tively transferred to 15 ml centrifuge tubes with methylene
chloride, and evaporated to dryness using a gentle stream of
clean, dry air at 40°C. The extract was immediately dissolved
in an accurately measured volume of hexane and a portion was
withdrawn and analyzed by gas/liquid chromatography under the
following conditions:

Instrument: Beckman Model 45 GC gas/liquid chromatograph
Detector: Helium-arc electron capture

Column: 183 x 2 mm 1.D. glass containing 5% Dexsil on 80/100
mesh Supelcoport

Carrier gas: High purity helium at 55cc/min.

Detector gas: High purity helium at 1l0cc/min. and carbon
dioxide at 3cc/min.

Inlet: 225°C, Column: 200°C, Outlet: 240°C, Detector: 260°C
Recorder: mv full-scale, chart speed 1.3 cm/min.

Atrazine was eluted in 4.2 minutes, and 10 ng gave full-scale
pen deflection at an electrometer attenuation of 8 x 10-10
amperes. Linearity of response was not assumed during the
study, rather a graph of peak height versus ng of atrazine
injected was constructed prior to analyzing each batch of
atrazine samples. The amount of atrazine in each sample was
then determined by graphical interpolation. The recovery of
atrazine from spiked water samples using the above methods
was essentially quantitative and no adjustments were neces-
sary for recovery (Table 3).

Fish tissue was extracted by the column method of Hesselberg
and Johnson (1972). The extraction solvent was diethyl ether.
The extract was analyzed by gas/ liquid chromatography using
conditions previously described for water analysis. No clean-
up of the fish extract was performed prior to analysis. The
chromatographic column (5% Dexsil 300 GC) was cleansed of
contamination by fish oils and other extractable interferences
by heating overnight at 325°C. The next morning the tempera-
ture was reduced to 250°C, and injection of atrazine was made
to condition the column, and finally the oven temperature was
lowered to 200°C and atrazine analyses were resumed with no
change in efficiency. The recovery of atrazine from spiked
tissue samples was again essentially quantitative (Table 3).

11


Ee

TABLE 12. SURVIVAL AND GROWTH OF FATHEAD MINNOWS (Pimephales promelas) EXPOSED 30
DAYS, 60 DAYS, AND 43 WEEKS TO VARIOUS ATRAZINE CONCENT ONS

Mean Measured Atrazine Concentration mg/liter

0.2L 0.112 0.054 0.033 0.015, Control
Item A ry B A B A B K ry 3B

30_ DAYS .
Survival (%) | 86 89 | 86 86 | 80 69 {100 97 | 63 83 | 60 47

Total Length

16 1 16 ww {4 13 Ww 13 14 13 20 19
($3) (3e9)" (21M 368) (345](302) (328)| (2-8) (35 ](lb.5) (3.84.2) 3.9)

60 DAYS
Survival (%)"| 80 86 | 71 69 | 69 66 90 70 | 57 67 | 60 47

tote yeneth 21 22 18 21 19 20 20 21 19 21 24 26
($.D.) (3.2) (3039(3.9) (408) (36) (4.0)] (3.5) (505) (605) (5e1)(4.2) (lhe)

43 WEEKS

Survival (%)% 93 67 |87 67] 80 80 | 87 80 | 80 87 |93 93

#¢ removed 0 1 te) 1 1 1 2 3 0 3 () 2

“fe at term, 8/6 3/6 |3/10 3/6 14/7 2/9 15/6 4/5 |4/8 4/6 15/9 2/9

ote el 6s 63 | 6670 | 636s «| GC | 62) 69 | BC
5452 | 52H | 4B Ok | 57) 53: | 500 55 | 5255

2.4 2.01 2.7 3.3] 2.5 2.5] 2.4 2.81 1.8 3.0) 2.4 2.0

Tote Velent
&
1.8 1.8} 1.0 1.5] 009 1.3 1.5 1.0) 1.3 1.3] 1.1 1.3

standard Deviation
Dsurvival based on 40 fish/duplicate tank,
°survival based on 15 fish/duplicate tank after thinning on day 60 of exposure,


Complete reproductive impairment of surviving adult gammarids
was observed among groups exposed to 0.94 mg/l atrazine, and
the number of progeny produced per female appeared reduced
among groups exposed to 0.49 mg/l atrazine. Additionally,
the survival during the first 30 days of development of
young produced by gammarids exposed to 0.49 and 0.24 mg/l
atrazine appeared to be less than that of young produced by
gammarids exposed to 0.14 and 0.06 mg/l atrazine (Table 9).

The growth of second generation gammarids during the 30 day
period following shedding by the females was significantly
reduced by continuous exposure to concentrations of atrazine
as low as 0.14 mg/l. None of the young organisms exposed to
0.49 mg/l atrazine developed beyond sixth instar stage

and half of them never developed beyond the fifth instar. Of
the young gammarids continuously exposed to 0.24 mg/l and 0.14
mg/l atrazine, only 75% developed to seventh instar, while
100% of the control organisms and 93% of those exposed to

0.06 mg/l developed to seventh instar.

The small number of young gammarids produced among control
groups was due to the fact that in these aquaria males
greatly outnumbered females. Consequently, we occasionally
observed moribund gravid females apparently due to continued
pursuit by various males and repeated copulation and separa-
tion. Also, nearly 60% of the gravid females isolated during
the experiment failed to yield young gammarids.

Based on a statistical evaluation of the above data, and
consideration of the effects of continuous exposure to
atrazine on the survival and reproductive potential of adult
gammarids, and on the morphological development of second
generation gammarids, we estimate the maximum acceptable
toxicant concentration of atrazine for Gammarus fasciatus is

>0.06 and <0.14 mg/l.
Lepomis macrochirus

Survival and growth of bluegill exposed to atrazine for 6

and 18 months was similar for all concentrations (Table 10).
Spawning activity occurred between days 594 and 716 of
exposure. Although spawning activity was too sporadic to be
conclusive, the fact that egg numbers in the highest concen-
tration (.095 mg/1) were comparable to the controls suggests
that atrazine had no effect on spawning within the range of
concentrations tested. An attempt was made to induce

further spawning from bluegill by administering interperitoneal
injections of carp pituitary extract on test day 716. All

28


same treatment but indicated that about half of the eggs
spawned successfully developed to this stage. Incubation in
degree-days was significantly reduced for brook trout eggs
spawned and incubated in 0.72 and 0.45 mg/l of atrazine.

Survival of brook trout fry after 30 days exposure was
similar for all treatments. After 60 and 90 days exposure,
survival of fry was reduced for brook trout exposed to
0.72, 0.45 and 0.24 mg/l of atrazine (Table 16).

As was observed during the first generation exposure, mean
length and weight of fry, including those from unexposed
parents, was significantly reduced after 90 days exposure
to 0.72, 0.45 and 0.24 mg/l of atrazine.

As in parental fish, the lethargy and reduction in feeding
activity appeared to be the underlying causes for the
measured response.

Based on the available evidence relating to pesticide-induced
reduction in growth of yearling brook trout, we estimate that
the maximum acceptable toxicant concentration of. atrazine for
brook trout is between 0.065 and 0.12 mg/1.

RESIDUE ANALYSIS

Three samples of muscle tissue from adult bluegill and brook
trout taken at the end of the exposure period were analyzed to
determine the concentration of atrazine residues in fishes
exposed to the highest concentration of atrazine in each chronic.
The small size of the individual fathead minnows prompted the
use of three samples of pooled eviscerated carcasses of
adults exposed to the highest concentration of atrazine. In
all cases the results indicated that the concentrations of
atrazine in tissues were below the minimum detectable limits.
Brook trout exposed to a mean measured concentration of 0.74
mg/l atrazine for 44 weeks contained <0.20 mg/kg atrazine in
the muscle tissue. Bluegill exposed to 0.094 mg/l for 78
weeks contained <0.20 mg/kg atrazine in the muscle. Fathead
minnows exposed to 0.21 ng/L atrazine for 43 weeks contained
<1.7 mg/kg atrazine in the eviscerated carcass. These data
clearly indicate that the fishes tested do not bioconcentrate
atrazine significantly.

37


INVERTEBRATES

1. Chironomus tentans (Chironomidae, midge)
2. Daphnia magna (Cladocera, water flea)
3. Gammarus fasciatus (Gammaridae, scud)

FISHES

1. Lepomis macrochirus (Centrarchidae, bluegill)
2. Pimephales promelas (Cyprinidae, fathead minnow)
3. Saivelinus fontinalis (Salmonidae, brook trout)

A reason for selecting both invertebrate and fishes is that
the susceptibility of fishes to a chemical should be compared
to the susceptibility of fish-food organisms to that same
chemical. An understanding of both these phenomena will be
invaluable in establishing realistic and meaningful water
quality criteria and standards.


Clemens, H. P., 1950. Life cycle and ecology of Gammarus
fasciatus, Say. Ohio State Univ., Franz Theo. Stone
Inst. Hydrobio-Contrib, 12, 63 p.

Cope, 0. B., 1966. Contamination of the freshwater
gsosystem by pesticides. J. Appl. Ecol. 3 (Suppl.):

Devel, C. R., D. C. Haskell, D. R. Brockway and 0. R. Kingsbury,
1952. New York State fish hatchery feeding chart, N.Y.
State Conserv. Dept. Fish. Res. Bull. No. 3. 26 p.

Drummond, R. A. and W. F. Dawson, 1970. An inexpensive
method for simulating diel patterns of qapnr ine in the
laboratory. Trans. Am. Fish. Soc., 99:434-435,

Duncan, D. B., 1955. Multiple range and multple F-tests.
Biometrics 11:1-42.

Eaton, J. G., 1970. Chronic malathion toxicity to the
bluegill (Lepomis macrochirus, Rafinesque). Water
Research 4:673-684.

Eisler, R., 1973. Annotated bibliography on biological
effects of metals in aquatic environments. EPA,
Ecol. Res. Ser. EPA-R3-73-007, Washington, D.C., 287 p.

FWPCA, 1968. Water Quality Criteria. Report of the National
Tech. Adm. Comm. to Secr. of the Interior Fed. Water -
Poll. Contr. Adm. U.S.D.I., 234 p. -

Hall, J. K., M. Pawlus and E. R. Higgins, 1972. Losses of
atrazine in run-off water and soil sediment. J.
Environ. Qual. 1:172-176.

Halter, M. T. and H. E. Johnson, 1974. Acute toxicities of
polychlorinated biphenyl (PCB) and DDT alone and in
combination to early life stages of coho salmon
(Qncorhyncus kisutch). J. Fish. Res. Bd. Canada

Hansen, D. J., P. R. Parrish, J. I. Lowe. A. J. Wilson, Jr.
and P. D. Wilson, 1971. Chronic toxicity, uptake and
retention of Aroclor 1254 in two estuarine fishes.
Bull. Environ. Contam. Toxicol. 6:113-119.

Henderson, N. E., 1963. Influence of light and temperature
on the reproductive cycle of the eastern brook trout,
Salvelinus fontinalis. J. Fish. Res. Bd. Canada

: - .

47


information suggested response to atrazine exposure in the
range of 5-10 mg/l (Walker, 1964; FWPCA, 1968; Sanders,
1969). Based on field observation Walker (1964) suggested
that atrazine concentrations of 0.5-1.0 mg/l had no effect
on fishes and only temporarily affected benthic forms.
Hiltibrand (1967) suggested that 10 mg/l atrazine had no
discernible effect on bluegill and green sunfish fry during
8 days post-hatch exposure. All of these data suggest safe
concentrations of atrazine generally an order of magnitude
greater than the estimated MATC values for the six species
tested in chronic studies. Although the time and effort is
considerably greater in developing and estimating MATC
values for chemicals based on chronic exposure, it appears
to be the only currently available method for generating the
information required to establish meaningful and realistic
water quality criteria.

Mount and Stephan (1967) have reported the utilization of
application factors to estimate chronic toxicity of
chemicals to fish based on acute toxicity data, and consider-
able information based on the chronic exposure of a variety
of fishes to pesticides and heavy metals supports this
hypothesis. The similarity of the limits of the application
factors for each of the fishes and two of the three
invertebrates tested with atrazine lends confidence to the
general validity of the application factor concept.

45


this species than any other (Mount, 1968; Brungs, 1969; Mount
& Stephan, 1969; Hermanutz et al. 1973) and procedures for
utilizing this species in long-term laboratory assays are
well defined. The results of our fathead minnow chronic
study in terms of performance of the test organisms are
excellent and indicate again that subtle long-term effects of
chemicals can be evaluated using this technique.

The number of spawnings per fathead minnow female, the number
of eggs per spawn, the percent hatchability, and the survival
of fry during the first 60 days development are as good or
better than that generally observed in previous studies.

In view of these facts, and the fact that the fathead minnow
procedure represents the only "true chronic bioassay" of the
three species tested (i.e. at least one complete life cycle)
we feel strongly that fathead minnows should be one of the
freshwater fish species of choice until methods suitable for
using other species are sufficiently defined.

We feel the brook trout offers significant potential for

use in “partial chronic bioassays". Generally the numbers

of fish participating in spawning and the number of eggs
spawned were adequate. Although admittedly variable, the
percent fertilization of eggs, the percent hatch and survival
through the first 30 days Cio swim-up) were generally good.

We feel that valid information can be generated utilizing
this species and these procedures. However, obvious critical
areas in the performance of such assays exist. The accidental
exposure of parent brook trout to increased day length at a
time which coincided with the period of late gametogenesis

and the onset of spawning activity verified Henderson's (1963)
observation that functional maturity in brook trout is delayed
or inhibited by increasing day length. Additionally we
suggest that separate systems, free of light, be provided

for egg incubation as Letritz (1959) has suggested that both
trout and salmon eggs are sensitive to direct light.

We also suspect that the decreased survival of brook trout
fry observed in all groups during the period 30-90 days
after hatch may be related to the stress associated with
handling fry at 30 and 60 days to count and measure them.
We suggest that a single measurement after 60 days exposure
would provide the same useful information regarding
potential effects of chemicals on second generation trout
yet would minimize possible adverse effects of handling.
We have recently conducted several similar studies with
brook trout eggs and fry and achieved excellent success

in hatching and fry survival by shielding the developing

43


isolated individually in 400 ml beakers containing the
corresponding test solutions. Some females were maintained
for up to 28 days in beakers with the test solution being
replaced every 48 hours with fresh test solution from the
appropriate experimental aquarium. Twenty to fifty young
collected from isolated females in the same adult chamber

on the same day were placed in the respective larval

growth chamber and exposure continued for 30 days. At the
end of 30 days, survival and growth were recorded by the
procedure of Clemens (1950). After a period of 17 weeks, the
exposure of the original gammarids to atrazine was terminated.

Lepomis macrochirus

In December 1971, chronic exposure of bluegills to atrazine
was initiated using 7 to 10 cm fish obtained from a commercial
hatchery in Wisconsin. Bluegills were acclimated to the test
water for three months, after which twenty fish were randomly
distributed to each test chamber. Bluegills were fed ad
libitum the largest commercially prepared trout pellet which
they would take twice daily. All tanks were siphoned twice
weekly to remove fecal material, excess food, and detritus,
and were brushed when algal growth became excessive. Total
length and weight of each individual fish was measured at the
initiation of exposure, after 90 days, and at thinning (185
days) using 100 mg/l of tricaine methanesulfonate (MS-222) to
lightly anesthetize the fish.

During the first year, when secondary sex characteristics
were well developed, fish in each tank were separated into
groups of males, females and undeveloped fish, using the shape
of the urogenital opening as the main criterion for sexual
differentiation (McComish, 1968). Sexually mature fish were
randomly reduced to 3 males and 7 females per duplicate tank
and all other fish were discarded after examination of gonadal
development and measurement of total length and weight. At
this time, two spawning substrates similar to those described
by Eaton (1970), were placed into each duplicate tank.
Substrates were 30.5 x 40 x 5 cm with a bowl-shaped depres-
sion 25 cm in diameter and 4 cm deep. Although *“"egills
developed secondary sex characteristics and exhibived
territorial behavior characteristic of spawning bluegill
populations under natural conditions, no spawning occurred
among either exposed or control fish during the first year.
Consequently, substrates were removed and the exposure of
bluegills to atrazine was continued a second year, repeating
the photoperiod schedule.

15


ee

TABLE 13. SPAWNING RESULTS, EGG HATCHABILITY, SURVIVAL AND GROWTH OF OFFSPRING
AFTER 30 AND 60 DAYS, FOR FATHEAD MINNOW (Pimephales promelas) CONTINUOUSLY
EXPOSED TO ATRAZINE

Mean Measured Atrazine Concentration mg/liter
O.2l 0.112 0.054 0.033 0.015 Control
Item A B A B A B A B A B A B
Spawnings/- 1.6 768 [5.5 10.8]3.5 53 |O.2 0 [4.3 7.7 [3.5 99
EES
spawned/ Q 133 1208 1757 1255 |399 1160 [7.5 0 | 450 862 |446 1284
Eggs/Spawmm |100 154 |138 116 13 217 | 45 o |120 122 {143 130
% Hatch@ 82 76 | 77 81 | 7 87 - - 76 84 | 83 74
(N) 5 27 22 27 8 23 0 ie} 11 22 19 37
30_ DAYS
Survival (%)] 56 64 68 73 65 60 - - 50 69 62 89
Length {mm) 12 10 11 10 13 12 - - 11 11 9 10
(S.D.) (2.63) (207}(165) (265}(2.6) (2.3) - - | (1.8) (2.7}(1.7) (2.1)
60 DAYS
Survival (%)| 45 28 | 57 69 | 48 55 - - 25 67 | 62 84
Length (mm) 17 21 17 16 20 19 - - 15 18 16 19
(S.D.) g(2s9) (3.7 (3.3) (4.5)(3.8) (3.6) - - | (4.0) (4.4)(4.0) (4.5)
# fry groups 1 3 2 4 2 2 ° (e) 2 4 3 2

ach sample contained 50 one day old eggs.
Dstandard Deviation.

°Bach group contained 40 one day old fry.


ABSTRACT

Representatives of the aquatic invertebrate species of water
flea (Daphnia magna), midge (Chironomus tentans), and scud
(Gammarus fasciatus); and the fish species bluegill (Lepomis
macrochirus), fathead minnow (Pimephales promelas), and
brook trout (Salvelinus fontinalis) were chronically exposed
to various concentrations of atrazine in separate flowing-
water systems.

Maximum acceptable toxicant concentrations (MATC) of atrazine
for the selected species in soft water were estimated using
survival, growth, and reproduction as indicators of toxic
effects. The MATC was estimated to be between 0.11 and 0.23
mg/l for midges, between 0.14 and 0.25 mg/l for water fleas,
and between 0.06 and 0.14 for the scud. For fishes the MATC
was estimated to be between 0.09 and 0.50 mg/l for bluegills,
between 0,21 and 0.52 mg/l for fathead minnows, and between
0.06 and 0.12 mg/l for brook trout. The incipient-LC50 for
fishes and the 48-hour LC50 for invertebrates was estimated
from acute exposures and was used to calculate application
factors (MATC limits/LC50). For aquatic invertebrates and
atrazine the estimated application factors were between

0.15 and 0.32 for midges, between 0.02 and 0.04 for water
flea, and between 0.01 and 0.02 for scud. Application
factors were estimated between 0.01 and 0.07 for bluegills,
between 0.01 and 0.03 for fathead minnows, and between 0.01
and 0.02 for brook trout. “

This report was submitted in fulfillment of Project Number
18050 HQH, Contracts 68-01-0092 and 68-01-1844, under the
sponsorship of the Water Quality Office, Environmental
Protection Agency.

iii


TABLE 8. MEAN® PRODUCTION OF YOUNG PER FEMALE Daphnia magna
CONTINUOUSLY EXPOSED TO ATRAZINE FOR DAYS

Mean measured Generation” I Generation? It Generation” IIT

conc. (mg/1) r Day - 5 Day 5 pat ;

Control 16.3 40.0 10.8 15.8 16.6 11.8
0.06 24, 33.0 6.3 12.2 6.3 9.7
0.14 19.0 30.5 4.9 144 10.4 14.9
0.25 14.6 18.3 3.6 10.1 4.8) 3.3
0.55 18.8 24.8 8.8 10.1 6.6 8.8
1.15 13.7 8.0 3.7 13.2 6.3 8.2

28ach value represents the mean of four replicates.

Ppuration of exposure for generations I, II, and III were days
1-22, 22-43, 43-64, respectively.

The results of this research indicate that reproduction of
Daphnia mgana is a much more sensitive measure of species
susceptibility to atrazine than is survival. Some of the
adult daphnids in each generation were males, and data on
reproduction was adjusted to represent that portion of the
population, in each experimental unit, directly involved in
production of progeny. The incidence of males induced from
parthenogenetic forms appeared to be higher in gf#oups exposed
to atrazine than in the control groups. Hutchinson (1967)
considered the presence of males in test populations a reac-
tion to an environmental stress, which atrazine may be in this
case.

Based on the statistical analysis of the survival and repro-
ductive success of daphnids continuously exposed to atrazine
for three successive generations, the maximum acceptable
toxicant concentration of this chemical for Daphnia magna is
estimated to be >0.14 and <0.25 mg/l.

Gammarus fasciatus

Statistical analysis indicated that survival of original
arids continuously exposed to a mean measured concentra-
tion of 0.94 mg/l atrazine for 30 days was significantly less
than all other experimental groups (Table 9). No significant
difference between survival of control groups and gammarids
continously exposed to 0.49 mg/l for 119 days was observed.

26


TABLE 3. PERCENT RECOVERY OF ADDED ATRAZINE FROM WATER
(ug/l) AND WHOLE FISH (ye/g)

Sample Type Added Mes sured Recovery (%)
(ppm) (ppm)
Water 0.05 0.052 104
0.05 0.049 98
0.05 0.050 100
0.50 0.46 92
0.50 0.51 102
0.50 0.50 100
5.0 4.6 92
5.0 4.9 98
5.0 5.0 100
98.4 + 4,1
Whole Fish 0.380 0.374 98
0.494 0.488 99
0.392 0.384 98
3.82 3.94 101
3.52 3.4y 98
3.49 3.46 99
98.8 + 1.1%

4standard deviation.

During the chronic exposures, total hardness, alkalinity,
pH and acidity were generally measured bi-weekly in the
control and one atrazine concentration according to Standard
Methods for the Examination of Water and Wastewater (APHA,

. emperature and dissolved oxygen concentrations were
measured in selected tanks each day using a YSI dissolved
oxygen meter with a combined oxygen-temperature probe.

12


‘percentage of the initial average biomass per tank and was
adjusted after 90 days and 136 days when total weights of
fish were again measured. Feeding rates were from a New

York State fish hatchery feeding chart (Deuel et al., 1952).
Total length and weight of individual fish were measured at
the initiation of exposure and after 90 days exposure,
utilizing 100 mg/l of tricaine methanesulfonate (MS-222) to
quiet the fish during measuring. All tanks were siphoned
twice weekly to remove fecal material, excess food, and
detritus, and were brushed when algal growth became excessive.

Secondary sexual characteristics were observed on test day
210 and males, females and undeveloped fish were identified
in each tank and the number of sexually mature fish was
randomly reduced to 2 males and 4 females per tank. All
other fish were discarded after being measured and examined
for gonadal condition. At this time, two stainless steel
spawning substrates, similar to those described by Benoit
(1974), were placed in each duplicate tank. The bottom of
these box-like substrates (25 x 25 x 15 cm) contained a
gridwork of 2.5 cm cubicles made of 20-mesh stainless steel
screen to retain the eggs where they were deposited. A

25 x 25 em square of 4-mesh stainless steel screen was placed
on top of the egg retainer and silicone adhesive was used to
attach 1.3 - 2.5 cm diameter stream gravel 2 cm apart on the
sereen.

Substrates were checked after 1:00 p.m. each day and eggs

were recovered by removing the gravel screen and egg retainer
and pipetting the eggs into a pan containing the test water.
Eggs from each spawn were counted and two egg cups containing
a group of 50 and 100 eggs, respectively were attached to the
rocker apparatus (Mount, 1968) for incubation in their respec-
tive test aquaria. Dead eggs were removed and recorded daily
and the number of fry hatching each day in the incubation cups
with 50 eggs was recorded. After 15 days, the group of 100
eggs was removed and the number of eggs developing a neural
keel was observed to determine percent fertilization.
Hatchability (percent of 50 eggs) and mean time to hatch
(degree days) were determined.

Twenty: five fry from the earliest two spwans in each tank
with at least 50% live hatch were placed in the respective
growth chambers. Fry from all other spawns were discarded
after determining percent hatchability. Cumulative mortality
and total length of live fry were determined after 30, 60

and 90 days using the photographic method of McKim and Benoit
(1971). Brook trout fry were fed five times daily ad libitum
with a commercially prepared trout starter food.

18


Daphnia magna

Laboratory stocks of Daphnia magna were obtained from the
University of New Hampshire, Durham, New Hampshire, and
successfully cultured in the laboratory according to the
methods of Biesinger and Christensen (1972).

Typically, 10 daphnids (<24 hours old) were placed into each
of four replicate experimental units, resulting in a total

of 40 animals per concentration. A food supply consisting of
trout starter and dry-powdered Cerophyl (2: ay was prepared

in an aqueous suspension (12.5 mg/ml) and delivered from a
Mariotte bottle via a volumetric delivery system to a mix-
ing chamber each diluter cycle. The diluted food suspension
was subsequently transferred to the food cells from which

25 ml (0.1 mg/ml) were delivered to each test container
during each diluter cycle.

Survival and reproduction of daphnids were recorded after
one, two and three weeks. Reproduction was measured by
recording the number of young in each experimental chamber
weekly and discarding the progeny after weeks one and two.
At the end of the third week, the number of original animals
remaining was recorded, the specimens discarded, and 10
daphnids (<24 hours old) were randomly selected from each
chamber to begin the second generation exposure. The

above procedures were followed for the second and third
generation, after which the experiment was terminated.

Gammarus fasciatus

The test organisms were collected in October 1972 from the
raceway outlets at the National Fish Hatchery, North
Attleboro, Massachusetts. Sexually mature adults were
acclimated at 17 + 1°C in the laboratory for a period of
three weeks. After the acclimation period, females were
isolated and their progeny collected. When sufficient

1 to 22-day old gammarids were available, the chronic
exposure to atrazine was initiated by placing 30 specimens
into each replicate aquarium. During the exposure, the
gammarids were fed pre-soaked maple leaves, water cress, and
Elodea. Brine shrimp (Artemia) nauplii and Daphnia were
also fed to the gammarids weekly.

Survival was recorded once each month by siphoning

contents of each test chamber into a pan and counting the
gammarids. At the onset of reproduction, all adult chambers
were checked daily for gravid females which were then

14


LZ

TABLE 9.

ATRAZINE FOR 17 WEEKS

PERCENT SURVIVAL AND REPRODUCTIVE SUCCESS OF Gammarus fasciatus EXPOSED TO

Mean Measured Atrazine Concentration (mg/1)

0.94 0.49 0.24 0.14 0.06 Control
A B A B A B A B A B A B
satay Survival
Day 30 uo 4o 58 60| 64 82| 56 66| 7% 66) 64
60 20 18} 30 42 32 60 26 Ay 26 58 24 38
91 16 16 24 32) 24 56] 16 34) 16 42] 30 30
119 4 lo} 24 4 14 34 12 32 16 32 12 18
Total # young - -| 18 1) 33 4o|] 39 43] 56 4] 18 -
Survival after
30 days (%) - -) 22 o| 52 38] 82 65 | 86 43) 33 -



Chironomus. In addition, ultra-violet lights (24 watt) were
placed over the headbox and water cells of each invertebrate
diluter to minimize the introduction of fungus and pathogens
into the test system.

Illumination was provided by a combination of Duro-test
(Optima FS) and wide-spectrum Grow-Lux fluorescent lights
fixed centrally above the test chambers in all experimental
units. Incandescent bulbs (100 watt) simulated a 15-minute
dawn or dusk light intensity change (Drummond and Dawson,
1970). A constant 16-hour light, 8-hour dark photoperiod
was controlled by an automatic timer in the Chironomus and
Daphnia experiments.

The photoperiod for Gammarus, bluegills, and brook trout
followed the normal daylight hours of Evansville, Indiana
(representative of U.S. daylength), and was adjusted the
first and fifteenth of each month. The photoperiod for
fathead minnows followed Evansville daylengths but was
started with the daylength for December 1st on day one of
the experiment (Nov. iy. All experimental units were
screened with black polyethylene curtains to prevent
unnecessary disturbance of test organisms and the influence
of extraneous lighting on the intended photoperiod.

ACUTE TOXICITY PROCEDURES

Static acute toxicity bioassays were conducted with inverte-
brates and atrazine to estimate the 48-hour LC50 and its 95%
confidence interval. Five organisms in three or four
replicate containers at each concentration were exposed at
20 + 11°C. First instar Chironomus tentans, Gammarus fasciatus,
and <24-hour old Daphnia magna were the organisms exposed.

A linear regression equation was calculated after converting
test concentrations and corresponding percent mortalities to
logarithms and probits, respectively, and this equation was
utilized to estimate the 48-hour LC50's and confidence
intervals.

Acute toxicity studies with trout and bluegill were conducted
in flow-through systems using proportional diluters (Mount
and Brungs, 1967). The incipient LC50 was estimated when

no additional significant (>10%) mortality of the test
organisms was observed for 48 hours among fishes exposed to
any concentration. At this time, the exposure was terminated
and a linear regression equation was calculated by converting
atrazine concentrations and corresponding mortalities into
logarithms and probits, respectively. This equation was
utilized to estimate the incipient LC50 and 95% confidence
interval. Due to the solubility of atrazine in water, a
static bioassay was used to estimate the acute toxicity of
atrazine to fathead minnows.


SECTION I
CONCLUSIONS

Estimates (LC50) of the acute toxicity of atrazine generally
ranged from 4.9-15.0 mg/l for all the species tested except
the midges for which the estimated 48 hour LC50 was 0.72 mg/l.

All six species were similar in their susceptibility to
chronic exposure to atrazine with the lower limit of the
estimated MATC concentrations for all species ranging from
0.06-0.21 mg/l.

Continuous exposure of chironomids through two successive
generations to a mean measured concentration of 0.23 mg/l
atrazine resulted in reduced hatching success, larval
mortality, developmental retardation, and a reduction in the
number of organisms pupating and emerging.

Continuous exposure of daphnids to atrazine through three
successive generations indicates that reproduction is a

much more sensitive measure of species susceptibility than

is survival during acute or chronic exposures. Although
continuous exposure to a mean measured atrazine concentration
of 1.15 mg/l had no significant effect on survival, continuous
exposure to 0.25 mg/l significantly reduced production of

progeny.

Morphological development of Fy gammarids is a more sensitive
indication of species susceptibility to chronic exposure to
atrazine than is survival. Although continuous exposure of
parental gammarids to 0.49 mg/l atrazine for 119 days had no
effect on survival, and continuous exposure to 0.14 mg/l for
30 days had no effect on survival of Fl gammarids, the number
of Fy gammarids exposed to 0.14 mg/l atrazine which success-
fully developed to the seventh instar form was reduced 25%
when compared to lower treatments and controls.

Growth of brook trout is the most sensitive indication of
species susceptibility to chronic exposure to atrazine.
Although continuous exposure to a mean measured concentration
of 0.72 mg/l atrazine for 44 weeks had no effect on survival
of parental fish, continuous exposure during the period to
0.12 mg/l atrazine significantly reduced growth of fry when
compared to lower treatments and controls.


TABLE 6. SUMMARY OF THE EFFECT OF VARIOUS CONCENTRATIONS OF
ATRAZINE ON HATCHABILITY, PUPATION AND EMERGENCE
OF Chironomus tentans CONTINUOUSLY EXPOSED FOR
TWO GENERATIONS

Mean measured . Generation I Generation II
mg/l Rep Hatch Pupa Adult Hatch Pupa Adult
¢ ) % Bi
Control A 91 89 89 90 87 87
B 93 95 93 83 83 80
0.12 A o4 91 90 95 93 89
B 90 83 80 90 81 90
0.23 A 86 75 70 89 71 65
B 90 63 59 93 69 56
0.42 A 91 31 30 70 11 10
0.78 B 86 264 26 63 29 26
B 86 of - - - -
1.33 A 84 FD - - - -
B 92 10) - - - -

Aonly late second-instar larvae present after 37 days
continuous exposure.

Ponty late first-instar larvae present after 38 days
continuous exposure.

Egg masses for the second generation exposure were obtained
from those groups where first generation adults emerged
during the same period as the controls (day 33-36 of exposure).
Continuous exposure of second generation eggs to 0.42 mg/l of
atrazine significantly reduced hatchability of eggs. Reduced
hatchability was not observed during the first generation
exposure to 0.42 mg/l and is indicative of a cumulative
effect of atrazine on the midges during the exposure of
successive generations. As was observed during the first
generation exposure, continuous exposure of midges to 0.42
and 0.23 mg/l of atrazine significantly reduced the number

of midges pupating and emerging. The data do not indicate

a cumulative toxic effect on these processes due to exposure
to these two concentrations of atrazine.

The results of continuous exposure to C. tentans to mean
measured concentrations of atrazine of 0.23 mg/l and greater
through two successive generations resulted in reduced
hatching success, increased larval mortality, developmental
retardation, or reduction in the number of organisms pupating
and emerging. Exposure to 0.11 mg/l atrazine had no observ-
able effect on the growth and development of these organisms

24


WATER CHEMISTRY

The results of the chemical analyses of water samples taken
during the various chronic exposures indicate that hardness,
alkalinity, acidity, pH and dissolved oxygen varied minimally
within any one chronic exposure. Statistical analysis showed
no significant differences for any of the above parameters
between treatments within a chronic; therefore, only means
and ranges for the various parameters are presented (Table 4).

The results of gas chromatographic analyses of water samples
taken periodically during chronic exposure of the fishes and
aquatic invertebrates to atrazine indicated that mean measured
atrazine concentrations closely approximated nominal concen-
trations (Table 5).

CHRONIC EXPOSURE
Chironomus tentans

The continuous exposure of first generation chironomid eggs to
mean measured concentrations of atrazine as high as 1.33 mg/l
had no significant effect on hatchability (Table 6). However,
about 60% mortality was observed among first instar larvae
developing from eggs exposed to 1.33 mg/l atrazine. Further-
more those first instar larvae surviving exposure to this
concentration of atrazine never developed beyond late first
instar-early second instar during 37 days exposure. Larvae
hatched from eggs exposed to a mean measured atrazine concen-
tration of 0.78 mg/l did not experience significant first
instar larval mortality but the majority of these larvae never
developed beyond late second instar-early third instar.

Larvae that experienced developmental retardation when com-
pared to controls were considered affected by chemical exposure,
and no assessment of the potential for emergence of these
retarded forms was made. All data regarding pupation and
emergence represent comparable data for all groups during 37
days exposure since pupation and emergence were completed
among control organisms within that time. Based on these
eriteria and an analysis of variance of comparable data, we
observed a significant reduction in the number of adults
emerging among those groups exposed to mean measured atrazine
concentrations of 0.42 and 0.23 mg/l. No significant differ-
ences in hatchability, survival, pupation and emergence were

observed between midges exposed to a mean measured atrazine
concentration of 0.12 mg/l and controls during the first
generation exposure (Table 6).

21


1.

5e

6.

7.

9.

10.

11.

TABLES
PAGE

Physical characteristics of test chambers
utilized to evaluate the chronic toxicity of
atrazine to fishes and fish food organisms. 7

Chemical analysis of the diluent water utilized
during chronic aqueous exposure of fishes and
aquatic invertebrates to atrazine. 8

Percent recovery of added atrazine from water
(ug/l) and whole fish (pg/g). 12

Mean and range of measured concentrations of
hardness, alkalinity, acidity, dissolved oxygen

and pH (range only) from water samples taken
periodically during chronic exposure of aquatic
invertebrates and fishes to atrazine. 22

Nominal and measured atrazine concentration (mg/1)
in water during chronic exposure of aquatic
invertebrates and fishes to atrazine. 23

Summary of the effect of various concentrations

of atrazine on hatchability, pupation and

emergence of Chironomus tentans continuously

exposed for two generations. 24

Mean percent survival of Daphnia magna continuously
days.

exposed to atrazine for 6 25

Mean production of young per female Daphnia magna
continuously exposed to atrazine for days. 26

Survival and reproductive success of Gammarus
fasciatus exposed to atrazine for 17 weeks. 27

Survival and growth during 6 and 18 months, and
results of spawning activity of bluegill

(Lepomis macrochirus) continuously exposed to
atrazine. 29

Hatchability, survival, and growth of bluegill
(Lepomis macrochirus) fry exposed to atrazine. 31

vi


CONTENTS
SECTION

I Conclusions

II Recommendations

III Introduction

IV Methods and Materials
Chronic Exposure Systems
Acute Toxicity Procedures
Chemical Methods
Statistics
Chronic Exposure

Chironomus tentans

Daphnia ma magna
Gammarus fasciatus
Lepomis Tepouts macrochinis

Pimephales promelas
Salvelinus. fontinalis
V_ Results
Acute Bioassays
Water Chemistry

Chronic Exposure
Chironomis tentans

Daphnia ma;

Gammarus fasciatus
Lepomis macrochirus
Pimephales promelas
Salvelinus fontinalis

Residue Analysis

Calculation of Application Factors

VI Discussion
VII References


The chronic exposure concentrations for both invertebrates
and fishes were selected by evaluating observed mortality

and no-effect concentrations from acute studies. The 48-hour
LC50 values for invertebrates and the incipient LC50 values
for fishes (except minnows) were used to estimate application
factors describing the relationship between acute and

chronic toxicity.

CHEMICAL METHODS

Toxicant concentrations and basic water quality characteristics
were initially monitored in each aquarium each week to
establish that the toxicant concentrations and water quality
characteristics were constant with minimum variability.

After the determination of these concentrations and parameters
in the experimental systems, a minimum monitoring effort was
conducted to measure variability and detect changes from the
established means. Generally, atrazine concentrations were
determined once each week during each of the chronic exposures
by taking 500 ml water samples from each aquarium. Stock
solutions of. atrazine (Sif aci., Ciba-Geigy Chemical Co.) in
nanograde dimethyl sulfoxide (DMSO) were delivered to the
dilution water from 50 ml glass syringes through stainless
steel needles. The solvent (DMSOY was not added to the
control water in any of the chronic exposures and the amount
added to the highest atrazine concentration of each chronic
test ranged from 12 pg/l during the brook trout chronic to

65 pg/l during the Daphnia chronic. In each case, these
concentrations were determined to be less than 1/500 of the
48-hour or 96-hour LC50 of the solvent for each species.

Water samples were taken in amber-glass bottles with teflon-
lined caps. The volume of water was accurately measured and
transferred to a separatory funnel. The sample bottle was
rinsed with 50 ml of methylene chloride to remove any adsorbed
atrazine and this solvent was added to the separatory funnel
containing the water sample. The separatory funnel was shaken
for two minutes to extract atrazine from the water, allowed

to stand until phase separation occurred, and the solvent
drained into a beaker. The extraction was repeated three
times using 25 ml of methylene chloride per extraction and

the extracts combined in the beaker.

The solvent extract was passed through a 20 x 50 mm column of
sodium sulfate and the column was rinsed with an additional
50 ml portion of methylene chloride. The solvent was
evaporated to approximately 5 ml on a stream bath, quantita-

10


ACKNOWLEDGEMENTS

During the period of time devoted to this research effort,

we were fortunate to have laboratory assistance from Mark
Lindberg and Gerald LeBlanc, who maintained exposure systems

and were significantly involved with routine aspects of the

fish chronics. Gratitude is extended to Krogh Derr, Pat Costa
and Karen Frey for their involvement and expertise in conducting
the invertebrate chronics. Also, our appreciation is directed to
Mr. Curtis Hutchinson for construction of all diluter systems
utilized throughout this study, and to Rod Parrish and Dr. Sam
Petrocelli for constructively reviewing the final report.

We are grateful to Mr. Richard Griffith, Northeast Regional
Director, U.S. Bureau of Sport Fisheries, Boston, Mass. for

his assistance in obtaining disease-free brook trout used in

the chronic exposure and to Mr. Quentin Pickering, Newtown Fish
Toxicology Station, EPA, Cincinnati, Ohio, for providing fathead
minnow eggs used in the study.

Finally, our sincere appreciation is extended to Mr. John Eaton,
Project Officer, Environmental Protection Agency, Environmental
Research Laboratory-Duluth, Duluth, Minnesota, for his guidance
and constructive advice during the performance of these studies.

viii


During the second year of bluegill exposure, when males
demonstrated territorial behavior, the substrates were
replaced in the tanks and total length and weight was
determined for each fish. Substrates were checked for eggs
after 1:00 p.m. each day and eggs were brushed loose from

the substrate under a stream of test water. Eggs were also
siphoned from the tank bottom after each spawn. Two

hundred eggs were randomly selected from each spawn and
placed in an egg cup which oscillated in the respective test
water by means of a rocker arm apparatus (Mount, 1968). Eggs
from each spawn were counted volumetrically by first counting
a subsample of exactly 1 ml and multiplying that number by
the total measured volume (ml) of spawned eggs. Eggs were
allowed to settle for two minutes in the graduated cylinder
before measuring. Hatchability was expressed as the percent
of live fry hatching out of 200 eggs.

Thirty bluegill fry from the earliest two spawnings in each
tank with at least 25 percent live hatch were placed in the
growth chambers. Fry from all other spawns were discarded
after hatchability was determined. Fry in the growth chambers
were fed live zooplankton from mixed laboratory cultures of
copepods, rotifers, protozoans, and brine shrimp ad libitum
three times daily. Cumulative mortality and total lengths of
fry were determined after 30, 60 and 90 days using the photo-
graphic method of McKim and Benoit (1971). Parental bluegills
were sacrificed after spawning had ceased in all tanks for
three weeks. Total length, total weight, sex, and gonadal
condition were determined for each fish and samples cf muscle
were retained for residue analysis.

Pimephales promelas

Chronic exposure of fathead minnows to atrazine began in
September 1971 with 18-day old fish obtained as eggs from the
Newtown Fish Toxicology Station, Newtown, Ohio. Forty fish
were randomly distributed to each test chamber. Cumu. ative
mortality and total length of live fish were determined after
30 and 60 days using the photographic method of McKim and
Benoit (1971). At 60 days, the number of fish in each test
chamber was randomly reduced to fifteen. Fathead minnows
were fed ad libitum twice daily with a commercially prepared
trout starter food which was supplemented with daphnids and
brine shrimp nauplii. All tanks were siphoned twice weekly
to remove fecal material, excess food, and detritus, and were
brushed when algal growth became excessive.

The discovery of bacteria and external parasites on a few

16


EPA-600/3-76-047
May 1976 Ecological Research Series



CALCULATION OF APPLICATION FACTORS

A summary of the estimated LC50 value, the maximum acceptable
toxicant concentration, and the application factor derived
therefrom for atrazine and all species studied is presented
(Table 17). The MATC for each species except bluegill and
fathead minnows is estimated between the highest mean measured
concentration having no significant effect and the lowest
mean measured concentration significantly affecting the
organism during chronic exposure. The MATC for bluegill

and fathead minnows is estimated between the highest mean
measured concentration applied during chronic exposure and
the minimum concentration which produced harmful effects
during acute exposure. Application factors describing the
relationship between the acute and chronic toxicity of
atrazine for the species studied are calculated using
concentrations bracketing the MATC and the 48-hour LC50 for
invertebrates. Similar application factors are calculated
for fishes utilizing the values bracketing the MATC and the
incipient LC50 which Eaton (1970) suggests to be a better
measure of acute toxicity for this type of calculation.

Application factors are similar for five of the six species
tested. Application factors for the fishes are remarkably
similar. However, the factors calculated for one of the
invertebrates (midges) are an order of magnitude greater
than those calculated for the fishes, and the other two
invertebrates.

39


SECTION II
RECOMMENDATIONS

Midges (Chironomus tentans) were more susceptible to atrazine
than any of the other Species studied. A variety of sublethal
effects were observed. These observations, combined with the
relatively short life history of midges, and the adaptability
of these organisms to the laboratory, suggests the species as
a desirable bioassay organism for evaluating the chronic
toxicity of chemicals to aquatic organisms.

Concerning chronic toxicity studies with bluegill, we feel
that more information must be developed on the laboratory
conditions necessary to induce and maintain spawning activity
and on the procedures necessary to handle and feed newly
hatched bluegill fry, in order to allow successful completion
of bluegill chronic toxicity studies.

Based on the lack of an acceptable level of spawning activity
among several groups of fathead minnows in which the number
of males was essentially equal to or greater than the number
of females, we suggest that care be taken throughout a
fathead minnow chronic to insure that during the spawning
period the ratio of females to males is greater than 1.5:1.

We recommend that the fathead minnow be considered the fish
species of choice, among those tested, for chronic toxicity
bioassays. This recommendation is based on the fact that an
adequate level of spawning activity can be induced under
laboratory conditions, successful handling of eggs and larvae
is possible, and excellent survival of fry is obtained. The
combination of these factors provides opportunities for
statistical and biological evaluation of possible toxic
effects due to chronic exposure to chemicals. Finally,
studies with this species represent the only "true chronic"
(at least one complete life history) of the fishes studied.


The data indicate the tendency for all three fishes to
bioconcentrate atrazine from water is very low when compared
to other pesticides. Chemical analysis of fish tissue
samples indicated that residue concentrations after prolonged
exposure were below minimum detectable limits for all the

fishes analyzed.
The similarity of the limits of the application factors for

each of the fishes, and two of the three invertebrates,
tested supports the general validity of the application

factor concept.


se

TABLE 14, MEAN INCREASE IN LENGTH AND WEIGHT OF YEARLING BROOK TROUT (Salvelinus
fontinalis) DURING 90 AND 306 DAYS CONTINUOUS EXPOSURE TO ATRAZINE
Mean Measured Atrazine Concentration mg/liter

0.72 0.45 0.24 0.12 0.065 Control
Item A B A B A B A B A B A B
90_ DAYS
Mean increase
in length 10 5 10 16 12 16 18 22 17 24 27 26
(mm)
Mean increase .
in weight(g) 22 8 20 36 24 39 40 46 44 52 60 61
306 DAYS
Mean increase
in length 70 57 51 70 54 64 66 78 87 86 | 101 92
(mm)
Mean increase
in weight(g) 149-122 100 157 106-155) {153 180 214 204 | 253 216



TABLE 5. NOMINAL AND MEASURED ATRAZINE CONCENTRATIONS (mg/1)
IN WATER DURING CHRONIC EXPOSURE OF AQUATIC
INVERTEBRATES AND FISHES TO ATRAZINE

Species and Measured Concentration mg/liter
Nomin. Conc. Mean _S.D. Range of Samples
Chironomus
tentans
° 1.33 + 0.930 0.69 - 3.3 20
1.00 0.78 + 0.310 0.24 - 2.8 20
0.50 0.42 + 0.170 0.13 - 0.63 19
0.25 0.23 + 0.062 0.15 - 0.33 20
0.12 0.11 ¢ 0.025 0.082- 0.19 20
Daphnia
magna
20 1.15 + 0.370 0.41 - 1.70 21
0.60 0.55 + 0.122 0.35 - 0.79 21
0.30 0.25 + 0.057 0.14 - 0.35 18
0.15 0.14+ 0.036 0.08 - 0.21 15
0.08 0.06 + 0.012 0.05 - 0.09 16
Gammarus
Lasciatus
T.00 0.94 + 0.167 0.50 - 1.30 40
0.50 0.49 + 0.208 0.29 - 0.80 40
0.25 0.24 + 0.069 0.16 - 0.53 38
0.12 0.14 + 0.029 0.08 - 0.21 40
0.06 0.06 + 0.011 0.03 - 0.08 35
Lepomis
macrochirus
. 0.095 + 0.026 0.034 - 0.17 106
0.050 0.049 + 0.011 0.031 - 0.084 63
0.025 0.025 + 0,008 0.011 - 0.053 58
0.012 0.014 + 0.004 0.005 - 0.031 58
0.006 0.008 + 0.004 0.004 - 0.022 57
Pimephales
rome Las
0.250 0.213 + 0.075 0.11 - 0.41 35
0.125 0.112 + 0.044 0.05 - 0.23 31
0.062 0.054 + 0.017 0.031 - 0.084 28
0.031 0.033 + 0.016 0.011 - 0.057 35
0.015 0.015 + 0.008 0.004 - 0.045 30
Salvelinus
ontinalis
° —_ 0.72 * 0.278 ; 0.18 - 1.10 46
0.05 0.45 + 0.156 0.12 - 0.66 28
0.25 0.24 * 0.042 0.17 - 0.32 23
0.12 0.12 + 0.039 0.03 - 0.20 28
0.06 0.065+ 0.013 0.021- 0.081 21

23


TABLE 17. SUMMARY OF CONCENTRATIONS OF ATRAZINE (mg/1)
PRODUCING ACUTE AND CHRONIC TOXICITY TO AQUATIC
SPECIES, AND CALCULATED APPLICATION FACTORS
DESCRIBING THE RELATIONSHIP BETWEEN ACUTE AND
CHRONIC TOXICITY. (MATC/LC50)

Common Limits on

Species Name Lc502 MATC application
factor

Chironomus 0.72 b
tentans midge (0.36-1.4) >.11<.23 | 0.15&0.32
Daphnia 6.9
magna water flea/(5.2-8.1) >.14<.25 | 0.02&0.04
Gammarus 5-7
fasciatus scud (3.6-8.0) >.06<.14 | 0.01&0.02
Lepomis 6.7
mMacrochirus |bluegill | (5.4-8.4) >.10<.50 | 0.01&0.07
Pimephales {fathead 15
promelas minnow (11-20) >.21<. 52 | 0.01&0.03
Salvelinus brook 4.9
fontinalis trout (4.06.0) >.06<.12 | 0.01&0.02

348-hour LC50 for invertebrates, incipient LC50 for fishes.
95% confidence interval.

40


rx

TABLE 4, MEAN AND RANGE OF MEASURED CONCENTRATIONS OF HARDNESS,. ALKALINITY, ACIDITY,
DISSOLVED OXYGEN AND pH (RANGE ONLY) FROM WATER SAMPLES TAKEN PERIODICALLY

DURING CHRONIC EXPOSURE OF AQUATIC INVERTEBRATES AND FISHES TO ATRAZINE
Acidi D.O. HH

Hard Alkalini
Species araness taal) (ne/13 (ng/i) ,

Chizonomus

‘Sean S.D. 49 ah] 39,0888 4,841,
Range 4o-¥5 (36-40 (4.35.
# of Samples 5 5

Daphnia

magna

ean + S.D. | 32.2+0.81 30.3+1.0 +.280.45 6.740

Range (27-35) (24-35) (1.9-6.0 (6.4-7
# of Samples 18 18 18 to)

Gammarus

fasciatus
Mean + S.D. Sac e i 32
Range (29-39 (
# of Samples 42

Lepomis
macrochirus

M £S.D. 9th, 32.743.
Range acraet (3885
# of Samples 20 20

Pimephales
romelas
Mean + S.D. 363282 33 34.642.7 4
Range (33-40 (27-48) (2.9
# of samples 13 13
Salvelinus
fontinalis

ean +t 8.D. 3 5749 08 34 64? 4,541.8 8,140.8 -
R O= 23-46 1,.9-7. ~9-11.2 6,4-7,4
# of Samples (38: ?) (238 j (2.3070?) (i gre2| (6-474)

840.75 -
7-6) (6.4-7.2)
he 120

«80,
27-46

) | (6.3-7.4)
30

6,940.7 :
) (3.4-10.2)} (6.5-7.3)
238 WwW



12.

13.

14,

15.

16.

17.

PAGE

Survival and growth of fathead minnows
(Pimephales promelas) exposed 30 days, 60

days, and 43 weeks to various atrazine
concentrations. 32

Spawning results, egg hatchability, survival

and growth of offspring after 30 and 60 days,

for fathead minnows (Pimephales promelas)
continuously exposed to atrazine. 33

Mean increase in length and weight of yearling
brook trout (Salvelinus fontinalis) during 90
and 306 days continuous exposure to atrazine, 35

Results of spawning activity of yearling brook
trout (Salvelinus fontinalis) during continuous
exposure to atrazine. 36

Survival and growth of second generation brook
trout (Salvelinus fontinalis) during the first

90 days of development of fry continuously
exposed to atrazine. 38

Summary of concentrations of atrazine (mg/1)
producing acute and chronic toxicity to aquatic
species, and calculated application factors
describing the relationship between acute and
chronic toxicity (MATC/LC50). 40

vii


STATISTICS

Means of the biological parameters measured in each duplicate
container during chronic exposure were subjected to

analysis of variance according to Steel and Torrie (1960).

When treatment effects were indicated by analysis of variance,

the means of these effects were subjected to Duncan's Multiple
Range Test (1955) to determine which treatments were different
from the controls. Statistical significance was accepted at P=.05.

CHRONIC EXPOSURE
Chironomus tentans

Stock cultures of Chironomus tentans were obtained from
Michigan State University, East Lansing, Michigan. Cultures
were maintained in 60 1 glass aquaria utilizing a substrate
prepared by placing 50 grams of paper hand towel (Nibroc,
Brown Co.) in a blender with 5 grams of high protein chicken
feed and 1 liter of water to cover the mixture. The mixture
was homogenized for three to five minutes. This substrate
was used to cover the bottom of each aquarium to a depth of
1.5 cm. Emergent adults were allowed to mate within the
screened aquaria and provided a ready supply of egg masses.

Egg masses were selected from the stock cultures according to
uniformity of age, and one egg cup of 100 eggs was suspended
in each of the replicate experimental aquaria. Egg cups were
made from 20 ml glass vials with the bottoms removed and
replaced with 40 mesh nylon screen. After hatching was com-
pleted in all treatments (3-5 days), the number of surviving
first instar larvae were counted and carefully placed on the
substrate.

After 26 to 29 days, the number of emergent adults, pupal
exuviae, and dead pupae were observed and recorded. Mating
of adults was observed, and egg masses produced by mating
pairs in each replicate aquaria were collected and utilized
to conduct second generation exposures according to the above
methods. At the initiation of the second generation exposure,
new substrate was placed in the experimental aquaria which
would receive first instar larvae. The number of emergent
adults, pupal exuviae, and dead pupae was observed in each
replicate concentration and the experiment terminated.

13


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

7” REPORT NO. 2 3. RECIPIENT'S ACCESSION NO.
EPA-600/3-76-047

[4 TITLE AND SUBTITLE Is. REPORT DATE
Chronic Toxicity of Atrazine to Selected Aquatic May 1976 (Issuing Date)
Invertebrates and Fishes 6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S) ls. PERFORMING ORGANIZATION REPORT NO.

Kenneth J. Macek, Kenneth S. Buxton, Scott Sauter,
Sarah Gnilka, Jerry W. Dean

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.
Bionomics 1BA608
EG & G Inc. TT. CONTRACT/GRANT NO.
790 Main Street 68-01-0092 and 68-01-1844
Wareham, Massachusetts 02571
12, SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
Environmental Research Laboratory-Duluth Final
Office of Research and Development 14. SPONSORING AGENCY CODE
U.S. Environmental Protection Agency EPA/ORD

Duluth, Minnesota 55804
15. SUPPLEMENTARY NOTES

16, ABSTRACT
Representatives of the aquatic invertebrate species of water flea (Daphnia
magna), midge (Chironomus tentans), and scud (Gammarus fasciatus); and the fish
species bluegill (Lepomis jmacrochirus) , fathead minnow (Pimephales promelas) ,
and brook trout (Salvelinus fontinalis) were chronically exposed to various
concentrations of atrazine in separate flowing-water systems.

Maximum acceptable toxicant concentrations (MATC) of atrazine for the
selected species in soft water were estimated using survival, growth, and
reproduction as indicators of toxic effects. The MATC was estimated to be between
0.11 and 0.23 mg/1 for midges, between 0.14 and 0.25 mg/l for water fleas, and
between 0.06 and 0.14 for the scud. For fishes the MATC was estimated to be
between 0.09 and 0.50 mg/l for bluegills, between 0.21 and 0.52 mg/1 for fathead
minnows, and between 0.06 and 0.12 mg/1 for brook trout. The incipient-LC50 for
fishes and the 48-hour LC50 for invertebrates was estimated from acute exposures
and was used to calculate application factors (MATC/LC50). For aquatic invertebrates
and atrazine the estimated application factors were between 0.15 and 0.32 for
midges, between 0.02 and 0.04 for water flea, and between 0.01 and 0.02 for scud.
Application factors were estimated between 0.01 and 0.07 for bluegills, between
0.01 and 0.03 for fathead minnows, and between 0.01 and 0.02 for brook trout.

V7. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS lb. IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Toxicity Chronic toxicity oer
Fishes Tissue residues
Invertebrates Flowing water system
Pesticides Application factors
Atrazine
18. DISTRIBUTION STATEMENT. 19, SECURITY GLASS (This Report) 21. NO, OF PAGES
Unclassified
Release unlimited 7 SGROTLSRERL EE PRT PR PRICE
EPA Form 2220-1 (9-73) 50

‘Yr U.S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5441 Region No. 5-1!


fish were injected with 0.5-1.0 cc of a 200 ug/100 ml

solution of carp pituitary in physiological saline. The
injections failed to induce the desired increase in spawning
and may have been administered too late in the spawning period
to be effective. Parental fish were sacrificed on test day
736 and samples of muscle were retained for residue analysis.

The percent of eggs successfully hatching among groups of
eggs exposed to each of the four highest concentrations of
atrazine tested was similar to that observed among control
groups and indicates that continuous exposure to mean
measured concentrations of atrazine as high as 0.095 mg/l
did not significantly affect hatchability of eggs (Table 11).
Survival of bluegill fry was significantly lower during 30
days exposure to 0.095 and 0.049 mg/l than to the other
three concentrations of atrazine, suggesting a toxicant
related effect. Unfortunately, survival of fry in control
groups was very low and it is impossible to confirm whether
fry survival was toxicant related. Survival of fry in all
groups during this period appeared to be related to an early
acceptance of the appropriate available food rather than
toxicant related. This interpretation is based on the
observation that survival in all groups was excellent during
the final 60 days of exposure when feeding habits became
well established and food was readily accepted.

Total lengths of bluegill fry at 30 days did not vary
significantly between controls and any concentration. Total
lengths at 60 and 90 days were significantly lower among

fish exposed to 0.049 mg/l of atrazine, however these were
the only fry groups remaining in the experiment when a

heater malfunction caused a decrease in temperature in the
experimental system. The malfunction could not be corrected
soon enough to bring growth rate of these fish back to a

rate comparable with other groups. Total lengths of bluegill
fry in all other tanks were similar after 60 and 90 days
exposure, and we conclude that continuous exposure of
bluegill fry to atrazine concentrations as high as 0.095 mg/l
for 90 days had no significant effect on growth (Table 11).

Based on the data obtained from continuous exposure, it
appears that the maximum acceptable toxicant concentration is
eater than the highest measured atrazine concentration
0.095 mg/1) to which bluegill were exposed during the chronic
test. Since we observed loss of equilibrium among bluegill
continuously exposed to 0.50 mg/l atrazine for 28 days, the
maximum acceptable toxicant concentration of atrazine for
this species is estimated to be between 0.095 and 0.50 mg/l.

30


TABLE 11. HATCHABILITY, SURVIVAL, AND GROWTH OF BLUEGILL
(Lepomis macrochirus) FRY EXPOSED TO ATRAZINE

Mean Measured Atrazine Concentration (mg/1)
ITEM 0.095 0.049 0.025 0.01 0.00 Control
Hatchability(%) , 91 90 87 81 66 84
# of egg groups 4 3 6 12 3 7
Survival %%)
30 days 18 22 45 36 48 22
60 days 18 20 45 35 46 22
90 days 18 20 42 35 43 22
Mean Total Length
+ S8.D.
30 days 2042.9 1741.8° 2243.2} 2043.2 | 2142.4 |18+2.0
60 days 3043.1 (1842.0 |3046.0] 3043.1 | 2543.7 |2943.1
90 days 4o+5.9 |18+2.0 [3848.0] 3844.9 | 3543.5 [4047.2
# of fry
groups¢ 1 1 2 4 2 2

@pach group contained 200 one day old eggs.
Pstandard deviation,

CRry groups exposed to lower water temperature due to heater
malfunction.

drach group contained 50 one day old fry.

Pimephales promelas

Statistical analysis of data on survival and growth of
fathead minnows after 30 and 60 days exposure to atrazine
indicated that survival was significantly less and growth
significantly greater among controls than all treated
groups (Table 12). We believe these to be related
phenomena. Although no explanation for the lower survival
among controls exists, we suggest the greater growth among
fish remaining in these tanks is related to reduced
competition for food. This hypothesis is supported by the
observation that during the period of exposure from 9-43
weeks no significant differences in growth and survival
among any of the experimental groups were evident.

Spawning activity of fathead minnows in all experimental
groups occurred between days 164 and 280. Virtually no
spawning activity occurred among fathead minnows exposed to
0.033 mg/l atrazine, and spawning among fish exposed to
0.213 mg/l in replicate A was reduced (rable 13). These
were the only groups in which the number of males was equal

31


SECTION VII
REFERENCES

American Public Health Association, 1971. Standard Methods
for the Examination of Water and Wastewater, 13th ed.

Am. Public Health Assoc., New York, 874 p.
Arthur, J. W., 1970. Chronic effects of linear alkylate
sulfonate detergent on Gammarus pseudolimnaeus,

Water Research

Gampelone decisum and Physa integra.

Arthur, J. W., A. E. Lemke, V. R. Mattson and B. J. Halligan,
1973. Toxicity of sodium nitrilotriacetate (NTA) to the
Water Research

fathead minnow and amphipod in soft water.
8:187-193.

Arthur. J. W. and rea Leonard, 1970, Effects of copper on
Gammarus pseudolimnaeus sa integra and Campeloma
decisum in soft water. "ys. Fish. es. Bd. canada 27:

Benoit, D. A., 1974. Artificial laboratory spawning substrate
for brook trout (Salvelinus fontinalis, Mitchill).

Trans. Am. Fish. Soc. 3
Biesinger, K. E. and G. M. Christensen, 1972. Effects of

various metals on survival, growth, reproduction, and

metabolism of Daphnia magna. J. Fish. Res. Bd. Canada

29:1691-1700.
Bioassay Committee, 197la. Recommended bioassay procedures
for fathead minnow (Pimephales promelas, Rafinesque)
chronic tests. National Water ity Laboratory, Duluth,
Minnesota, 13 p.
Bioassay Committee, 1971b. Recommended bioassay procedures
‘for bluegill (Lepomis macrochirus, Rafinesque) partial
National Water Quality Laboratory, Duluth,

chronic tests.

Minnesota. 12 p.
Bioassay Committee, 1971c. Recommended bioassay procedures

for brook trovt (Salvelinus fontinalis, Mitchill) partial
chronic tests. National Water Quality Laboratory, Duluth,
Minnesota, 12 p.
Chronic toxicity of zinc to the fathead

Prunes tnnow Pi ephal promelas, Raf T
mnow Pime) es promelas inesque. ‘rans. Am.
Fish. Soc. 3 - ° °

46


After spawning had ceased in all tanks for a period of three
weeks, parental fish were removed and the condition of the

gonads was observed. Total lengths and weights were deter-
mined for each individual fish before obtaining muscle

samples for residue analysis and discarding the remaining
fish.

19


Mount, D. I. and W. A. Brungs, 1967. A simplified dosing

apparatus for fish toxicological studies. Water Research
1:21-29.

Mount, D. I. and ©. E. Stephen, 1967, A method for establishing
acceptable toxicant limits for fish-malathion and the
putoxyethanol ester of 2,4-D. Trans. Am. Fish. Soc.

Mount, D. I. and C. E. Stephan, 1969. Chronic toxicity of
copper to the fathead minnow (Pimephales promelas) in
soft water. J. Fish. Res. Bd. Canada 36 340927057.

Pickering, Q. H. and T. 0. Thatcher, 1970. The chronic toxicity
of linear alkylate sulfonate (LAS) to Pimephales
promelas, Rafinesque. J. Water Pollut. Control Fed. 42:

Sanders, H. 0., 1969. Toxicity of pesticides to the crustacean
Gammarus lacustris. U.S. Bur. Sport,Fish Wild, Tech. Rep.

75. 18 p.

Steel, R.G. D. and J, H. Torrie, 1960. Principles and procedures
of statistics. McGraw Hill, New York. 481 p.

Walker, C. A., 1964. Simazine and other s-triazine compounds
on aquatic herbicides in fish habitats. Weeds 12:134-139.

49


DISCLAIMER

This report has been reviewed by the Environmental Research
Laboratory-Duluth, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the
U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or
recommendation for use.

ii


sé

TABLE 16, SURVIVAL AND GROWTH OF SECOND GENERATION BROOK TROUT (Salvelinus fontinalis)
DURING THE FIRST 90 DAYS DEVELOPMENT OF FRY CONTINUOUSLY EXPOSED TO ATRAZINE

Mean Measured Atrazine Concentration mg/1

0:72 o.58 0.38 0.12 0.065 Control
Item rN BI Xx B | x B |x Bx B| i 8
No. groups? 1 33>) 12> 3 | 2 2 | 2 2 | 2 2| 2 2

Survival (%)
30 days | 91 95 hoo 85 186 92 |78 96 [ay 84 | 96 88

60 days 9 39 «| 68 27 172 16 | 32 76 166 52 | 80 74

90 days 4 15 148 9 | 32 8 |22 58 146 38 | 52 50
Mean Lengt,

(mm)

30 days | 21 23 124 20 | 21 20 |j2l 23 |22 21 | 24 25

60 days | 22 25 126 23 | 24 22 | 25 27 | 26 24 | 27 27

90 days | 25 28 | 29 28 | 27 25 {31 33. «134 32 | 34 35
Mean Worent

&
90 days |0.10 0.11/0.14 0.11)0.11 0.10]0.20 0.28/0.30 0.24 0.27 0.30

a

arry groups contained 25 one day old fry.
Dery from unexposed parents,


9¢

TABLE 15. RESULTS OF SPAWNING ACTIVITY OF YEARLING BROOK TROUT (Salvelinus fontinalis)
DURING CONTINUOUS EXPOSURE TO ATRAZINE a

Mean Measured Atrazine Concentration mg/l

0.72 0.45 0.20 0.12 0.065 Control
Item ny 3B x 3B K B nN B nN B A B
o/e 2/4 2/4 | 2/4 3/3 | 2/4 1/3 | 2/4 3/3 | 2/h afb | 3 3 2/h
t ming vt vs 4 2 4 3 4 3 vt vt 43 a4
# spawns/? 2 2 1 2 2 3 2 2 2 2 2 3
Total # e
j seamed 1485 589 | 531 814 |21790 1710 [1606 1506 hae rye |ri22 ange
eggs
spawned/? 371-295 | 133 407 | 448 570 | 402 502 | 353 446 | 562 373

Neural keel
developed(%) 26 40 0 83 74 63 27 66 65 50 70 AL

Incubation time

(degree days) | 387 405 432 432 450 4h7
Mean Hatchability

(4%) 58 8 - 48 67 26 62 78 25 34 65 3?
(Nn)? 1 1 0 3 5 2 1 3 2 2 2 1

'Indicates the number of groups of 50 eggs in which at least half successfully
developed a neural keel,


SECTION IV
MATERIALS AND METHODS

The thethodology for chronic testing of fishes generally
followed the recommended bioassay procedures issued by the
Environmental Protection Agency's National Water Quality
Laboratory, Duluth, Minnesota (Bioassay Committee 1971a,
1971b, 1971c). Acute bioassay procedures were bascially

those recommended in Standard Methods for the Examination of
Water and Wastewater (APHA, 1971). Chronic testing procedures
for invertebrates were determined through communication of
Bionomics staff members with personnel at the National Water

Quality Laboratory.
CHRONIC EXPOSURE SYSTEMS

Proportional diluters (Mount and Brungs, 1967), with a
dilution factor of 0.5 and a syringe injector, delivered the
test water and toxicant, dissolved in dimethyl sulfoxide, to
the mixing chamber, mixing cells, and ultimately to the test
chambers. Five atrazine concentrations and a control flowed
to mixing containers and into separate giass delivery tubes
leading to the replicate test chambers. In the test system
for Daphnia magna, baffles were inserted in each of the
quadruplicate test chambers to minimize turbulence of influent
water. The diluter was also modified to include food cells
which delivered a measured amount of food along with the
toxicant and diluent water. All other exposure systems
utilized duplicate test chambers of varying construction and
flow rates as summarized in Table 1.

Two growth chambers 21 x 20 x 15 cm (height x length x width)
with a water depth of 16 cm were provided for the young Gammarus
and received test water at a flow rate equal to that in the
adult test chambers. Fathead minnow test chambers were
subdivided to provide space for two growth chambers 25 x 20.5

x 12.5 cm, and test chambers for brook trout contained a

shelf which supported two growth chambers 25 x 25 x 12.5 cm
with a water depth of 12.5 cm. Water was delivered directly

to both the adult test chamber and growth chambers through

a glass, flow-splitting chamber calibrated to provide equal
flow rate to all chambers. The bluegill test chambers

supported two 18 x 28 x 12 cm growth chambers with a water
depth of 10.5 cm, and air was utilized to pump water from the
test chamber to the growth chambers at a rate of 6 chamber
volumes every 24 hours. All fish growth chambers had 40-mesh
stainless steel screen affixed to one end to allow water to
flow out while retaining the young fish. Bluegill and brook
trout test chambers were aerated with oil-free air in order

to maintain dissolved oxygen levels above 60 percent saturation.

6



Hermanutz, R. 0., L. H. Mueller amd K. D. Kempfert, 1973.
Captan toxicity to fathead minnows (Pimephales
Fi: ) and brook

romelas), bluegills (Lepomis macrochirus
trout (Salvelinus fontinalis). J. Fish. Res. Bd.
Canada 7

Hesselberg, Ro J. and J, L. Johnson, 1972. Column extraction
of pesticide from fish food and md. Bull. Environ.
Contam. Toxicol. 7:115-120.

Hiltibrand, R. C., 1967. Effects of some herbicides on
fertilized fish eggs and fry. Trans. Am. Fish. Soc.

Hutchinson, G. E., 1967. A Treatise on Limnology, Vol. 2
John Wiley & Sons, New York, 1115 p.

Johnson, D. W., 1968. Pesticides and fishes - A review of
selected literature. Trans. Am. Fish. Soc. 97:398-424.

Leitritz, E., 1959. Trout and salmon culture. State of
California, Dept. Fish & Game, Fish Bull. No. 107,

169 p.

Macek, K. J. and S. Korn, 1970. Significance of the food
chain in DDT accumlation by fish. J. Fish. Res. Bd.
Canada 27:1496-1498,

Macek, K. J., K. S. Buxton, S. K. Derr, J. W. Dean and S. Sauter,
1975. Chronic toxicity of lindane to selected aquatic
invertebrates and fishes. EPA, Ecol. Res. Series
(In Press).

Mayer, R. L., P. M. Merhle and W. P. Dwyer, 1975. Toxaphene
effects on reproduction, growth and mortality of brook
trout. EPA, Ecol. Res. Series (In Press).

McComish, T. S., 1968. Sexual differentiation of bluegills
by the urogenital opening. Prog. Fish. Cult. 29:28

McKim, J. M. and D. A. Benoit, 1971. Effects of long-term
exposures to copper on survival, growth, and reproduction
of brook trout (Salvelinus fontinalis). J. Fish. Res.
Bd. Canada 28:655-662.

Mount, D. I., 1968. Chronic toxicity of copper to fathead
minnows (Pimephales romelas, Rafinesque). Water
Research 2: =2232

48


Diluent water was pumped from a 400 foot subterranean well to
a cement holding tank. Results of the chemical analysis of
the diluent water are summarized in Table 2.

TABLE 2, CHEMICAL ANALYSIS OF THE DILUENT WATER UTILIZED
DURING CHRONIC AQUEOUS EXPOSURE OF FISHES AND
AQUATIC INVERTEBRATES TO ATRAZINE

Parameter . mg/liter Parameter mg/liter
Calcium 6.0 Chloride 17.6
Magnesium 2.1 Fluoride 0.5
Potassium 1.1 Cyanide <0.005
Sulfate 11.6 Iron <0.01
Nitrate <0.05 Copper 0.004
Nitrite <0.05 Zine 0.01
Ammonia <0.1 Cadimum 0.001
Phenol <0.001 Chromium <0.001
Chlorine <0.01 Lead <0.01

Diluent water was delivered through PVC pipes to the exposure
systems. Unheated well water was mixed with heated well water
from a glass-lined heater to provide a constant temperature
of 2841°C before delivery to the bluegill experimental unit.
Test chambers in the fathead minnow and brook trout units
were maintained in circulating water baths at the desired
test temperatures. Water circulating in the water baths of
the fathead minnow unit was heated by two immersion coil
heaters controlled by a mercury column thermoregulator which
maintained a temperature of 25+1°C. Water in the baths of
the brook trout unit was cooled by recirculating through a
mechanical chiller to temperatures of 15+1°C from May through
August, 12+1°C during September, and 941°C from October
through the end of juvenile exposure. Prior to entering
experimental units for the invertebrate studies, diluent
water was delivered to a stainless steel headbox where
immersion coil heaters and thermoregulators maintained a
temperature of 19+1°C for Daphnia and Gammarus and 23+1°C for



EPA-600/3-76-047
May 1976

CHRONIC TOXICITY OF ATRAZINE TO
SELECTED AQUATIC INVERTEBRATES AND FISHES

by

Kenneth J. Macek
Kenneth S. Buxton
Scott Sauter
Sarah Gnilka
Jerry W. Dean

Bionomics, EG&G Inc.
Wareham, Massachusetts 02571

Contract No. 68-01-0092
68-01-1844

Project Officer

John Eaton
Environmental Research Laboratory
Duluth, Minnesota 55804

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804


62

TABLE 10. SURVIVAL AND GROWTH DURING EXPOSURES OF 6 AND 18 MONTHS, AND RESULTS OF

SPAWNING ACTIVITY OF BLUEGILL? (Lepomis macrochirus) CONTINUOUSLY EXPOSED
TO ATRAZINE

Mean Measured Atrazine Concentration (mg/liter)

0.095 0.049 0.025 0.014 0.008 Control
Item rN B x B ry B K B K B ry B
6 MONTHS
Survival (%) 100 85 | 95 80] 100 95] 95 100 | 95 851 75 95
Total Length
(mm) 124 129 |127 128} 126 128 | 129 130 }130 126} 133 129
Total Weight(g 35 4o | 40 40] 37 38 | 41 bo | 41 37, 43 4)
18 MONTHS
Survival (%) 90 80 | 90 100 | 100 90 | 100 100 {100 70} 90 100
o/e 3/6 3/5 13/6 = 8/5} 3/7 ~— 2/7 43/7 = 46 [3/7 = 2/5] 3/6 3/7
Total nyen
mm
g 185 199 |200 193 | 172 179 | 192 192 {198 199} 182 207
g 157 153 1155 153 | 158 166 | 158 161 |156 165} 160 158
Total Weight(g)
¢ 120 146 |165 147 | 102 112 ot 138 |146 163] 122 172
3 77 69 | 62 63] 73 86] 7 76 | 72 78| 76 72
# of Spawnings 4 3 (0) 4 8 (e) v4 0 7 8 2
Eggs/? 2920 2432 - 7331 [5153 - 6703 8218 - 15254]16890 580
Eggs/Spawn 19380 4053 - 9164 |4509 - 6703 6163 - 10895}2668 2031

@vean initial length and weight 100 mm, 15 g.

Pgased on 10 fish per aquarium after thinning on day 185 of exposure.


fish prompted the use of flush treatments of tetracycline
hydrochloride (4% mg/l active ingredient) and a combination
of malachite green and formalin (25 pl/l of formalin contain-
ing 3.7 g/l malachite green crystals). These treatments

were concentrated between days 70 and 129 of the experiment.

In order to establish the appropriate ratio of male fish to
female fish, some of the males were removed after secondary
sexual characteristics were well developed. Five spawning
sites of halved, 3-inch transite drain tiles were placed in
each tank when fish were released from growth chambers at 60
days. The tiles were placed concave surface down at locations
that minimized the chance of encounters by separate egg-
guarding males. When spawning began, eggs were removed and
counted after 1:00 p.m. each day. Fifty eggs from each spawn
were oscillated in their corresponding test waters ey means
of an egg cup and a rocker arm apparatus (Mount, 1968). Dead
eggs were removed and counted each day until hatching was
completea (3-5 days at 25°C). Percent hatch was based on the
number of live fry from 50 eggs.

Forty fry from the earliest two spawns in each tank with at
least 80% live hatch were placed in the respective growth
chambers. Cumulative mortality and total length of live fish
were determined at 30 and 60 days by the photographic method
of McKim and Benoit (1971). Fry from all other spawns were
discarded unless a growth chamber was later made available
by termination of 60-day old fry. Finely ground starter

food and brine shrimp nauplii were fed three times daily to
fry in the growth chambers.

Parental fish were sacrificed after all spawning had ceased
for one week. Total length, weight, sex and gonadal condition
were determined for each fish and three samples per concentra-
tion of eviscerated fish were retained for residue analysis.

Salvelinus fontinalis

Chronic exposure of brook trout to atrazine began in May 1972,
with yearling fish obtained from the National Fish Hatchery,
Manchester, New Hampshire. Distribution of fish to the test
chambers was delayed by the discovery of external parasites

on a few fish. The parasites were controlled by flush
treatments of malachite green and formalin (25 pl/l of formalin
containing 3.7 g/l of malachite green). These treatments were
repeated shortly after the distribution of 15 fish to each
duplicate tank to insure control of the parasites. Brook trout
were fed twice daily with a measured ration of the largest
trout pellet they would take. The feeding rate was based on a

17


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:

Environmental Health Effects Research

Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine 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.

VO PONn>

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


SECTION VI
DISCUSSION

The estimated 48-hour LC50 value of 6.9 mg/l determined for
Daphnia is higher than the previously estimated value of

3-6 mg/l (FWPCA, 1968). The difference may be attributable
to any number of variables (e.g. diluent water quality, age
of test organisms, temperature, etc.). No previous estimates
of the acute toxicity of atrazine to chironomids and
gammarids are available.

The 96-hour LC50 of atrazine to rainbow trout (Salmo gairdneri),
pased on the results of a static bioassay, was previously
estimated to be 12.6 mg/l (FWPCA, 1968). The difference
between this value and our 4.9 mg/l estimated incipient LC50
for brook trout is probably due to species differences and/or
the fact that the rainbow trout were tested under static
conditions while the brook trout were tested in a dynamic
system. Our estimate of the acute LC50 of 6.7 mg/l for
pluegill compares favorably with a previously estimated 96-
hour LC50 of about 6 mg/l, (i.e. 12 mg/l 50% Wettable Powder
Formulation) reported by Walker (1964).

The results of these investigations indicated that the three
invertebrate species were very similar in their susceptibility
to chronic exposure to atrazine despite the fact that there
were order of magnitude differences in their susceptibility

to acute exposure. It is interesting to note that this
similarity existed despite the fact that the MATC for each
species was estimated on the basis of different criteria.

The most sensitive criterion for determining the effects of
atrazine on these invertebrates was developmental retardation
for Chironomus, production of young for Daphnia, and survival
of young for Gammarus.

The fishes tested were not only similar to each other in

their susceptibility to chronic exposure to atrazine but also
were remarkably similar to the invertebrates studied. Only

one other study has been reported describing the susceptibility
of the above species to chronic exposure to a pesticide

(Macek et al., 1975). In that study, we found that although
the estimated MATC of lindane to the fishes tested were similar,
the MATC estimates for two of the three invertebrates tested
were significantly lower.

Except for Gammarus, little other previous information is
available to provide a basis for making generalizations

about the relative susceptibility of fishes and invertebrates
to chronic éxposure to chemicals in water. However, much of

41


when compared to control groups. Based on these data, the
maximum acceptable toxicant concentration of atrazine for
Cc. tentans is estimated to be >0.11 and <0.23 mg/l.

Daphnia mgana

Statistical analysis of the mean percent survival of original
Daphnia from each of three successive generations indicated
that continuous exposure to mean measured concentrations of
atrazine as high as 1.15 mg/l had no significant effect on
survival (Table 7). Also, these data indicate that no
atrazine-induced cumulative effects on mortality were observed
in second or third generation daphnids.

TABLE 7. MEAN® PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO ATRAZINE FOR 64 DAYS

Mean measured Generation” I Generation” II Generation” IIt

conc. (mg/1) Da; Day Day
8B 15 70 | 29 38 43} 50 57 65

Control 89 61 49 | 92 88 84} 69 65 55
0.06 81 72 51 | 95 92 91| 70 64 55
0.14 84 75 65 | 82 79 78) 39 36 36
0.25 78 = =«75 60 | 84 82 80} 78 72 59
0.55 68 5h 34 | 64 56 52] 50 45 4h
1.15 88 85 82 | 91 89 71) 82 76 7h

@fach value represents the mean of four replicates.

Ppuration of exposure for generations, I. II and III were
days 1-22, 22-43, 43-64, respectively.

Analysis of variance indicated that the mean number of young
produced per female daphnid during the first generation was
significantly reduced by exposure to atrazine (Table 8).
Continuous exposure of the first generation Daphnia to mean
measured concentrations of 1.15, 0.55, and 0.25 mg/l atrazine
significantly reduced the production of young. Although
reproduction among second and third generation daphnids exposed
to these concentrations was generally less than other groups,
variability in the data precluded ascribing statistical
significance to these observations.

We are, at this time, unable to explain the declining rate of

progeny production, observed in every experimental group.
uring the successive generation experiment. It is obviously
not toxicant related, but is apparently related to inadequacies

in the laboratory life-support systems.

25


to or greater than the number of females and suggests that
such sex ratios may encourage competition among males and
discourage spawning by females. With the exception of these
groups, there were no significant differences in the mean
number of eggs produced per female, and the mean number of
eggs per spawn indicating that continuous exposure to atrazine
had no effects on these parameters. Hatchability, survival
and gown of second generation fathead minnows through 30

and 60 days was similar among all treatments.

The available evidence, based on the chronic exposure of
fathead minnows to atrazine, indicates that the maximum
acceptable toxicant concentration is greater than 0.213 mg/l.
Since we observed 25% mortality among 3 to 5 day old fathead
minnow fry during 96 hours exposure to 0.87 mg/l atrazine in
a static system, the estimated maximum acceptable toxicant
concentration of atrazine for the fathead minnow is

between 0.21 and 0.87 mg/l.

Salvelinus fontinalis

Continuous exposure for 44 weeks to mean measured atrazine
concentrations as high as 0.72 mg/l had no significant effect
on survival of brook trout. Continuous exposure to 0.72,
0.45, and 0.24 mg/l atrazine for 90 days significantly reduced
the weight and total length of brook trout when compared to
controls. Total lengths and weights of brook trout fry were
significantly reduced by 306 days exposure to concentrations
of 0.72, 0.45, 0.24 and 0.12 mg/l atrazine (Table 14). Brook
trout in these tanks appeared lethargic when compared to fish
in the controls and those exposed to 0.065 mg/l atrazine, and
did not feed as well.

Spawning activity of yearling brook trout in all experimental
units was delayed approximately one month due to a malfunction
in the lighting system which exposed fish in all tanks to dim
lighting during scheduled night hours. After correcting the
problem, spawning occurred in all tanks between days 222 and
302 of exposure. Total number of eggs spawned, number of eggs
per female, and percent fertilization and hatchability appeared
to be unaffected by exposure to all concentrations of atrazine.
Variability between replicate tanks of the same concentration
precluded ascribing statistical significance to values which
appeared reduced at higher atrazine concentrations ffable 15).
The percentage of eggs developing a neural keel after 15

days was extremely variable between replicate tanks of the

34


SECTION V
RESULTS
ACUTE BIOASSAYS

Analyses of the results of acute static bioassays with the
invertebrate species indicated the 48-hour LC50 (95% confidence
interval) of atrazine for Chironomus, Daphnia, and Gammarus

was 0.72 (0.36-1.44), 6.9 (5.2-8.1), and 5.7 (3.6-8.0) mg/I,
respectively. Mortality was observed with Chironomus exposed

to 0.5 mg/1, with Daphnia exposed to 3.0 mg/I, and with Gammarus
exposed to 2.4 mg/I. ased on these data, the highest nominal
concentrations selected for the chronic exposures were 2.0 mg/1
(Chironomis), 1.2 mg/l Daphnia and 1.0 mg/1 (Gammarus).

A 7-day continuous-flow bioassay conducted at 19+ 1°C with
6.5-gram bluegill indicated the 96-hour LC50 for atrazine and
bluegill was +8.0 mg/l, and the incipient LC50 was 6.7 (5.4-8.4)
mg/l. Fathead minnows were not killed in 8 days by 8 mg/1, the
highest concentration of atrazine that could be maintained in

test chambers in a flow-through test. A 120-hour acute bioassay
conducted at 19 + 1°C with 1.8-gram fathead minnows and utilizing
the 24-hour renewal technique indicated that both the 96-hour

and incipient LC50 values were 15 (11-20) mg/l. An 8-day
continuous-flow bioassay at 13 + 1°C with 52-gram brook trout
indicated the 96-hour LC50 for atrazine and brook trout was

6.3 (4.1-9.7) mg/1, and the incipient LC50 was 4.9 (4.0-6.0)

mg/l. In each of these tests, fish appeared darkened and stressed
at concentrations as low as 1.4 mg/l. In order to select
concentrations for chronic studies which would include safe
concentrations, other acute studies were performed on each species.
Bluegill (15 3) exposed to a nominal concentration of 0.5 mg/1
atrazine for 28 days in a flow-through test became letnargic,

fed poorly, and exhibited partial loss of equilibrium. Toxicity
was dose-related since effects occurred earlier and were more
severe at higher concentrations and no effects were observed at
lower concentrations nor in the controls. We observed 25 percent
mortality among fathead minnow fry (approximately 3-5 day old)
statically exposed to a measured concentration of 0.52 mg/1
atrazine for 96 hours. Mortality was dose-related with more
occurring at higher concentrations and none at lower ones or in
the controls. Based on these data, the highest nominal concentra-
tions selected for chronic exposures were 0.1 mg/1 (bluegill),
0.25 mg/1 (fathead minnow) and 1.0 mg/1 (brook trout).

20


SECTION III
INTRODUCTION

The current concern regarding the protection of aquatic life
in surface waters has prompted evaluation of the effects of
chemicals on aquatic invertebrates and fishes. Much of the
toxicological research with aquatic biota has been limited

to the development of acute toxicity values as a measure of
the effect of chemicals on the biota. More recently,
utilization of the chronic exposure of fishes and aquatic
invertebrates to chemicals has received particular attention
due to the numerous parameters that can be evaluated as
indices of toxic effects (Mount, 1968; Eaton, 1970; Arthur,
1970; McKim and Benoit, 1971; Arthur et al., 1973; Macek

et al., 1975) The "Laboratory Fish Production Index

TULFPI)" as defined by Mount and Stephan (1967) reflects toxic
effects on reproduction, growth, spawning behavior, egg
hatchability, and fry survival. Somewhere between the highest
observed toxicant concentration that has no effect on these
parameters during continuous chronic exposure and the lowest
effect concentration is a theoretical value termed the maximum
acceptable toxicant concentration (MATC).

The occurrence and persistence of chlorinated hydrocarbon
insecticides and heavy metals in surface waters has resulted
in widespread interest in the effect of these chemicals in
aquatic ecosystems. As a result, much information on the
effect of these materials on aquatic organisms has been
generated (Cope, 1966; Johnson, 1968; Eisler, 1973). However,
much less is known of the effects of less persistent
chemicals despite the fact that many of these are utilized
extensively in agriculture. One of the most widely utilized
of these non-persistent chemicals is the herbicide atrazine
(2-chloro-4-ethlyamino-6-isopropylamino-s-triazine) with more
than 100 million pounds being applied annually to agricultural
lands in the United States (Hall et al., 1972).

Past studies with atrazine have been few and consisted of
short-term aqueous exposures which generated acute toxicity
values (Walker, 1964; FWPCA, 1968; Sanders, 1969). Due

to the extensive use of this chemical and lack of information
concerning its long-term, subtle effects on aquatic organisms,
this study was undertaken to determine the MATC of atrazine
for selected aquatic invertebrates and fishes based on
continuous chronic exposure. The organisms selected for this
research effort were:


the previously reported information leads one to the conclusion
that invertebrates generally are more susceptible to chronic
exposure than fishes. Pickering and Thatcher (1970) reported
the MATC of LAS for fathead minnows was >0.63 <1.2 mg/1,
while Arthur (1970) reported the MATC of LAS for Gammarus
seudolimnaeus to be >0.2 <0O.4 indicating that this inverte-
brate species may be about 3 times more susceptible to LAS
than the fathead minnow. Mount and Stephan (1969) reported
the MATC of copper in soft water for fathead minnows was
>10.6 <18.6 nels while Arthur and Leonard (1970) reported
the MATC of copper in soft water for G. pseudolimnaeus was
>4.6 <8.0 indicating again that the invertebrate species may
be at least 2 times more susceptible than the minnow. More
recently, Arthur et al. (1973) have reported that gammarids
may be 3 times more susceptible to chronic exposure to NTA
than were fathead minnows.

Only one similar chronic toxicity study with bluegill has
been previously described (Eaton, 1970). In comparing the
data from our study with those previously reported, it
appears that the bluegill in our study were significantly
larger and older than those in Eaton's work. As a result,
when spawning did occur in our study, we observed more spawns
per female and greater egg production per female than reported
in the previous work. It is extremely unfortunate that the
factors influential in stimulating or inhibiting spawning
activity in our study could not be adequately identified.

The percent hatch of bluegill eggs which we observed
(generally >80%) was comparable to that observed by Eaton
(generally>72%). As in our previous study with lindane
(Macek et al. 1975) we experienced difficulty in maintaining
and feeding bluegill fry during the initial 30-day developmental
period. We attribute this primarily to an inability to
provide adequate amounts of suitable food forms during the
various stages of fry development. Eaton (1970) experienced
the same difficulties as described above and suggested that
lack of suitable food was the primary factor responsible for
poor fry survival. Before additional effort is expended to
investigate chronic toxicity of chemicals to bluegill,
definitive information to determine what are suitable food
sources for developing bluegill fry, and how adequate numbers
of these can be effectively made available to the fry, must

be developed.

The results of our investigation of the chronic toxicity of
atrazine to fathead minnows, though not without its problems,
reinforces our opinion that the species provides the best
opportunity for evaluating chronic toxicity of chemicals to
freshwater fishes. Certainly more work has been reported with

42


eggs from light and minimizing handling of fry. We have
observed that continuous exposure of developing brook trout
eggs to 0.72 and 0.45 mg/l atrazine significantly reduced
incubation time (degree-days). A similar phenomenon has
recently been reported by Halter and Johnson (1974). These
investigators observed that exposure of coho salmon to
Aroclor 1254 resulted in premature hatching of eggs.

The results of the analysis of fish tissue samples to
determine the concentration of atrazine residues showed
that residues in fish tissues were below minimum detectable
limits in all cases. Certainly such findings preclude
concern over bioconcentration of atrazine by fishes, a
subject of considerable concern for other types of pesti-
cides. -These data are, in fact, consistent with other
studies that we have conducted which suggest that biocon-
centration of triazine herbicides by fish is relatively
low (Bionomics, unpublished data).

During the last two years, we have had the opportunity to
investigate the bioconcentration of some 50 pesticides by
bluegill during a minimum of 30 days continuous exposure
(Bionomics, unpublished data). With all but three of the
pesticides studied, we observed bioconcentration factors of
at least 10X, and with more than half of those studied we
observed bioconcentration factors >50X. The absence of
detectable concentrations of atrazine residues from our
experiments suggest that fishes do not bioconcentrate
atrazine to the extent they do many pesticides. Certainly,
bioconcentration of atrazine does not appear as important

a phenomenon as it appears to be for certain chemicals

which have recently been the cause of great concern among
regulatory agencies and environmentalists. For example,
Macek and Korn (1970) reported brook trout continuously
exposed to 3 1 DDT for 120 days concentrated the chemical
approximately 8500X. Dr. R. Reinert, U.S.D.I., Great Lakes
Research Lab., Ann Arbor, Michigan has found that rainbow
trout bioconcentrate methylmercury by as much as 8000X
(personal communication). Hansen et al. (1971) reported
marine fish exposed to polychlorinated biphenyls concentrated
the material 30,000X. Mayer et al. (1975) reported brook
trout exposed continuously to toxaphene concentrate that
chemical 5,000-16,000X.

The value of estimating the MATC for a chemical and an
aquatic organism is clearly demonstrated in the case of
atrazine. All of the previously reported acute toxicity


TABLE 1, PHYSICAL CHARACTERISTICS OF TEST CHAMBERS UTILIZED TO EVALUATE THE CHRONIC
TOXICITY OF ATRAZINE TO FISHES AND FISH FOOD ORGANISMS
Material Dimensions® Water Volume Flow Rate
Species (shape) (cm) Depth (liters) (tank vol./
(om) 24 hours)
Chironomus lass
tentans rectangular) 21x26x18 16.0 7.5 6
Daphnia lass
Magna cylindrical) 17x13.5 14.0 1.8 2
Gammarus lass
fasciatus rectangular) 25x40x21 19.0 16.0 3
Lepomis stainless steel
macrochirus (rectangular) 40x180x30.5 30.5 168.0 7
Pimephales lass
promelas rectangular) 30.5x90x30.5 15.0 41.0 7
Salvelinus stainless steel
fontinalis (rectangular) 40x90x30.5 30.5 84.0 7

Dimensions are height x length x width for rectangular chambers and height x diameter
for cylindrical chambers. .