29, 1977).seeceeeececresceees

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO DINOSEB IN FRESHWATER AND SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (FEB. 22-MAR. 10, 1977).....

SURVIVAL OF YEARLING COHO SALMON EXPOSED TO DINOSEB IN
FRESHWATER AND SUBSEQUENT SURVIVAL FOLLOWING TRANSFER

TO SEAWATER (APR. I19-MAY 16, 1977). cececececeereeeceereeeee
ACUTE TOXICITY OF DIQUAT TO VARIOUS FISH SPECIES...........

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO DIQUAT IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (APR. 19-MAY 5, 1977) 2.00

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO DIQUAT IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER. ..-..-seeeeeeceseeceeereeeee


Much of the fish toxicity work on the phenoxy herbicides concerns the
PGBE esters of 2,4-D or 2,4,5-T, but little has been done on mixtures of
these compounds. Hughes and Davis (1962b) found that both the 24 and 48-h
LC50's for bluegill] sunfish exposed to the PGBE ester of 2,4-D were 29 mg/L
under static conditions in water with a mean pH of 6.9 and a mean hardness
of 29 mg/L. Meehan et al. (1974) observed that the 96-h no-effect level
for coho salmon fingerlings exposed to the PGBE ester of 2,4-D was less
than 1 mg/L. There was a mean fry mortality of 26.7% after 96 h of
exposure to 1 mg/L of the herbicide in water that ranged in hardness from
10.0 to 33.6 mg/L as calcium plus magnesium. A 48-h LC50 of 1.1 mg/L PGBE
ester of 2,4-D was reported by Cope (1966) for rainbow trout (no water
quality given). Butler (1965) observed that the 48-h LC50 for the
estuarine longnose killifish was 4.5 mg/L in seawater.

Studies on the toxicity to fish of the PGBE ester of 2,4,5-T have been
reported by Kenaga (1974). This herbicide appears to be generally more
toxic than the corresponding PGBE ester of 2,4-D. All rainbow trout died
within 24 h at 0.13 mg/L (a.e.) of the PGBE ester of 2,4,5-T, and all fish
were dead after 3 h of exposure to 0.67 mg/L of the herbicide (no water
quality given). After 7 h of exposure to 0.13 mg/L of the (PGBE) ReddonR
formulation, all bluegill sunfish were dead. Bluegills exposed to Esteron
245 (PGBE) had 24- and 48-h LC50's of 17 mg/L in static water with a mean
pH of 6.9 and a mean hardness of 29.0 mg/L. €xposures of fathead minnows
to different formulations of the PGBE ester of 2,4,5-T [0.13 to 1.33 mg/L
(a.e.)] all resulted in 100% mortality within 72 h (no water quality

given).

Bioconcentration of the PGBE ester of 2,4-D has not been observed in
fish tissues. No detectable residues of the herbicide were found in
bluegill sunfish exposed to 10 mg/L PGBE ester of 2,4-D (Cope 1966).

Matida et al. (1975) noted that when a mixture of 2,4-D and 2,4,5-T as
the butoxyethanol ester (commercially called "Brush Killer'') was aerially
spread over 9.5 hectares of forest at a rate of 150 kg/ha, no appreciable
change was noted in the aquatic community. The authors were unable to
detect the chemical in the stream during the 48 h observation period
following spraying. Similarly, fishes (cherry salmon and dace fingerlings)
showed no mortality nor abnormal behavior and the standing crop of in-
vertebrates appeared unchanged. Ina later laboratory study Matida et al.
(1976) found that "Brush Killer" (mixture of 2,4-D and 2,4,5-T) exhibited
toxic effects on aquatic sow bugs Asellus hilgendorffii and cherry salmon
fry and dace tingertings. The 96-h LC50 for the groups tested were 1.6
(sow bug), 0.6 (cherry salmon), and 1.3 {dace) mg/L. Exposures of cherry
salmon fingerlings to concentrations of 0.47 and 0.62 mg/L "Brush Killer"
for 96 h caused histological changes of liver parenchyma which the authors

considered a nonspecific response to a toxic agent.

37


Number

1

TABLES

Page
HERBICIDES TESTED FOR ACUTE TOXICITY TO YEARLING COHO
a 8
CHEMICAL CHARACTERISTICS OF TEST WATER DURING STATIC
TOXICANT EXPOSURE, . 0.0.00... cece cee cece ce cceeecececceveneres 12
ACUTE TOXICITY OF ACROLEIN TO VARIOUS FISH SPECIES.......... 16

SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING COHO SALMON
EXPOSED TO ACROLEIN IN FRESHWATER AND THE SUBSEQUENT
SURVIVAL FOLLOWING TRANSFER TO SEAWATER (MAY 10-27, 1977).. 18

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO AMITROLE-T IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (JAN. 17-FEB. 16, 1977)

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO ATRAZINE IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (MAR. 2-19, 1977)... cece eee 24

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO DICAMBA IN FRESHWATER AND SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (JAN. 6-23, 1977)

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO KRENITE IN FRESHWATER Al

ND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (FEB. 2-17, 1977)... eee eee 27

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED
TO PARAQUAT-CL IN FRESHWAT

ER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (MAR. 22-APR. 7, 1977)

SURVIVAL AND GILL ATPASE ACTIVITY OF YEARLING COHO SALMON
EXPOSED TO 2,4-D IN FRESH

WATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER (DEC. 29, 1976-JAN. 14,
1977)

TUTTE ee eee eect tenet tee ener tenes 3h

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO 2,4,5-T (WEEDAR) IN FRESHWATER AND THE SUBSEQUENT
FURVIVAL FOLLOWING TRANSFER TO SEAWATER (MAR. 31-APR.
17, 1977)...e0e0e :

TUN e eee eee eee eee e ence teeeeeee 35

viii


2,4-D

Review of Literature: Toxicity to Fish and Behavior in the Environment

The chlorine-substituted phenoyxacetic acids, 2,4-D (2,4-dichlorophenoxy-
acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) were introduced
as selective herbicides following investigation of their growth-regulating
and herbicidal properties during World War II (Templeman 1955). They are
effective against many broadleaf weeds but not against graminaceous weeds.
The herbicide 2,4-D is registered for use on forests, range and pasture
grasses, rye, wheat, barley, oats, corn, asparagus, apples, pears, grapes,
potatoes, blueberries, cranberries, non-cropland uses, turf, and certain
aquatic sites.

Most phenoxyalkanoic acid herbicides are formulated as the salt or
ester form. Amines of 2,4-D are the most commonly used salt forms of
2,4-D, although the sodium, potassium and ammonium salts are also used.

The di-and tri-substituted amines are particularly important; they are
highly soluble in water and are used in the formulation of water soluble
concentrates. Normal rates of application of 2,4-D amines are as follows:
1/4 to 1-1/2 lb/acre (a.i.) for most crops; 2 to 3 lb/acre (a.i.) for
forest spraying; and 0.25 to 0.50% (a.i.) for the control of emergent and
floating weeds. The ester formulations of 2,4-D are extremely important
and are used extensively in forestry and agriculture for vegetation control.
The ester formulations are 10 to 100 times more toxic to fish and other
aquatic organisms than the dimethylamine salt, but are often chosen for use
because of desirable physical properties such as: control of droplet size,
limited solublity in water, good spread of herbicide upon contact with
vegetation being controlled, and persistence in the environment.

The salts of 2,4-D have very low volatility. When dissolved in hard
water they form insoluble and essentially inactive calcium, magnesium, or
tron salts of 2,4-D. Leaching of 2,4-D in soil is dependent upon soil type
and herbicide solubility in water. Decreased leaching occurs in clay and
organic soils due to adsorption by soil colloids.

Crosby and Tutass (1966) found that 2,4-D decomposed rapidly in the
presence of water and ultraviolet light or sunlight, and they identified
the decomposition products. Audus (1950) demonstrated the importance of
microbial degradation of phenoxyacetic acid herbicides. In a warm, moist
loam, 2,4-D can be expected to disappear within 2 to 3 weeks (Loos 1976).
Norris (1970) noted that 94% of 2,4-D was degraded in 35 days when applied
in a red alder forest floor.

Wojtalik et al. (1971) found no harmful or distinguishable response or
accumulation in zooplankton, phytoplankton or macroinvertebrates following
treatment of water with 20 to 40 Ib per acre (acid equivalent, a.e.) of
DMA-2,4-D. The authors believed there was little danger of biomagnification
of the 2,4-D in contrast to chlorinated hydrocarbon pesticides. Wojtalik
et al. reported residues (in water) greater than 0.02 mg/L at only 2 of 19
stations 4 weeks after treatment. The authors stated that disappearance of
DMA-2,4-D from water is rapid compared with the herbicides dichlobenil and
fenac which persisted at detectable levels for up to 160 days.

31


Figure 2. Exposure tanks with diluter

in background.

In addition, smaller groups of fish (about
flowing toxicant in 0.61-m fiberglass tanks. Flows (5 L/min, 95% replacement

every 1.5 h) to the 120 L tanks were Provided by a siphon from mid-depth of

the 1.54-m tanks used for chronic exposure. Al] tanks were covered to
Prevent loss of fish.

50/tank) were exposed to

An activated carbon filter
remove herbicides from the
laboratory.
is considered
of waste pesti

(Marking and Piper 1976) was set up to
effluent waters prior to discharge from the .
The carbon filter was changed every 8 days and incinerated. This

one of the better methods for destruction of small quantities
cide chemicals (Kennedy et al. 1969)

WATER QUALITY

Water for the stud
istics of the water have been monitored
McPherson 1977). Alkalinity, hardness,
the static exposure tanks were
2). In the flow-through system

y came from several wells. The chemical character-

during the past years (Lorz and

dissolved oxygen, pH and ammonia in

measured routinely at the laboratory (Table
the dissolved oxygen was always >8.5 mg/L,


This report presents data on the effects of selected herbicides on
survival, seawater adaptability, (Na,K)-stimulated ATPase levels, and
downstream migration of yearling coho salmon following acute and chronic
herbicide exposure. The report includes a detailed description of
methods, a results and discussion section arranged on a compound by
compound basis, and a series of appendices. The results and discussion
section includes reviews of pertinent literature relevant to toxicity
characteristics, and the environmental behavior of each compound at
recommended application rates.=/

/application rates from Oregon Weed Control Handbook 1977 unless otherwise
noted,


CONTENTS

DISCLAIMER. . 0... cece cece eee e cece eee e enter eee cence seeeeeeses
FOREWORD... eee eee
ABSTRACT.
FIGURES..

1 CONCLUSIONS. ...... see eeer eens tere were ewe e eee eeeee
11 RECOMMENDATIONS. .
Hil INTRODUCTION....

IV METHODS. 0... eee c cece cee ete e cece reece renee
SELECTION OF TOXICANTS AND EXPERIMENTS. .
EXPERIMENTAL FISH. ...cceeseeeecseee o aes
EXPOSURE TO TOXICANTS...

ToxicantS........eeeee

Static exposure tests.

Flow-through tests....
WATER QUALITY..... .

CHEMICAL ANALYSIS...
GILL ATPASE ACTIVITY. .
TOLERANCE TO SEAWATER............-+0..-
ASSESSMENT OF COEFFICIENT OF CONDITION.
HISTOLOGICAL EXAMINATION........ se eeeee
DOWNSTREAM MIGRATION...
V RESULTS AND DISCUSSION...

ACROLEIN...----. scene
Review of Literature.

Experimental Results.
AMITROLE-T.. ese eee eereee tee
Review of Literature....
Experimental Results.
ATRAZINE... ee cece cece eeeee
Review of Literatur
Experimental Result
DICAMBA. . .
Review o erature...

Experimental Results.

10


TABLE A-8. PERCENT MIGRATION (TO JULY 6,1977) OF YEARLING COHO SALMON
RELEASED INTO A SMALL COASTAL STREAM FOLLOWING ACUTE AND
CHRONIC EXPOSURE TO DIQUAT (RELEASED MAY 26, 1977).

Percent migration
Nominal concentration Days post release

mg/L 1-5 6-10 11-20 21-30 31+

1. Acute exposure - 96h

Control 66.0 71.8 72.8 72.8 72.8
0.5 47.6 58.1 58.1 58.1 58.12/
1.0 54.5 60.4 62.4 62.4 62.4
2.0 38.8 46.9 47.9 47.9 47.92/
3.0 41.0 53.0 53.0 53.0 53.02/

Control 55.2 62.3 63.4 63.4 63.4
0.5 43.2 53.9 - 55.5 55.5 55.5
1.0 38.7 47.5 48.1 48.1 48.124
2.0 25.6 41.7 42.2 42.2 42.22/
3.0 16.9 26.4 26.4 26.4 26.42/

2/migration rates significantly lower than controls (P = 0.05).

102


Flow-through exposure--

Additional tests were conducted with diquat because it is used as a
prophylactic treatment in fish culture and for control of aquatic vege~
tation, and affects seawater survival of coho smolts. Following tank
acclimatization, 150 yearling coho salmon per tank were exposed to diquat £
starting May 13, 1977. After 144 h and 312 h of diquat exposure, groups °
yearling coho were transferred to seawater. Only one fish died (from the
highest toxicant concentration 3 mg/L for 312 h) during the seawater
challenge (Table 23). The toxicant concentrations of our composite water
samples were close to the desired concentrations. No apparent effect of
diquat on the (Na,K)-stimulated ATPase activity was noted (Table 23).

TABLE 23. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO DIQUAT IN FRESHWATER AND THE SUBSEQUENT SURVIVAL
FOLLOWING TRANSFER TO SEAWATER.

Concentration

Percent
(mg/L) Percent surviva
nominal measured survival?/ Gill ATPase®/ — (268-h SW)

A. 144-h exposure

Control 0 100 100 (21)&/
0.5 0.58 100 100 (19)
1.0 1.07 100 100 (20)
2.0 2.04 100 100 (20)
3.0 3.06 100 100 (20)

B. 312-h exposure (240-h SW)
Control 0 100 4.93 (20) 100 (20)
0.5 0.44-0.58 100 100 (29)
1.0 0.92-1.04 100 5.83 (20) 100 (20)
2.0 1.91-2.04 100 100 (31)
3.0 2.86-3.06 100 4.28 (20) 95 (19)

3/300 fish exposed per concentration. \

— (Na,K)-stimulated ATPase activity of gill; mean with sample size in

arentheses.

2/Number of fish tested in parentheses.

Exposure to diquat for 285 h showed
factor of yearling coho salmon (Table A-7). Generally, the fish appeared
to feed well in all tanks, although those fish exposed to 3 mg/L exhibited
extensive exophthalmia and Opaqueness of the eyes.

No apparent effect on the condition

A release of 10 groups of yearling coho salmon exposed to diquat for
285 h and 5 groups exposed for 96 h was made on May 26, 1977. An inhibitory
effect on migration occurred in all concentrations of diquat tested, in
both the acute and chronic exposures (

Fig. 12). The coho salmon exposed

60


SECTION V

RESULTS AND DISCUSSION

From November 1976 to June 1977 we completed static toxicity tests
with 13 herbicides. These tests included determinations of acute lethality,
(Na,K)-stimulated ATPase activity of the gills, and tolerance to seawater
following exposure to herbicides in freshwater. In addition, tolerance to
seawater, (Na,K)-stimulated ATPase activity, and migratory disposition
were investigated using yearling coho salmon previously exposed to Tordon
101, dinoseb and diquat ina flowing freshwater toxicant system. The

results of tests with each herbicide are discussed below.

ACROLEIN

Review of Literature: Toxicity to Fish and Behavior in the Environment

e common name for acrylaldehyde or 2-propenal, and is
name of Aqualin®. {t is registered for use

canals, and irrigation ditches) for the

ds. Acrolein has recently
uatic herbicide and is also used as a molluscicide
(Tweedy and Houseworth 1976).

Acrolein is th
manufactured under the trade
in flowing water only (rivers,
control of submerged and
found use as a contact aq
for control of river-dwelling snails

Acrolein ts reported to be hydrolyzable and volatile. It polymerizes
slowly in the presence of air, producing an insoluble white precipitate.
Macek et al. (1976a) referring to some unpublished work by Battelle (1970)
noted acrolein's extreme volatility. When acrolein was applied at 0.7
mg/L tn an irrigation ditch flowing at approximately 130 cfs, its concen-
temperature of 17.8°C

tration decreased by 98% over a 19-mile section at a
62% was lost in a 27-mile stretch.

(64°F), while at 8.9°C (48°F) only
Moen (1961) found that acrolein quickly disappeared from a pond when it
was applied at 0.5 mg/L.

Acrolein ts very toxic to fish (Tal
2h-h LCSO's of 80 ug/L and 65 ug/L acro
tshawytscha) and rainbow trout (Salmo gairdneri), respectively, under
static conditions. These findings were similar to 2h-h LC5O values of 75 ug/L
) and 46 yg/L acrolein for

for fingerling brown trout (Salmo trutta fario,
H $ d by Burdick et al. (1964) using

bluegills (Lepomis macrochirus) as reporte
a continuous flow-through system. Folmar (1976) reported the 96-h LC5O
for rainbow trout to be 140 ug/L acrolein.

ble 3). Bond et al. (1960) reported
lein for chinook salmon (Oncorhynchus

15


SECTION 1

CONCLUSIONS

Acrolein and dinoseb were the most toxic of the herbicides tested
having 96-h LC50 values of 68 and 100 ug/L, respectively.

Atrazine, diquat and picloram were moderately toxic in freshwater
with 96-h LC50 values ranging from 10-30 mg/L.

Fish exposed to Amitrole-T, diquat, and paraquat in freshwater exhibited
dose-dependent effects in subsequent seawater entry tests.

Freshwater exposure to sublethal concentrations of diquat (5 mg/L for
144 h) resulted in some deaths when fish were challenged with seawater.
Diquat exposures of 0.5-3.0 mg/L for 96 h resulted in reduced downstream
migration following release of fish into a natural stream.

No apparent effects on the (Na,K)-stimulated ATPase activity of the
gills were observed with any of the herbicides tested.

The herbicide formulations tested appeared to have no effect on
smolting of yearling coho salmon except for the direct toxicity of
acrolein and dinoseb, and the effect of diquat on seawater survival

and downstream migration. Therefore, application of these formulations
at their recommended levels of use (Oregon Weed Control Handbook,

_1977) should not affect smolting.

Misuse of chemicals has led to direct loss of fish, and this suggests
that stricter enforcement of regulations and the possible need for
training and refinement in licensing of applicators is required.

The data indicate that some bioconcentration from the water occurred
in the fish exposed to dinoseb and Esteron; however, it is low
compared to pesticides (e.g. DDT).

Some of the chemicals tested produced pronounced histopathological
effects in exposed fish; the tissue effects were similar to those
produced by toxic agents.


Riepma (1962) found that soil microorganisms decomposed amitrole
rapidly and that the decomposition rate increased with increasing amounts
of soil organic matter. Grzenda et al. (1966) observed a tight adsorption
of amitrole to the hydrosol and a fairly rapid decomposition of amitrole in
a pond. Norris (1970) found that the common brush control herbicides,
2,4-D, amitrole, 2,4,5-T, and picloram were all degraded in the forest
floor although the rates of degradation varied considerably. In red alder
(Alnus rubra) forest floor material, 80% of amitrole was degraded in 35
days.

Amitrole's persistence in water, following aerial application to
forest areas for brush control in Oregon, has been studied in several
investigations (Marston et al. 1968, Norris 1967, Norris et al. 1966, and
Tarrant and Norris 1967). In a study of a stream in a coastal, municipal
watershed sprayed with 2 }tb/acre amitrole for control of salmonberry, the
maximum concentration of herbicide, 155 ppb (in water),was observed 30 min
after application (Marston et al. 1968). Only 26 ppb was detected after 2
h and none after 6 days. Norris (1967) monitored amitrole concentration in
a stream at a spray site treated at 2 Ibs/acre. Five min after spray,

422 wg/L amitrole was detected; 10 hours later the concentration had
decreased to 4 ug/L and none was found after 3 days.

The toxicity of Amitrole-T is thought to be similar to that of ammonium
thiocyanate (House et al. 1967). Russian studies have indicated that
ammonium thiocyanate is lethal to fish at 200 mg/L (Demyanenko 1941).
Amitrole-T is believed to be relatively non-toxic to fish (Meyer 1966).
Two-inch bluegills tolerated 10 mg/L Amitrole-T for 100 h (U. S. Fish and
Wildlife Service 1963). Sanders (1970) noted bluegills survived concen-
trations of >100 mg/L Amitrole-T for 48 h.

The literature contains a number of publications on the toxicity of
amitrole formulated without ammonium thiocyanate. Bond et al. (1960) found
the 48-h LC50 for coho salmon to be 325 mg/L amitrole under constant flow
conditions and water pH 7.5 to 7.7 and 41 to 71 mg/L total alkalinity.
Largemouth bass were able to survive 1,000 mg/L amitrole under static con-
ditions, but in flow-through apparatus all test fish died at this concen-
tration in 6 days. A 24-h LC50 of 1,200 mg/L amitrole was established for

bluegill sunfish by Hughes and Davis (1962a) in water of 29 mg/L hardness
and pH 6.9.

Hiltibran (1967) investigated the effects of selected herbicides on
fish reproduction. Survival of fertilized eggs and fry of bluegill, green
sunfish (Lepomis cyanellus), smallmouth bass (Micropterus dolomieu) and

lake chub sucker (Erimyzon sucetta) were not affected by 50 mg/L amitrole
under static conditions.

Experimental Results

The 96-h LC50 of Amitrole-T was approximately 70 mg/L for yearling
coho salmon (Table 5). There was no apparent effect of the herbicide on
the (Na,Kk)-stimulated ATPase activity of the gills, however, upon transfer
to seawater there appeared to be a dose-dependent mortality effect (Fig. 4,
Table 5). Our 96-h LC50 value is one-third of that reported in the Russian

20


APPENDIX It.

EFFECT OF VARIOUS HERBICIDES ON HISTOLOGY OF YEARLING COHO
SALMON .

BY DR. J. D. HENDRICKS, OREGON STATE UNIVERSITY.
ACROLEIN

Control, 144 h (3 fish) - All tissues were normal.

50 ug/L_acrolein, 144 h (3 fish) - The livers had occasional exfoliated,
necrotic cells, but were otherwise normal. One of the kidneys was normal,
one had extensive vacuolation of the collecting duct cells, while the other
had protein precipitate and cellular debris Present in Bowman's capsule and

various tubular regions. Both hypertrophy and hyperplasia were seen in the
gill epithelium of fish examined.

100 yg/t acrolein, 44 h (3 fish) - Most of the liver cells were delineated
and separate from adjacent ones, some were necrotic. All the kidneys had
considerable debris and precipitate in tubule lumens, and one kidney had
extensive necrosis of both segments of the Proximal tubule. All kidneys
were engorged with blood. The gill epithelium from all fish was totally
destroyed, necrotic and sloughed. Two hearts were normal. A section of
esophagus exhibited massive necrosis of all 4 tunics.

AMITROLE-T

Control, 144 h (3 fish) - There was extensive Peripheral, coagulative
necrosis due to bile spillage and several foci of peribiliary necrosis
within the substance of the liver. The remaining liver cells were normal,
so | assume both of these lesions were the result of poor post-mortem
handling of the livers. Kidney and gill tissues were normal.
100_mg/L_Amitrole-T, 144 h (1 fish) - All liver cells showed either hydropic
degeneration or coagulative necrosis. The necrosis was diffuse; approxi-
mately 25% of the Parenchymal cells were affected. There was extensive
coagulative necrosis of all regions of the nephrons as well as most of the
hematopoietic tissue of the kidney. The lamellar epithelium of the gills
was hydropic and separated from the underlying pillar cells on many of the
lamellae. On the basis of the effec

ts on these 3 tissues, this treatment
was severe and the changes would be incompatible with survival.

200_mg/L_Amitrole-T,60 h (3 fish) - Changes in the liver were of the same
type as in the 100 ppm exposure but not as extensive. One of the fish had
considerable diffuse coagulative necrosis of liver cells, but the others
showed predominantly hydropic degeneration. Kidneys exhibited similar
necrotic changes in the tubules and hematopoietic tissue, but not quite as
extensive. Epithelial cells of the gills were hypertrophied, disrupting
normal lamellar architecture. Although not as severe as the previous
treatment, these tissue changes would probably not permit longtime survival.

90


4. Procedure:

a. Extraction: Measure 400 mL of water sample into 500 mL separatory
funnel fitted with teflon stopcock. Shake sample with three successive
75 mL portions of methylene chloride combining the three portions in
a 250 mL beaker. Evaporate the methylene chloride to near dryness
using a stream of nitrogen. Add 20 mL methanol and evaporate to
near dryness again to ensure all methylene chloride is removed.

Transfer to volumetric flask with methanol.
b. Gas Chromatography: Gas chromatograph parameters:
Coulometer settings: 250 2, 250 mV bias, low gain mode.

Column: 4h mm 1.D. x 1.2 m glass packed with 5%
OV-1 on 80/100 Gas Chrom Q.

Temperatures

Column 175°C
Inlet 205°C
Transfer line 225°C
Combustion furnace: 800°C
Gas flows: Carrier gas 60 ml nitrogen/min,

oxygen 50 mL/min.
Sweep gas 20 mL nitrogen/min.

Recorder: 0.1 mV/in (0.04 mV/em), 1/2 in/min
(1.27 cm/min) chart speed. Equipped
with a disc integrator.

Retention time of
atrazine: 2.8 min.

The amount of atrazine in the samples is determined by injecting an
aliquot of the methanol solution into the gas chromatograph. Compare
peak chart areas with areas for known amounts of atrazine.

DICAMBA, PICLORAM, 2,4-D, and 2,4,5-T
Residues in water

1. Summary of method: Samples are collected and stabilized with sodium
hydroxide. The samples are adjusted to a pH <2, extracted with
ether and alkylated to form the methyl ester. Detection and quantification
is accomplished using microcoulometric gas chromatography (MCGC) in

the halogen mode.

83


SECTION II

RECOMMENDATIONS

The techniques and methodology developed in this and prior studies
(Lorz and McPherson 1977, Lorz et al.

1978) are recommended for the
determination of pollutant effects On anadromous fishes. The Seawater
Entry Test (Lorz et al. 1978) is a quick method for determining if
pollutants in freshwater could be potentially harmful to the seawater
life history phase.

Additional research should be conducted to determine if ofl-soluble
herbicide formulations are detrimental to smolting. Present
methodology needs to be reviewed, however, and Probably development of
new exposure methods and improved analyses for chemicals in the aquatic
environment is required.

Other life stages (egg, alevin, and fr
for toxicity and sublethal effects (
especially with herbicide
periods.

y-fingerling) should be tested
Possibly behavioral studies),

s that persist in the environment for extended


able to determine concentrations of diquat by direct readings of the water
at 310 nm and for paraquat at 256 nm. Concentration was determined from a
Standard curve.

89


APPENDIX 111. METRIC AND ENGLISH EQUIVALENTS

English-to-Metric Metric-to-English

‘V inch = 2.5400 cm 1 centimeter = 0.3937 in

1 inch3 = 16.3871 cm3 1 centimeter? = 0.0610 in?

1 foot = 0.3048 m 1 meter = 3.2808 ft

1 foot? = 0.0929 m2 1 meter? = 10.7639 ft?

1 chain = 20.1207 meter 1 meter = 0.0497 chain

1 acre = 0.4047 ha 1 hectare = 2.4710 ac

1 foot?/acre = 0.2296 m2/ha 1 meter?/hectare = 4.3560 ft2/ac

T gallon(U.S.liquid) = 3.7853 L 1 liter = 0.2642 gallon (U.S. liquid)
1 pound = 453.5924 gms 1 gram = 0.0022 pounds

Degrees Fahrenheit

9/5 (°C) + 32 Degrees Celsius = 5/9 (OF - 32)

1 meter = 100 centimeters
1 meter = 1,000 millimeters
1 hectare = 10,000 m2

94


9g

ACUTE TOXICITY OF DIQUAT TO VARIOUS FisH species!’

TABLE 21.
(e307 ‘AlkaTintey Hardness

Species Toxicity test {ag/LJ pH Temperature (mg/t_as CaC03) Reference
Carassius auratus static, field 35 (96 h) 82 48-890F 233 280 ilderhus (1967)

goldF isi conerete pond
Esox luctus static, field 16 (96 h) 82 48-89°F 233 280 Gilderhus (1967)
“Trorthera pike) concrete pond
Fundulus similis Flowing, lab. 1.0 wre (48 n)3/-4/ Butler (1965)

Tsh

Jctalurus punctatus static, lab. 10.0 wre (11TH) 8.4 23.3-25.8°C 5 8 Jones (1962)
““fehanne! catfish-fry)
1. punctatus static, lab 10.0 NTE (96 n)2/ 75°F Lawrence et al, (1965)
Lepomis eyane! lus static, plastic 4.0 wtee/ 8.3-8.9 120-189 Yeo (1967)

(green sunfish poot
+ macrochirus static, lab. 10.0 NTE (12 d) 22-25% Hiltibran (1967)

~(pTuegiTT-Fry) 4.0 NTE (96 h)

25.0 (30 h) 8.4 23,3-25.8°C 15 78 Jones (1962)
macrochirus static, lab. 19 (48 5) 24° Cope (1966)

L. macrochirus static lab. 525 (24 h) 6.9 25°C 40 29 Hughes and Davis (1962)
(Finger lings) 150 (48 h)
macrochirus static, fil 35.0 (96 h) 8.2 48-89°F 233 280 Gilderhus (1967)

concrete pond

‘L. macrochi rus static, lab. 9-10 UD. (97 n)2 75°F Lawrence et al. (1965)

Le macrochirus static, lab. 72 (96 h) Teh 25oc 22 22

140 (96 h) Br 25°C 32 34 Surber and Pickering
(1962)
i i ; y
Micropterus dolomieul static, plastic 0.5 NTE’ 8.3-8.9 120-209 Yeo (1967)
{enol Touth bass) poo! 4.0 LC), (96 h)
static lab. 10.0 LD). (96 h) 75°F Lawrence et al. (1965)
M. dolomieui static, lab. 2.5 (2h h) 22-25°C Hiltibran (1967)
ry
salmoldes static, lab. 1.0 NTE (96 h) 22-23°C 6 B Jones (1962)
‘(largemouth bass-fry)
static, lab. 7.8 (96 h) 8.3 25°C 20 2 Surber and Pickering
(1962)
static, lab. 11.0 (48h) Mui rhead-Thompson
(sz)

Horone saxatilis static, lab 1.0 (26-96 h) 70°F Hughes (1973)
(striped bass-larvae) ° ,
(Fingerlings) static, lab 35.0 (24 h)

(fry) 10.0 (96 h) 70°F Huches (1973)
(fingertings) static, lab. 315.0 (24 h)
80.0 (36 h) 8.2 2ec ey 35 Wellbora (1969)


for 285 h showed a dose-dependent inhibition of their migration. Less
than 30% of the fish exposed to 3 mg/L diquat migrated the 6.4 km down-
stream compared to 63% of the controls. The 1, 2, and 3 mg/L groups all
showed significantly less migration than the controls (P = 0.05). In
the 96-h exposure groups the percent migration varied from 48% (2 mg/L)
to 72% (controls), but the group responses were not dose-dependent. The
0.5, 2 and 3 mg/L (96-h exposure) groups all exhibited significantly

less migration than the controls. The controls showed the best migratory
response following both acute and chronic diquat exposure (Table A-8)-

Histological examination of di
necrosis of the liver, kidne:
coho salmon (Appendix II).
exhibited the most degenerat
necrosis (Appendix II).

quat-exposed fish showed degenerative
ys, and gill lamellae of chronically exposed
In acute exposures, however gill tissues
jon although the liver did show limited

No bioconcentration of diquat was found in muscle, kidney, or liver
tissue, although difficulty was encountered in the extraction procedure.
The literature generally indicates that diquat may enter a fish's body
but is not accumulated since the herbicide is metabolized or excreted
soon after it disappears from the water (Beasley et al. 1965, Calderbank
1972, Newman 1970). However, Gilderhus (1967) found diquat residues in
bluegills 6 weeks after treatment with 1.0 ppm. Calderbank 1972,

Hiltibran et al. (1972) and Berry et al. (1975) showed that most, if not
all, diquat residues in fish were located in skin, gills, and viscera
and not in edible flesh.

Considering that diquat is u:
in fish culture for treatment of
gation appears warranted.

sed for both aquatic weed control and
bacterial gill disease, further investi~
Information concerning the effect of reduced

f recovery following exposure would permit a
better evaluation of diquat's possible adverse effects where migrating
salmonids are concerned.

62


eggs obtained from the Oregon Department of Fish and Wildlife (ODFW) Fal}
Creek Salmon Hatchery, Alsea River, Oregon, under conditions similar to those
reported by Lorz and McPherson (1977). Steelhead fry (Salmo gairdneri) from
Big Creek Salmon Hatchery (ODFW) were’ used in the Esteron tests in addition

to yearling coho salmon.

EXPOSURE TO TOXICANTS

Toxicants

Twelve water-soluble and one water-emulsifiable herbicides were tested
under static exposure conditions. The herbicide formulation tested, its
manufacturer, chemical name, and summary of registered uses are listed in
Table 1.

Static exposure tests

The static toxicity tests were carried out in 0.6]-m diameter fiberglass
tanks. Water was continuously aerated and 85% of the 120 L was exchanged
Once per day. The fish were generally placed in the test tanks 3 days prior
to toxicant exposure for acclimatization and recovery from handling. Toxicant
solutions were mixed in a separate container prior to introduction into the
tanks. The daily exchange of toxicants always started with the control tanks
and went to successively higher toxicant concentrations; the mixing container
was rinsed following each concentration change. Upon completion of the daily
toxicant changes, the mixing bucket was rinsed several times and then flushed
overnight with running freshwater. Where possible, toxicant concentrations
tested were selected from published LC50 data for the particular herbicide.

A minimum of seven replicated concentrations, with 10 fish per test tank,
were used for each static toxicity test.

Flow-through tests

Four herbicides (Tordon 101, dinoseb, diquat and Esteron Brush Killer)
were used in a flowing water system. This system consisted of a gravity flow
diluter (Figs. 1 and 2) capable of delivering 12 L/min to each of 10 exposure
tanks (five duplicated concentrations). A volume of 1000 L was maintained in
each of the ten 1.54-m diameter fiberglass exposure tanks, and 95% of the
water was replaced every 3.7 h. Submersible pumps were used in each tank to
Provide additional current, aeration and mixing. Yearling coho salmon (210-
225/tank, except the Esteron study which utilized 50 fish/tank) were fin-
Clipped and allowed to acclimate at least one week before the toxicant
éxposure began. The concentrations were sublethal and based on prior static
bioassays. Water and toxicant flows in the diluter were checked at least
Once daily; only occasional minor adjustments were required.


ATRAZINE (AATREX)

15 mg/L AAtrex, 140 h (3 fish)- The livers and kidneys of these fish were

normal. There was hypertrophy of the gill epithelium in two of the fish,
while the third had several large aneurisms at the base of the filaments.

DICAMBA (BANVEL D)

100 _mg/L_dicamba, 144 h (5 fish) - All livers exhibited foci of peripheral
and/or peribiliary bile necrosis but were otherwise normal. Kidneys and
gills were also normal in these fish. The experimental treatment had

little if any effect on the tissues examined.

PARAQUAT

100 mg/L paraquat, 120 h (4 fish) - All the livers exhibited a low grade
hydropic degeneration, particularly in centrolobular regions and occasionally
in foci of peribiliary necrosis. One kidney was normal, the other 3 had
necrotic cells in the first and second proximal tubules. Two of the fish

had gills from which nearly all the epithelium had been sloughed; where it
remained, the epithelium was degenerate or necrotic. The other two fish had

less severe gill lesions.

ESTERON [BRUSH KILLER (2,4-D + 2,4,5-T PGBE ESTER)]

Control, 96h (5 fry)i/- Livers and kidneys were normal on all these fish,
but the gills were similar to the experimentals with regard to chloride cell

hyptertrophy. This response may be the result of something other than the
Esteron treatment. Its significance is not clear.

1200 g/L _Esteron, 28 h (5 fry)2/- Some peribiliary necrosis was present in
all livers and some diffuse cellular degeneration was observed in two of
them. Kidneys were normal in all fish, but gills had lesions similar to the
800 ug/L treatment, i.e. hypertrophied chloride cells and lamellae engorged
with blood.

1200 ug/L Esteron, 48 h (4 fry)2/- Livers were normal except some slight
legenerative changes in one. Kidneys were again normal and gills had
Similar lesions to the 2 previous treatments, i.e. hypertrophied chloride

cells and blood engorged lamellae.

1200 ug/L Esteron, 96 h + 24h rest, (5 fry)¥/- All tissues were normal
except for foci of peribiliary necrosis in 3 livers.

Control (yearling coho salmon), 96 h (3 fish) - All tissues were normal
except for some peripheral bile necrosis on the livers.

1
VV steelhead fry - Big Creek Salmon Hatchery.

91


ral

CHEMICAL CHARACTERISTICS OF TEST WATER DURING STATIC TOXICANT

TABLE 2.
EXPOSURE, AVERAGE VALUE WITH RANGE IN PARENTHESES.
Dissolved Ammonia Number
oxygen (mg/L Alkalinity Hardness of

Toxicant (mg/L) NH3-N) pha/ (mg/L as CaC03) analyses

Amitrole-T 10.5 b/ 7-5-7.6 79 100 4
(9.2-11.0) (75-82) (100-101)

Krennite 10.8 b/ 7.5-7.6 79 100 4
(10.7-11.0) (76-80) (99.5-100.5)

Dicamba 10.7 0.32 7-7-7.8 83 102 8
(10.2-10.9) (0.22-0.42) (82-84) (101-102)

Dinoseb 11.0 0.52 7.7-7.8 81 101 4
(10.7-11.2) (0.25-0.75) (77-83)

Tordon *101 10.0 0.67 7.4-7.6 81 102 10
(9.0-10.8) (0.65-0. 72) (80-83)

Tordon 22K (Picloram) 10.5 0.32 b/ b/ b/ 12
(8.6-11.2) (0.25-0.41)

Atrazine 11.1 0.29 7.4-7.6 84 101 8
(11.0-11.4) (0. 22-0.42) (100-101)

2,4-D 10.7 0.45 7.2-7.6 b/ 105 4
(10.4-10.8) (0. 40-0. 48)

2,4,5-T 11.0 7-5 77 99 4
(11.0-11.1) (0.5-<0.7) (76-81)

Esteron® Brush Killer 10.6 b/ 74 80 100 1
(10.2-10.8)

EY {PH value taken after 2 min of gentle stirring.

No data collected.


its dissipation under field conditions (NRCC 1974). This mobility is also
of environmental concern, as leached picloram may be transported to aquatic
ecosystems such as ponds, lakes, and streams. Residue levels in surface

runoff have reached 2 mg/L following application at 1.1 kg/ha (NRCC 1974).
However, studies have indicated that under most conditions only small pro-
portions of picloram (less than 5%) applied to a watershed are transported

in surface runoff.

Only negligible residues of picloram occur in streams, apparently due
to rapid dilution of the herbicide (Haas et al. 1971). Field plots
adjacent to the mouth of a small stream were treated with 1.1 kg/ha of
Picloram, and water samples were collected 0, 0.8, and 1.6 km downstream
from the plots following each rain for 5 months after application. Picloram
was detected in the stream samples only during the first significant runoff
(0.029 mg/L). No residues were found in subsequent samples (Haas et al.

1971).

Picloram contamination in lakes has not been reported, but levels in
farm ponds adjacent to plots treated with 1.1 kg/ha picloram reached | mg/L
(NRCC 1974). Dissipation of the herbicide in ponds has been shown to be
rapid. One study found an initial decline of 14 to 18% of picloram per
day, followed by a decline of less than 1% per day 15 weeks after appli-
cation (Haas et al. 1971). Residues of picloram in the pond-bottom sediments
48 ug/kg) immediately following application were only twice that in the
water (Kenaga 1973, in NRCC 1974). After 75 days, residues of 7 ug/kg
Picloram were detected in the pond-bottom sediments and 0.1 ug/kg picloram

was found in the water.

udies that the toxicity of picloram
to fish is influenced by its formulation and the water quality (NRCC 1974,
Woodward 1976, Sergeant et al. 1970). Technical grade picloram (a.i. 90%)
was found to be more toxic under alkaline conditions (Woodward 1976).

Increasing the pH from 6.5 to 8.5 increased the toxicity to cutthroat and
lake trout by a factor of 2 in both species. Increasing temperature did,
Toseynereasing hardness did not, lead to an increase in toxicity (Woodward

It is apparent from a number of st

The acute toxicity of picloram varied considerably with the formu-
lation and fish species. The isoocytl ester of picloram appears to be the
Most toxic commercial formulation (NRCC 1974, Sergeant et al. 1970, Kenaga
1969). LCS50's reported for this formulation are approximately | mg/L for
sensitive species. Toxicity levels of Tordon 22K (potassium salt) are
considerably lower as shown for several fish species in Table 14.

chronic picloram toxicity to fish is
(Woodward 1976, NRCC 1974). Longterm
to affect fish development and growth
(Woodward 1976), and swimming response and liver histopathology (Sergeant
et al. 1970). Woodward (1976) observed that the no-effect concentration of
technical grade picloram for lake trout was apparently <35 ug/L, as this
level of herbicide reduced fry survival and growth. Most mortalities
Cccurred during yolk absorption, which took 4-5 days longer in picloram-
treated fish.

. Based on available information,
not cumulative in terms of lethality
exposures, however, have been shown

4


Procedure:

Sample collection: Water samples are collected in 500 mL, wide-
mouth, glass bottles which contain 3 mL of 50% sodium hydroxide.
The samples are mixed to ensure complete hydrolysis of any DNBP
which may be present. The samples are transported to the laboratory

and stored at 2°C.

Sample extraction: Transfer 200 ml of water sample to a 500 mL
separatory funnel. Adjust to pH < 2 with concentrated sulfuric
acid. The acidified sample is extracted with three successive
aliquots of ether (100, 80, 60 mL). The ether extracts are collected
in a 250 mL beaker, evaporated to less than 20 mL. The ether layer
is carefully decanted away from any water into a 30 mL beaker. The
residual extract is rinsed with three small aliquots of ether adding
the rinsings to the beaker. The extract is evaporated to 2 ml with
a stream of dry nitrogen. This concentrated extract is transferred
with small aliquots of ether into a 10 mL volumetric flask, decanting
away any water and evaporated to dryness on a flash evaporator.

Derivation: The methyl ester of DNBP is formed using an ethereal
diazomethane reagent (Hopps 1970).

Gas chromatography: Gas chromatograph parameters:

Column: 4 mm |.D. x 1.2 m glass, packed with equal
amounts of 6% OV 210 and 4% SE 30 on
80-100 Gas Chrom Q.

Temperatures

Column 180°C

Inlet 200°C

Gas flow: : Carrier gas 80 mL nitrogen/min.

Recorder: 0.1 mV/in (0.04mV/em), 1/2 in/min (1.27cm/min)

chart speed.

Retention time of
ONBP methyl ester: 3.5 min.

The amount of DNBP in the samples is determined by injecting an
aliquot of sample solution in the gas chromatograph and comparing
peak heights with peak heights for known amounts of injected DNBP.

Residues in Fish

lL

Summary of Method: The method is based on Lane (1967) and Zweig and
Sherma (1972b). Whole body fish samples are digested with potassium
hydroxide and centrifuged. The supernate is adjusted to pH <2,
extracted with benezene and alkylated to form the methyl ester. The
ester is cleaned up with an alumina column. Detection and quantification
ts accomplished using electron-capture gas chromatography.

87


Herbicidal destruction of aquatic vegetation may expose small forage
fish to predation by large predacious fishes. Furthermore, fluctations in
oxygen tensions have frequently been shown to occur after application of
triazine herbicides. These fluctuations were associated with phytoplankton

blooms occurring in conjunction with decomposition of submerged vegetation
(Walker 1964).

Experimental Results

In our toxicity test, atrazine (AAtrex) appeared to produce a gradual
concentration dependent mortality in freshwater, with losses of 5 and 25%
at concentrations of 8 and 15 mg/L, respectively (Table 6). Dead fish
showed signs of severe edema. No apparent affect on (Na,K)-stimulated
ATPase activity occurred. McBride and Richards (1975) found that atrazine
significantly decreased sodium uptake of isolated perfused gills from carp
(Cyprinus carpio) but did not affect fluid flow rate at the concentrations
tested. The authors indicated the effect of atrazine on (Na,K)-ATPase
systems should be studied, because other pesticides (aldrin and DDT) have
been shown to inhibit (NatK)-ATPase (Cutkomp et al. 1971, Koch et al.
1971). When the survivors were transferred to seawater, the group that had
been exposed to 15 mg/L atrazine suffered a 25% mortality (Table 6). The
deaths occurred within the first 24 h and probably resulted from the poor
condition of the fish (due to toxicant exposure) and not osmoregulatory
failure. Histological examination of three fish exposed to 15 mg/L atrazine
showed no apparent affect on liver or kidney tissues but hypertrophy of
gill epithelium was evident in two of the fish (Appendix II).

TABLE 6. SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON
EXPOSED TO ATRAZINE IN FRESHWATER AND THE SUBSEQUENT
SURVIVAL FOLLOWING TRANSFER TO SEAWATER (MAR. 2-19, 1977).

Concentration Percent
(mg/L) Percent survival2/ Gill survival

nominal measured (144-h exposure FW) ATPase/ (264-h_sw)
Control 0 100 2.8 100
0.25  0.21-0.23 100 NTS/ 100
0.50 0.48-0.51 100 NT 100
1.0 1.31-1.38 100 3.4 100
3.0 3.85-4.25 100 NT 100
30 +7562 : 100 NT 100

0 10.65 95 2.4 hy
15.0 18.0-18.8 ort 2.30 7

a Twenty fish exposed per concentration.
B/ (wa /X) ~stimulated ATPase activity of gills; mean of 5 fish.
Y, lot measured.

Mean of 3 fish.

2h


TABLE |.

HERBICIDES TESTED FOR ACUTE TOXICITY TO YEARLING COHO SALMON2/.

Registered use(s)2/

Chemical_name

Manufacturer

Common or trade name

Acrolein
Anitrote-T(cytrat®)
Atrazine (AAtrex®)
Dicamba (Banvel®)

Dinoseb (Premerge 3)

Diquat (Ortho® Diquat Dibromide)

Esteron® Brush Killer
Krennite

Paraquat-CL
2,4-D (Amine 0)
2,4,5-T ("Weedar")
Tordon® 101

Tordon 22K (Picloram)

acrolein or acrylaldehyde

3-amino-1,2,4-triazole and ammonium
thiocyanate

2-chloro-4 ethylamino-6-isopropy|amino-
s-triazine

3,6-dichloro-o-anisic acid

2-sec-butyt-4,6-dinitrophenol

6,7-dihydrodipyrido (1,2-a:2',1'-c)
pyrazinediium ion

2,4-D propylene giycol butyl ether ester +
2,4,5-T propylene glycol butyl ether ester

Ammonium ethy! carbamoy|phosphonate

U1! -dimethy!-4,4'-bipyridinium ion

2,4-dichlorophenoxyacetic acid
(dimethylamine formulation)

2,4,5-trichlorophenoxyacetic acid
(triethylamine formulation)

4-amino-3,5,6~trichloropicol inic
acid + 2,4-dichlorophenoxyacetic acid
bath as the triisopropanolamine salts

4-amino-3,5,6-trichloropicolinic acid
(as potassium salt.

Shell O11 Co.-TexasS/
American Cyanamid Co.
Ciba-Geigy

Velsicol Chem. Co.

Dow Chemical 2’

Standard 011
(Ortho Div.)

Dow Chemical ®/

E.1. du Pont!’

de Nemours
Standard O14
(Ortho Div.)

Diamond Shamrock
Chem. Co.
Amchem. Prod. Inc.

Dow Chemical Co.

Dow Chemical Ca.

Control of submerged aquatic weeds
in flowing water.

Non-crop uses such as right-of-way,
industrial premises and ditchbanks.

Corn, sorghum, perennial ryegrass
and winter wheat.

Barley, corn, oats, wheat and
pasture and rangeland.

Both non-crop and food-crop uses.
Food crops include: alfalfa, cereal
grains, fruits, nuts and vegetabies.

Non-food use; seed crops of alfalfa,
clover and vetch plus canals, lakes

and ponds.

Pasture and rangeland, forest and
non-crop uses.

Brush control on non-cropland areas.

Preplant or directed spray on both
non-crop and food crops such as
alfalfa, fruits, nuts and vegetables.

Range and pasture grasses, vegetables,
fruit, grains, berries and certain —
aquatic sites.

On an extended basis for use on
pasture and rangeland, forests and

non-crop uses.

Pasture and rangeland

Pasture and rangeland

2/ commerical formulations of herbicides purchased from Wilbur Ellis Co. Portiand, Oregon unless otherwise noted.

D/oregon Weed Control Handbook, 1977.
S/one liter sample provided by Shell Oil Company, Houston, Texas.

sh
4/pow Chemical Company supplied 5 gal. Dow Premarge 3.

2/oregon State Dept. of Forestry (Astoria} provided 1 gal of Esteron Brush Killer.

£/pr. M. Newton, OSU School of Forestry, provided sample.


0-75 pg/L

0-100ug/L
I Ne le le le n= KX
~---Q 100 y9/U

PERCENT SURVIVAL

oe | ee es se Oe es ee |
72 120 24 72 120 168 216 240
Freshwater Seawater

EXPOSURE TIME. (hours)

Figure 9.

Percent survival of yearling coho salmon during exposure to
dinoseb (Dow Premerge) in freshwater and subsequent survival upon. transfer
to seawater.

TABLE 19.

SURVIVAL AND GILL ATPASE OF YEARLING COHO SALMON EXPOSED

TO DINOSEB IN FRESHWATER AND SUBSEQUENT SURVIVAL FOLLOWING
TRANSFER TO SEAWATER (FEB. 22-MAR. 10, 1977).

Concentration

Percent Percent
(ug/L) survival. Gill survival
nominal measured (144-h exposure FW) ATPase?/ (260-h SW)
Control 0 100 2.9 100
25 1.1- 12.0 100 2.7 100
50 3.0- 11.8 100 2.8 100
5 3.1- 13.1 100 3.7 100
100 16.6- 61.7 10 nTe/ 100
200 23.0- 65.4 0 - -
300 141.0-209.0 0 -
500 241.0-275.2

0

$ Twenty fish exposed per concentration.

2/ (Na,K)-stimulated ATPase activity; mean of 4 fish.
¢/Not measured.

52


Vi
vil
Vill
IX

PARAQUAT..... eee ee eee eeeeees
Review of Literature.

Experimental Results.
2,4-D..... dveceenne

Review of Literature..

Experimental Results..
Pl

Review of Literature.
aeview of Literature

Experimental Results.
ESTERON BRUSH KILLER......

Review of Literature.
Experimental Results.
TORDON 22 K (PICLORAM)....
Review of Literature.

Experimental Results.
TORDON 10T............ecee

Review of Literature.
ate or —iterature
Experimental Results..

Static exposure ..

Flow-through exposu
DINOSEB (PREMERGE)........ .

Review of Literature..
aN or =i terature.

Experimental -Results..

Static exposure....

Flow-through exposu

Review of Literature...

seem or aiterature.

Experimental Results....
Static exposure....

Flow-through exposu
GENERAL DISCUSSION.

REFERENCES

SPECIFIC HERBICI

re.

re

re

APPENDIX |. METHODOLOGY FOR ANALYSIS OF

DES............

APPENDIX I1. EFFECT OF VARIOUS HERBICIDES ON
HISTOLOGY OF YEARLING COHO SALMON
BY DR. J. DL HENDRICKS, OREGON

STATE UNIVERSITY
APPENDIX IIL. METRIC AND ENGL

vi

ISH EQUIVALENTS...

90
94


Temperatures
200°C for 2,4-D, 2,4,5-T, and picloram

Column
185°C for dicamba.
Inlet 205°C
Transfer line: 225°C
Combustion furnace: 825°C
Gas flow: Carrier gas 60 mL nitrogen/min,
Oxygen 50 mL/min.
Sweep gas 2] ml nitrogen/min.
Recorder: 0.1 mV/in (0.04mV/cm) 1/2 in/min

(1.27 em/min), chart speed. Equipped
with a dise integrator.

Retention time of
methyl esters: dicamba min (185°C)
2,4-D min (200°C)

2.0

2.0
2,4,5-T 3.0 min (200°C)
picloram 5.5 min (200°C)

The amount of herbicide in the samples is determined by injecting an
aliquot of sample solution in the gas chromatograph and comparing
peak chart areas with areas for known amounts of the herbicide

injected.

PICLORAM, 2,4-D, AND 2,4,5-T
Residues in Fish

Y Summary of method: Whole body fish samples are digested with potassium
hydroxide and centrifuged. The supernate is adjusted to pH <2,
extracted with ether and alkylated to form the methyl ester. Detection
and quantification is accomplished using microcoulometric gas chromato-

graphy (MCGC) in the halogen mode.

2. Reagents: Diethyl] ether (distilled over sodium)
Sulfuric acid (concentrated, reagent)

Potassium hydroxide (reagent)
Diazomethane
95% ethanol

Separatory funnels fitted with teflon stopcocks
Round=bottom boiling flask (250 mL)

Condenser (West, 24/40 3)

Volumetric flask, 10 mL

3. Apparatus:

85


Paraquat (cation) for a 96~h contact period (Lawrence et al. 1965). Davis
and Hughes (1963) reported a 48-h LC50 for bluegill sunfish as

100 mg/L paraquat (cation). Under static conditions (pH 7.6 to 8.0, hardness
210 to 290 mg/L), brown trout had a 48-h LC50 with paraquat of 82 mg/L
(Woodiwiss and Fretwell 1974). Alabaster (1969) reported a 48-h LC50 for
harlequin fish as 32 mg/L paraquat using a flow-through technique and water
of 20 mg/L hardness. Butler (1965) exposed the estuarine longnose killifish
(Fundulus similes) to 1.0 mg/L paraquat and found no effect.

Information on paraquat toxicity to fish under field conditions
is limited. Yeo (1967) reported that smallmouth bass and mosquito fish
were killed when placed in 180-gallon plastic pools with 1.0 and 3.0 mg/L
paraquat (pH 9.4). Blackburn and Weldon (1962), however, reported that
paraquat applied to a small Florida canal at 1.0 mg/L was not toxic to fish
(water temperatures 29 to 33°C, pH 7.6). Earnest (1971) noted that a
minimum of 34% of bluegills, placed in a Colorado farm pond, died within
48 h after treatment with 1.14 mg/L paraquat (surface temperature 18.9-
25.0°C, alkalinity 69-129 mg/L and pH 8.0-10.4). :

Newman and Way (1966) reported no direct effects of paraquat or diquat
on aquatic invertebrates in their experiments. They did, however, note
severe oxygen depletion at one location following decay of the treated
aquatic weeds. A low oxygen content is postulated to have caused the
deaths noted among Hirudinea, Isopoda, Odonata, Coleoptera, Trichoptera,
Gastropoda, Lamel]ibranchiata and captive trout, although free-living
coarse fish and trout appeared unaffected.

Experimental Results

In our study the 96-h LC50 of paraquat-Cl was 76 mg/L for yearling
coho salmon. Deaths occurred in a dose-dependent manner depending on
concentration and exposure time (Fig. 5). When surviving fish were
transferred to seawater, all coho salmon previously exposed to 50 mg/L died
during the first 40 h of exposure. Similarly, 64% of the fish previously
exposed to 10 mg/L died during the first 68 h in seawater (Table 9). Our
96-h LC50 value of 76 mg/L is slightly lower than the 48-h LCSO of 82 mg/L
given for brown trout by Woodiwiss and Fretwell (1974). The different
species and water quality, however, of the two test solutions could account
for this. The (Na,K)-stimulated ATPase activity of the gills was not
affected by exposure to paraquat-Cl. Histological examination of fish
exposed to 100 mg/L paraquat-C! for 120 h showed evidence of degenerative
damage to gills, and kidneys, and slight necrotic areas in the liver
(Appendix I1). Earnest (1971) similarly reported necrosis of liver tissue
following treatment of the Colorado farm pond with paraquat.

29


800 ug/L Esteron 96 h (3 fish) - Except for peripheral bile duct necrosis
livers were normal in all samples. Kidneys were normal with the exception

of large clear vacuoles in the collecting duct cells from two fish. The
gills had several abnormalities; 2 of them had curved gill filaments, the
basal, interlamellar epithelium had frequent hypertrophied chloride
(acidophillic) cells, some of which were exfoliated, and most of the lamellae
were engorged with blood, resulting in large aneurisms on some lamellae.

TORDON 22K (PICLORAM)

Control, 144 h (3 fish) - Livers had some foci of both peripheral and peri-
biliary bile necrosis. The hematopoietic tissue of the kidneys appeared

depleted and some of the gill filaments were crooked. Lamellar epithelium
appeared normal.

in nature and some similar to cyclo~
Propene fatty acid-induced damage. Cells were hypertrophied and contained
fiber-like strands in the cytoplasm. Kidneys were normal, while gills had
several abnormalities including hypertrophy of epithelial cells, and a
wrinkled appearance of many of the epithelial cells.

TORDON 101 (2,4-D + PICLORAM)

Control, 380 h (3 fish) - Livers wer.

bile necrosis. There were large clear vacuoles in the collecting duct
cells of one kidney, others were normal. The gill epithelium was wrinkled
as though it had been Stretched and then shrunk.

€ Normal except that one had peripheral

1.8 mg/L Tordon 101, 380 _h (4 fish) - Al
epithelium and resulting extensive
bile ducts were affected
gills were normal.

1 livers had degenerate bile duct
Peribiliary bile necrosis. Since the
» this may be due to the herbicide. Kidneys and

1,2 mg/L Tordon 101, 380 h (4 fish) - Al1 livers had degenerate bile duct
epithelium and peribiliary bile necrosis. All kidneys and gills were
normal except that there was considerable s

eparation of epithelial cells
from the pillar cells in the lamellae in one fish.

0.6 mg/L Tordon 101, 380 h (4 fish) - Peri
in all Tivers as above, kidneys and gills

biliary bile necrosis was present
were normal.

0.3 mg/L Tordon 101, 380 h (4 fish) - P
in all livers as above, kidneys and
Tordon 101 caused peribi
so it was assumed that i

i gills were again normal. All levels of
liary necrosis, this was not present in the controls
t was due to the treatment.

DINOSEB

ut otherwise they were normal
were normal.


Residues of the dimethylamine salt of 2,4-D (DMA-2,4-D) in hydrosol
and fish following pond application have been reported by Schultz and
Harman (1974a). Nine ponds in Florida, Georgia, and Missouri were treated
with either 2.24, 4.48 or 8.96 kg/ha of the herbicide. The highest de-
tectable level of 2,4-D in water (0.692 mg/L) was found 3 days after
application in a pond in Georgia that had been treated with 8.96 kg/ha
DMA-2,4-D. The herbicide had decreased to trace amounts (less than 0.005
mg/L) 28 days after application and was not detected thereafter. The
highest detectable level of DMA-2,4-D in mud occurred in the Georgia pond,
seven days after treatment. Largemouth bass, channel catfish, bluegill,
and redear sunfish, Lepomis microlophus, were held in live cages during
and after pond treatment; no Mortality was observed in any of the ponds.
Fifteen percent of the fish sampled contained detectable residues of DMA-
2,4-D (1.0 to 0.010 mg/kg). Radiometric measurements of the uptake,
metabolism, and dissipation of the DMA salts of ring-labeled C!4-2,4-pD in
water, hydrosol, and fish are in general agreement with these findings
(Schultz 1973). Catchable-size catfish, blue.
were exposed to 0.5 to 2.0 mg/L DMA-2,4-D for 84 days in plastic pools.
Muscle tissue of fish exposed to an initial treatment of 2 mg/L DMA-2,4-D
contained less than 0.06 mg/L 2,4-D, at the completion of the experiment
(84 days). Thirty-five days after treatment the highest detectable level

Norris and Moore (1971) reviewed the fate of chemicals in the aquatic

environment and Indicated that chemicals may be lost through volatilization,
adsorption on stream sediments, absorption by aquatic biota, degradation
by chemical, biological, or photochemical means, or by downstream movement.
They noted that the literature contains conflicting reports of the persis~
tence of pesticides in ponds and Streams. Aly and Faust (1964) reported
that 2,4-D esters were hydrolyzed to the free acid in 9 days in lake
water, but 2,4-D acid persisted up to 120 days. Schwartz (1967), however,
noted only 40% degradation of 2,4-D in water in 6 months under excellent
conditions for biological activity. Norris (1967) noted that herbicide
residues were found for only short periods of time in streams within or
adjacent to forest treatment areas. Concentrations approaching 1 mg/L
have been found in puddles which collected the first significant runoff
from an area treated one month earlier (Norris 1978). However, in 6 years
of monitoring spray operations for residues, phenoxy herbicides never
exceeded 0.1 mg/L in western Oregon streams (Norris and Moore 1971).

A review of the toxicity of DMA-2,4-p herbicides indicates that they

are relatively low in toxicity to fish, Folmar (1976) reported a 96-h
LC50 for rainbow trout at 100 mg/L. Davi

S and Hughes (1963) and Hughes
and Davis (1963) tested the toxicity of different formulations of 2,4-D to
bluegills. They found considerable vartation in the toxicity of different
formulations and even in the toxicity of a single formulation. The
researchers felt that these inconsistencies

could be attributed to the
different batch lots and/or manufacturing plants. The alkanolamine salt

32