above. Spawning was monitored for a 62-day period, during which
all possible pairs of unspawned fish in all duplicate aquaria
were spawned. Eggs that sank through a false bottom of 7-mm
square mesh #316 stainless steel screen were collected daily and
treated as described above.

CHEMICAL ANALYSES
Seawater

For each of the three acute tests, water was collected from
each aquarium at the beginning and end of the 96-hour exposure.
Water was collected from alternate duplicate aquaria weekly dur-
ing the chronic tests. Water samples were prepared and analyzed
as follows:

Methoxychlor--

Unfiltered seawater was extracted twice with two 50-mz por-
tions of Nanograde® (Mallinckrodt) dichloromethane. Volumés ex-
tracted were:

Nominal
concentration Volumes

(ug/x) (m2)
6 500
11 300
22 300
45 100
90 100

The combined extracts were dried by elution through anhydrous
sodium sulfate (heated at 100°C for 24 hours), concentrated to
approximately 1 még in a Kuderna-Danish evaporator, and solvent-
exchanged with Nanograde petroleum ether. The extract volumes
were adjusted to obtain a sensitivity of 0.05 ppm (nanograms

[Ing] per uk) by using a Perkin-Elmer Model 2100 gas chromatograph
equipped with a Ni6é3 electron-capture detector.

Operating conditions were:

Column (glass)--2 m X 4 mm ID 3% OV-101 on 80/100 mesh
Gas Chrom Q

Oven temp.--210°C Detector temp.--275°C

Injector temp.--250°C Carrier gas--Nitrogen

Malathion--
Unfiltered seawater samples of 500 mg were extracted twice

with two 50-m% portions of Nanograde dichloromethane. The ex-
tracts were dried by eluting through anhydrous sodium sulfate

9


TABLE 24. PERCENTAGE MORTALITY AND AVERAGE STANDARD LENGTH OF
30-DAY OLD SHEEPSHEAD MINNOW (CYRPINODON VARIEGATUS)
FRY WHICH WERE HATCHED FROM EGGS SPAWNED BY FISH EX-
POSED TO CARBOFURAN FOR 42-95 DAYS IN FLOWING, SYN-
THETIC SEAWATER. MORTALITY AND LENGTH ARE AVERAGES
OF FOUR GROUPS OF 40 FRY EXCEPT AS NOTED.

Concentration Number Mortality Length
(ig/k) of fry (8) (cm)
Control 160 5 1.4

6 160 2 1.3
15 160 8 1.3
23 120 122 1.4
49 80 412 1.4

Agignificantly different from the control.

APPLICATION FACTORS

Application factors were calculated from the results of the
acute and chronic toxicity tests (TABLE 25).

TABLE 25. CONCENTRATIONS (ug/2%) OF THREE PESTICIDES TOXIC TO
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) IN ACUTE
AND CHRONIC TESTS, AND THE RELATIONSHIP OF ACUTE TOX-
IcITY TO CHRONIC TOXICITY.

96~hour LC50

(95% confidence Application
Pesticide limits) MATC limits factor limits?
Methoxychlor 49 >12<23 0.24-0.47
(37-65)
Malathion 51 >4<9 0.08-0.18
(41-63)
Carbofuran 386 >15<23 0.04-0.06
(311-480)

aDerived by dividing the Maximum Acceptable Toxicant Concentra-
tion limits by the 96-hour LC50.

We find no application factors in the literature on which

to base a comparison of the sensitivity of sheepshead minnows
and any freshwater fish to methoxychlor or carbofuran. For

30


TABLE 16. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO MALATHION IN
FLOWING, NATURAL SEAWATER DURING TWO 10-DAY SPAWN-
ING PERIODS. FIVE UNSPAWNED FISH (2 MALES AND 3
FEMALES) WERE PLACED IN A SPAWNING CHAMBER IN EACH
DUPLICATE AQUARIUM, A AND B.

Concentration (ug/2)

Solvent
Day Control Control 4 9 18
A B A B A B A B A B

87-96 154% 747% 9777 626 4422 «445 341% 481 299 353
119-128 538 258 364 325 535 729 738 390 -B 3748

SUBTOTAL 692 1,005 1,141 951 957 1,174 1,079 871 299 727
TOTAL 1,697 2,092 2,131 1,950 1,026

@peath(s) occurred in spawning chamber.
bNo spawning chamber; 50% mortality had occurred.

TABLE 17. NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) EXPOSED TO MALATHION
IN FLOWING, NATURAL SEAWATER DURING TWO 10-DAY SPAWN-
ING PERIODS. FIVE UNSPAWNED FISH (2 MALES AND 3 FE-
MALES) WERE PLACED IN A SPAWNING CHAMBER IN EACH
DUPLICATE AQUARIUM, A AND B.

Concentration (ig/2)

Solvent
Day Control Control 4 9 18
AB AB A B A B A B
87-96 11 30 26 21 14 15 16 21 10 21
119-128 18 9 12 11 18 24 25 13 -2 19
Average of
duplicate 14 20 19 16 16 20 20 17 10 #16
Mean of
treatment 1729 18+7 1844 19+5 1445

8No spawning chamber; 50% mortality had occurred.

Malathion did not affect hatching success of fry from eggs

26


Mount, D.I. 1968. Chronic toxicity of copper to fathead minnows

(Pimephales promelas, Rafinesque). Water Research, 2:215-
223.

Parkhurst, Z.E., and H.E. Johnson. 1955. Toxicity of malathion
500 to Fall Chinook salmon fingerlings. The Progressive
Fish Culturist, 17(3):113-116.

Parrish, P.R., J.A. Couch, J. Forester, J.M. Patrick, Jr., and
G.H. Cook. 1973. Dieldrin: effects on several estuarine
organisms. Proceedings of the 27th Annual Conference of
the Southeastern Association of Game and Fish Commissioners.
p. 427-434.

Post, G., and T. Schroeder. 1971. The toxicity of four insecti-
cides to four salmonid species. Bulletin of Environmental
Contamination and Toxicology, 6(2):144-155.

Schimmel, S.C., and D.J. Hansen. 1975. Sheepshead minnows
(Cyprinodon variegatus): an estuarine fish suitable for
chronic (entire life-cycle) bioassays. Proceedings of the
28th Annual Conference of Southeastern Association of Game
and Fish Commissioners. p. 392-398.

Schimmel, S.C., P.R. Parrish, D.J. Hansen, J.M. Patrick, Jr., and
J. Forester. 1975. Endrin: effects on several estuarine
organisms. Proceedings of the 28th Annual Conference of the
Southeastern Association of Game and Fish Commissioners.

(In press.)

Sokol, R.R., and J.R. Rohlf. 1973. Introduction to Biostatis-—
tics. W.H. Freeman and Company, San Fransicso. 368 p.

Sprague, J.B. 1969. I. Review Paper: measurement of pollutant
toxicity to fish. I. Bioassay methods for acute toxicity.
Water Research, 3(11) :793-821.

U.S. Environmental Protection Agency. 1971. Method for organic
pesticides in water and wastewater. National Environmental
Research Center, Cincinnati, Ohio. 57 p.

U.S. Environmental Protection Agency. 1974. Analysis of pesti-
cide residues in human and environmental samples. Pesti-
cides and Toxic Substances Effect Laboratory, National
Environmental Research Center, Research Triangle Park,
North Carolina.

U.S. Environmental Protection Agency. 1975. Methods for acute

toxicity tests with fish, macroinvertebrates, and amphi-
bians. Ecological Research Series EPA-600/3-75-009. 61 p.

35


SECTION 2

CONCLUSIONS

Sheepshead minnows (Cyprinodon variegatus) are suitable test
animals for toxicity tests which include the reproductive portion
of the life cycle and the critical life stages (embryos and fry)
of the successive generation.

Tests with these saltwater fish are practical means of de-
termining maximum acceptable toxicant concentrations and appli-
cation factors because of (a) the amenability of sheepshead
minnows to laboratory culture and (b) the relatively short peri-
od of time required to reach sexual maturity and complete the
reproductive phase of the life cycle.

The application factor limits derived for sheepshead minnows
exposed to malathion are very similar to the application factor
limits derived for two freshwater fishes, indicating that this
saltwater fish may be used effectively to obtain data on which
to base water quality criteria.


Recorder--Leeds & Northrup dual pen, 0-1 mv range, 1.0 cm/min.
chart speed

Response--0.30 ng of malathion gave half-scale recorder pen
deflection using the N/P detector at an attenuation
of 16 X 1. Retention time was 2.0 min.

Three whole fish, weighing approximately 1 g each, were
spiked with 100 ng of malathion and were analyzed by the above
method. The average percentage recovery of malathion was 103+
6.8%. The analytical results were not corrected for recovery
which was considered quantitative.

Carbofuran--

Results of previous research in which fish were continu-
ously exposed to radiolabeled C14 carbofuran for 28 days indi-
cated that the maximum tissue concentrations were reached within
3-10 days, after which an equilibrium concentration was observed.
A concentration factor of 5-20X was calculated (FMC,1976). In
view of the rapid equilibrium, the low concentration factor, and
the absence of a routine gas-chromatographic analytical method
for fish tissues, fish from the carbofuran chronic test were not
analyzed for residues.

STATISTICAL ANALYSES

In the acute tests, the LC50's and 95% confidence limits
were calculated by linear regression analysis after probit trans-
formation (Finney, 1971).

In the chronic tests, differences between treatments were
determined by chi-square (X?) and analysis of variance (Sokol
and Rohlf, 1973). Differences were considered significant at
the 95% (P<0.05) confidence level. Post-hoc tests were con-
ducted on treatment means by using the Student-Newman-Keuls
range test (Keuls, 1952).

13


malathion, however, studies by Mount and Stephan (1967) with

fathead minnows and Eaton (1970) with bluegill show that the

application factors derived for all three fishes were similar
(TABLE 26).

TABLE 26. COMPARISON OF ACUTE AND CHRONIC MALATHION TOXICITY
TO TWO FRESHWATER FISHES AND A SALTWATER FISH.

Fathead Bluegills? Sheepshead
minnows? minnows
96-hour LC50
(g/2) 9,000 108 51
TC limit
g/t) 200-580 4-7 4-9
Application 0.02-0.06 0.04-0.06 0.08-0.18

factors limits

4From Mount and Stephan, 1967.
brrom Eaton, 1970.

SUMMARY

1. Carbofuran was less toxic to sheepshead minnows than were
methoxychlor and malathion in acute tests. Estimated 96-
hour LC50's, based on average measured concentrations in
water, were 386, 49, and 51 ug/k, respectively.

2. All three pesticides killed parental fish in concentrations
<50 ug/g during chronic tests, and the lowest concentrations
of the pesticides in which toxic effects were observed were
similar (TABLE 27).

3. The life stages of progeny from exposed parental fish that
were sensitive to each pesticide were: methoxychlor--embryo;
malathion--fry; and carbofuran--embryo and fry.

4. The relationship of acute toxicity and chronic toxicity for
sheepshead minnows exposed to malathion (as expressed by ap-
plication factors) was similar to that for two freshwater
fishes exposed to malathion.

5. Sheepshead minnows are a suitable estuarine fish for toxi-

city tests which include the reproductive portion of the
life cycle and the first generation.

31


FOREWARD

The protection of our estuarine and coastal areas from damage caused
by toxic organic pollutants requires that regulations restricting the
introduction of these compounds into the environment be formulated on a
sound scientific basis. Accurate information describing dose-response
relationships for organisms and ecosystems under varying conditions is
required. The Environmental Research Laboratory, Gulf Breeze, contributes
to this information through research programs aimed at determining:

the effects of toxic organic pollutants on individual species and
communities or organisms;

the effects of toxic organics on ecosystem processes and components;

the significance of chemical carcinogens in the estuarine and marine
environments.

This report describes effects of three insecticides in partial life-
cycle tests with an estuarine fish, the sheepshead minnow. The data will
be useful in establishing estuarine water quality criteria, and limiting
effluents containing carbofuran, malathion, or methoxychlor.

Thomas W. Duke
Director
Environmental Research Laboratory

iti


TABLE 12. CONCENTRATIONS OF METHOXYCHLOR IN SURVIVING ADULT
SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED
FOR 140 DAYS. MEAN TISSUE RESIDUES ARE WHOLE-BODY,
WET-WEIGHT. DUPLICATE ANALYSES OF EACH POOLED SAMPLE
(AT LEAST 2 FISH PER SAMPLE) WERE PERFORMED.

Concentration
Water Tissue Concentration Number
(ug/k) (ug/g) factor of samples
Control <0.1 - 4
Sol. control <0.1 - 4
3 0.34+0.24 113 6
5 1.32+0.24 264 6
12 1.38+0.25 115 6
23 3.18+0.53 138 6

TABLE 13. ACCUMULATION OF ORGANOCHLORINE PESTICIDES BY MARINE
FISHES. CONCENTRATION FACTORS WERE DERIVED BY DI-
VIDING CONCENTRATIONS IN FISH (WHOLE-BODY, WET-WEIGHT)
BY CONCENTRATIONS IN TEST WATER.

Concentration
Pesticide Fish factor Exposure Source
(maximum) (days)
DDT Atlantic 16,3004 21-35 Hansen and Wilson,

croaker 1970
Pinfish 40,0002 14

Dieldrin Spot 6,700? 35 Parrish et al.,

1973

Endrin Sheepshead 4,800) 33 Schimmel et al.,

minnows °1975

@Based on nominal water concentration.
Based on measured water concentration.

Malathion

Malathion affected survival of parental fish but did not
affect their growth or fecundity (TABLES 14-17). Exposure to
86 ug/& was lethal to 100% of the fish in one duplicate and 953

24


(heated at 100°C for 24 hours) and concentrated in a Kuderna-
Danish evaporator. The extract volumes were adjusted to obtain
a sensitivity of 1.0 ppm by using a Perkin Elmer Model 2100 gas
chromatograph equipped with a flame photometric detector oper-
ating in the phosphorus mode.

Operating conditions were:

Column (glass)--2 m X 4 mm ID 3% OV-101 on 80/100 mesh
Gas Chrom Q

Oven temp.--210°C Bead setting--500
Injector temp.--250°C Carrier gas--10% argon/methane
Carbofuran--

Unfiltered water samples (approximately 500 mz) were mea-
sured volumetrically in a graduated cylinder and placed in a 1-2
separatory funnel equipped with a Teflon® stopcock. The water
was extracted three times with separate 30-mg portions of Nano-
grade dichloromethane and the combined extract was passed through
an anhydrous sodium sulfate column to remove moisture from the
solvent. The sodium sulfate was rinsed with a portion of di-
chloromethane and the extract and rinse were placed in a Kuderna-
Danish evaporator equipped with a three-ball Synder column. The
solvent was evaporated to approximately 3 m& over an 80°C water
bath, the extract was transferred to a 15-mg% centrifuge tube with
a Teflon-lined cap, and evaporated to dryness at room temperature
by using a gentle stream of clean dry air. The extract was then
dissolved in an accurately known volume of Nanograde benzene and
stored in a freezer at 15°C prior to analysis by gas/liquid chro-
matography under the following conditions:

Instrument--Perkin-Elmer Model 3920 gas chromatograph
Detector--Nitrogen/phosphorus thermionic detection

Column--0.6 m X 2 mm ID glass packed with 20% SE-30 coated on 60/
80 Chromasorb W. The column was conditioned at 235°C for
two weeks prior to use. Several injections (3 X 50 uz)
of Silyl-8, a column-silanizing agent, were made ov@r the
two-week conditioning period.

Gas flows--38 cubic centimeters (cc) N,/minute (min.) carrier,
7 cc Ho/min. and 100 cc air/min. to the N/P detector.

Temperatures--Injection port: 225°C
Column: 155°C
Transfer line: 245°C

Recorder--Leeds & Northrup dual pen, 0-1 mV range, 5 mm/min.
chart speed

10


TABLE 3. NOMINAL AND MEASURED CONCENTRATIONS OF CARBOFURAN DUR-
ING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.

Concentration (ug/2)
Test Nominal Measured
OQ hour 96 hour Mean % of nom.

Acute Control <0.1 <0.1 - -
420 230 120 175 44
560 380 270 325 58
750 480 220 350 47
1,000 640 280 460 46
1,300 860 760 810 62
Mean S.D. Range % of nom. # samples
Chronic Control <0.1 - 2-12 - 7
Sol. Control -3 - - - -
31 6 +4 2-12 19 7
62 15 #11 1-29 24 10
125 23 +21 1-65 18 12
250 49 +44 2-150 20 12
500 100 +93 20-270 20 10

4Not analyzed.

TABLE 4. ANALYSIS OF PARENT AND HYDROLYZED CARBOFURAN IN SEA-
WATER SAMPLES COLLECTED FROM THE NOMINAL CONCENTRATION
OF 500 ug/2 DURING A 131-DAY EXPOSURE OF SHEEPSHEAD
MINNOWS (CYPRINODON VARIEGATUS) .

Parent
Test Carbofuran Hydrolyzed
Day (ug/tsppb) Carbofuran®

12 200 <1.0
27 120 <1.0
34 210 6.1
41 79 <1.0
55 29 <1.0
62 37 2.1
70 28 9.5
76 38 10.0
84 85 17.0
105 20 18.1

@The percentage recovery of the hydrolyzed carbofuran from water
is unknown. Therefore, these values are relative to each other.

16


receiver to dissolve the residue.

An aliquot of the extract was analyzed by gas chromatography
under the following operating conditions:

Instrument--Tracor Model MT-550 gas chromatograph
Detector--Electron capture with 15 millicuries of Ni63

Column--2 m X 2 mm ID glass packed with 3% OV-101 on 100/120
mesh HMDS-treated Supelcoport

Gas*flows--30 cc N2/min. carrier, 60 cc Nj/min. scavenger

Temperatures--Column: 200°C Inlet: 230°C
Transfer: 270°C Detector: 302°C

Recorder--Corning Model 841, 0-1 mV, 0.5 cm/min. chart speed

Response--2.5 ng of methoxychlor gave half~scale pen deflection
at an attenuation of 1.6 X 1079 amperes

Three fish, weighing approximately 1 g each, were spiked
with 100 ng of methoxychlor and were analyzed by the above
method. The average precentage recovery of methoxychlor was
10024.6%. The analytical results were not corrected for re-
covery which was considered quantitative.

Malathion--

Tissues were analyzed in the manner described for methoxy-
chlor except that malathion eluted from the florisil column
quantitatively in the 1:1 ethyl ether-in-petroleum ether frac-
tion. An aliquot of the extract was analyzed by gas chromatog-
raphy under the following operating conditions:

Instrument--Perkin-Elmer Model 3920 gas chromatograph

Detectors--Electron capture with 15 millicuries of Nié3 and
nitrogen/phosphorus thermionic detection

Effluent Splitter--10 parts to N/P and 3 parts to ECD

Column--2 m X 2 mm ID glass packed with 3% Dexsil 300 GC on 80/
100 mesh DMHS-treated Supelcoport

Gas flows--36 cc N,/min. carrier, 7 cc Nj/min. and 100 cc air/
min. to the N/P detector

Temperatures--Injection port: 250°C

Column: 220°C
Transfer line and splitter: 265°C

12


in the other after 5 days; exposure to 37 ug/2% was lethal to 100%
in one duplicate and 80% in the other after 30 days. Exposure

to 18 ug/2

was lethal to 50% of the fish in both duplicates af-

ter 86 days. No deaths occurred in any concentration or control
after day 90 of the 140-day study.

TABLE 14. PERCENTAGE MORTALITY OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO MALATHION IN
FLOWING, NATURAL SEAWATER FOR 140 DAYS. MORTALITY
IS THE AVERAGE FROM DUPLICATE AQUARIA AND DOES NOT
INCLUDE DEATHS WHICH OCCURRED IN THE SPAWNING CHAM-
BERS.

Concentration (ug/2)
Solvent

Day Control Control 4 9 18) 37 86

1-30 0 0 0 0 5 90 100
31-60 0 0 0 0 23 8 -
61-90 2 0 5 0 22 2 -
91-120 0) 0 0 0 0 - -
121-149 0 0 0 0 0 - -
Total 2 0 5 0 504 100% 100%

Asignificantly different from the control.

TABLE 15. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 140 DAYS TO MALATHION IN FLOWING, NAT-
URAL SEAWATER. MEAN STANDARD LENGTH AND STANDARD
DEVIATION ARE GIVEN IN CENTIMETERS AND WERE DETER-
MINED PHOTOGRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN
GRAMS AND WAS DETERMINED IN WATER.

Concentration Day 0 Day 61 Day 140
(ug/2) Length Wt. Length Wt. Length wt.
Control 1.4+0.2 0.1 2.840.4 0.7 4,140.4 1.4

Sol. control 1,340.2 0.1 2.740.6 0.8 4,140.5 1.4

4 1,440.2 0.2 2.740.3 0.7 3.940.4 1.2
9 1,340.2 0.1 2.840.3 0.7 3.940.4 1.3
18 1.340.2 0.1 2.640.4 0.6 3.740.3 -a
37 1,420.2 0.1 2,440.2 0.5 -b -
86 1.4#0.2 0.1 -b - - -

4n11 fish were used for acetylcholinesterase inhibition

analyses.

ball fish had died.

25


Carbofuran

Fish were obtained from the U. S. Environmental Protection
Agency's Gulf Breeze Laboratory, Sabine Island, Gulf Breeze,
Florida, where they had spawned naturally in outside earthern
ponds. Fish for the acute and chronic tests were 1.8-4.0 cm SL.

TEST METHODS
Acute Tests

All procedures followed methods of APHA et al. (1976) and
U. S. Environmental Protection Agency (1975), except as stated.
The 96-hour tests were conducted in an intermittent-flow system
by ‘using a proportional diluter (Mount and Brungs, 1967) con-
structed to deliver 1 %/cycle at a dilution ratio of 75%. The
average number of cycles was approximately 5/hour, providing 99%
replacement in 24 hours (Sprague, 1969). A mechanical injector
(manufactured by George Frasier, Duluth, MN), equipped with a
50-milliliter (mt) glass syringe and stainless steel needle,
pumped methoxychlor, malathion, or carbofuran stock solution
through polyethylene tubing to the mixing cell. Test containers
were 30 X 30 X 61-cm glass aquaria. Each contained 20 fish and
approximately 28 2% of water.

Chronic Tests

The tests were conducted as described above except that the
proportional diluter was constructed for 50% dilution. It de-
livered 1 %/cycle at a rate of approximately 5 cycles/hour. For
the methoxychlor and malathion tests, the diluter was modified
‘to include a solvent control wherein the same volume of solvent/
carrier (acetone) was added to methoxychlor- or malathion-free
seawater as was added to the highest pesticide concentration.

One injector, equipped with a 30-m glass syringe and a stainless
steel needle, metered the respective stock solutions through
polyethylene tubing into the mixing chamber. A second injector,
equipped with a 50-mg glass syringe with stainless steel needle,
metered acetone to each solvent control. Maximum solvent concen-
tration was 29 us/e (parts per million, ppm).

To begin each test, 20 acclimated fish were impartially se-
lected and placed in the test aquaria (a total of 40 fish per
treatment) after the toxicant delivery system had been opera-
tional for several days. Fish were daily fed flaked commercial
fish food (BiOrell® and Tetramin®) ad libitum. Salinity and dis-
solved oxygen were measured daily throughout the tests. Light
for all tests was provided by two 3.7-m fluorscent bulbs sus-
pended 46 cm above the test containers, providing approximately
1,100 lux incident to the water surface. Photoperiod was 16
hours light, 8 hours dark. Survival was monitored daily by vi-
sually inspecting each test container. Growth was monitored bi-
weekly according to the photographic method of McKim and Benoit

7


Number

21

22

23

24

25

26

27

Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 131 Days to Carbofuran in Flowing,
Synthetic Seawater .-.

Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Carbofuran in

Flowing, Synthetic Seawater.

Hatching Success of Fry from Eggs Spawned by Sheeps-
head Minnows (Cyprinodon variegatus) Exposed to

Carbofuran in Flowing, Synthetic Seawater.

Percentage Mortality and Average Standard Length of

30-Day Old Sheepshead Minnow (Cyprinodon variegatus)

Fry which were Hatched from Eggs Spawned by Fish

Exposed to Carbofuran for 42-95 Days in Flowing,

Synthetic Seawater .

Concentrations (ug/2) of Three Pesticides Toxic to
Sheepshead Minnows (Cyprinodon variegatus) in Acute
and Chronic Tests, and the Relationship of Acute

Toxicity to Chronic Toxicity .

Comparison of Acute and Chronic Malathion Toxicity

to Two Freshwater Fishes and a Saltwater Fish.

Summary of Significant Effects of Methoxychlor,
Malathion, and Carbofuran on Sheepshead Minnows
(Cyprinodon variegatus) duri..g Chronic Exposures

in Flowing Seawater.

viii

28

29

29

30

30

31

32


ez

TABLE 9.

NUMBER OF EGGS SPAWNED PER DAY PER FEMALE SHEEPSHEAD MINNOW

(CYPRINODON VARIEGATUS) EXPOSED TO METHOXYCHLOR IN FLOWING,

NATURAL SEAWATER DURING THREE 10-DAY SPAWNING PERIODS.

FIVE

UNSPAWNED FISH (2 MALES AND 3 FEMALES) WERE PLACED IN A SPAWN-
ING CHAMBER IN EACH DUPLICATE AQUARIUM, A AND B.

Day

54-63
101-110
116-125

Mean of
duplicate

Mean of
treatment

Concentration (yg/k)

Solvent
Control Control 3 5 12 23
AB A BR EBEABEAREAS
15 11 12 27 25 31 2 19 5 13 3 0
26 17 5 39 11 38 16 11 30413 6 19
16 #15 12 31 18 26 20 28 17 8 1 26
19 #14 10 32 18 #32 13 19 8 11 3 18
1745 21413 2549 16+9 10+5 11414



(96-hour LC50 108 ug/2) (Eaton, 1970). Similarly, sheepshead
minnows were more sensitive than were all but one of the seven
estuarine fishes tested under static conditions by Eisler (1970),
including the striped killifish and mummichog, for which the 96-
hour LC50's were 250 and 240 yg/2, respectively.

Carbofuran was of the same order of toxicity to sheepshead
minnows as to three freshwater fishes tested under static condi-
tions. Reported 96-hour TLM's (median tolerance limits; same
as LC50) for rainbow trout (Salmo gardneri), channel catfish
(Ictalurus punctatus), and bluegill were 280, 210, and 240 ug/2,
respectively (Anonymous, 1971).

CHRONIC TOXICITY

Methoxychlor

Methoxychlor affected parental fish in the 140-day study.
Exposure to 48 ug/% was lethal to 100% of the fish in one dupli-
cate after 10 days and after 15 days in the other. Mortality of
fish exposed to 23 ug/k was significantly greater than mortality
of control fish (TABLE 6).

TABLE 6. PERCENTAGE MORTALITY OF PARENTAL SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO METHOXYCHLOR IN
FLOWING SEAWATER. MORTALITY IS THE AVERAGE FROM DU-
PLICATE AQUARIA AND DOES NOT INCLUDE DEATHS WHICH OC-
CURRED IN THE SPAWNING CHAMBERS.

Concentration (ug/k)

Solvent

Da’ Control Control 3 5 12 23. 48

1-30 0 0) 0 2 0 10 100

31-60 0 0 0 0) 0 8 -

61-90 0 0 0 0 0 2 -

91-120 0 0 0 0) 0 0 -
121-140 0 0 0 0 0 0 -
“rotal == 0t~S 0 0 2 0 208 1008

4significantly different from the control.

Growth of parental fish exposed to methoxychlor was not
significantly different from growth of control fish. Although
growth was monitored biweekly, only measurements at the begin-
ning, middle, and end of the exposure are presented (TABLE 7).

19


CONTENTS

Foreword . 6 6 eee ee ee ee ee ee iii

Abstract . 6. 6 ee ee ee ee : : : : : iv
Tables . 6 1 ee ee ee ee ee ee ee ee ee UE
Acknowledgment . 2. 6. 2 1 2 ee eee ee ee ee ee ee

1. Introduction. . 2... 1. wee we ee ee ee ee
2. Conclusions... 2... wwe ee ee ee ee ee
3. Recommendations... . 1... ee eee ee ee eee
4, Materials and Methods

we

Test materials... 1. 1 wee ee ew ee ee 5
Test water... 1 wee ee ee ee eee ee 5
Test animals... 1... ee ee ee ee ee 6
Test methods... . 1 1 ee ee ee ew ee ee 7
Chemical analyses. . . 1. 1. - 1 ee ee ee eee 9
Statistical analyses ........+.-..+-+.. 213
5. Results and Discussion
Chemical analyses. . .....+-..+. ++ ees 14
Acute toxicity ... 1. 2 ee ee ee ee ew ee (18
Chronic toxicity... 1... 1 ee eee ee ee 619
Application factors. . . 1... 6 ee ee ee ee 30
Summary. 2. 6 6 6 ee ee ee ee ee ee ee ee

References . . 2. 6 6 6 ee ee ee ee ee ee ee eee ee 8B


ABSTRACT

Sheepshead minnows (Cyprinodon variegatus) were exposed to
each of three pesticides--methoxychlor, malathion, and carbofu-
ran--in flowing seawater to determine the acute and chronic (par-
tial life-cycle) effects. The calculated 96-hour LC50's and 95%
confidence limits, based on measured concentrations, were:
methoxychlor, 49 micrograms per liter (ug/2), 37-65 wg/%; mala-
thion, 51 ug/kz, 41-63 ug/2; and carbofuran, 386 ug/2, 311-480

ug/h.

Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of methoxychlor 223 yg/2z was significantly
(P<0.05) greater than mortality of control fish during the 140-
day study. Further, hatching success of fry from eggs spawned
by fish exposed to 23 ug/k was significantly less than hatching
success of control fry. The maximum acceptable toxicant concen-
tration (MATC) was estimated to be >12<23 ug/g and the applica-
tion factor limits were 0.24-0.47.

Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of malathion 218 ug/% was significantly
greater than mortality of control fish during the 140-day study.
Mortality of fry hatched from eggs spawned by fish exposed to 9
and 18 ug/% was significantly greater than mortality of control
fry. The MATC was estimated to be >4<9 yg/2 and the application
factor limits were 0.08-0.18.

Mortality of adult sheepshead minnows exposed to mean mea-
sured concentrations of carbofuran 249 yg/2 was significantly
greater than mortality of control fish during the 13l-day study.
Hatching success of fry from eggs spawned by fish exposed to 49
ug/& was significantly less than hatching success of control fry.
Also, mortality of fry hatched from eggs spawned by fish exposed
to 23 and 49 ug/k was significantly greater than control fry
mortality. The MATC was estimated to >15<23 ug/g and the appli-
cation factor limits were 0.04-0.06.

This report was submitted in fulfillment of Contract Number
68-03-0264 by EG&G, Bionomics Marine Research Laboratory, under
the sponsorship of the U.S. Environmental Protection Agency.
This report covers the period 23 May 1973 to 23 May 1975; work
was completed on 1 November 1976.

iv


(1971) and average weight was determined monthly by weighing
each group in water.

Effects of each pesticide on..spawning were determined after
fish began to exhibit signs of sexual maturity.

Methoxychlor and Malathion--

Monitoring of spawning activity was begun on day 54 of the
methoxychlor test and on day 87 of the malathion test. Spawning
chambers were constructed by lacing pieces of 6.5-millimeter (mm)
square mesh #316 stainless steel screen together with #316 stain-
less steel wire. The chambers were 30.5-cm square X 25.5-cm
high, and were supported by 5-cm high extensions of the screen
ends. Beneath each spawning chamber, a 29.5-cm square X 4.7-cm
high egg collection tray was placed to retain the demersal eggs
that sank through the bottom of the spawning chamber. The tray
was constructed of plate glass and silicone sealant, with a 4-cm
wide strip of 480-ym square mesh nylon screen along one side of
the bottom to facilitate consolidation of eggs. Spawning groups,
which consisted of two male and three female fish, were placed in
the spawning chambers for a 10-day period. All possible 2:3
ratios in each aquarium were spawned once and extra, unspawned
fish from each replicate aquarium were combined whenever possible
to form a 2:3 spawning group. Each day, one end of each spawning
chamber was lifted slightly and the egg collection tray was re-
moved from the aquarium. The eggs spawned during the previous
24 hours were washed with seawater, transferred by large-bore
glass pipette into glass Petri dishes, counted, and separated
into groups of 50 eggs. Each 50-egg group was placed in an egg
incubator cup (a 100-mg glass jar with the bottom cut off and
480-um square mesh nylon screen attached with silicone sealant).
Each egg cup was then placed in the same aquarium as the spawn-
ing group which produced it. The egg incubator cups were sus-
pended from a rocker-arm apparatus (Mount, 1968) which gently
oscillated them in the test aquaria. Eggs were removed from each
egg incubator by pipette daily, counted, and the cups washed with
bursts of freshwater to clean the screens. This procedure was
repeated until all living embryos hatched. Then, 40 fry were
placed in glass chambers (14-cm wide X 20.5-cm high X 26-cm long
with 381-ym square mesh #316 stainless steel screen over one end).
Survival was monitored daily and growth (standard length an@ av-
erage weight) was measured after 28 days. At least two groups
of fry per duplicate from each test concentration and controls
were monitored, except in the higher concentrations where toxi-
cant-induced mortality made it impossible to obtain spawning
groups and subsequent eggs and fry.

Carbofuran--

Effects on spawning were determined by monitoring spawning
activity of individual pairs of fish, beginning on day 28. Each
pair was placed in a 14 X 25 X 25-cm glass and #316 stainless
steel mesh spawning chamber similar to the fry chambers described


Number

10

ll

12

13

14

15

16

17

18

19

20

Hatching Success of Fry from Eggs Spawned by Sheeps-
head Minnows (Cyprinodon variegatus) Exposed to
Methoxychlor in Flowing, Natural Seawater. .....

Percentage Mortality, Average Standard Length, and
Weight (Determined In Water) of 28-Day Old Sheeps-
head Minnow (Cyprinodon variegatus) Fry Hatched
from Eggs Spawned by Fish Exposed to Methoxychlor
for 54-63 Days... . . 1. ee ee et

Concentrations of Methoxychlor in Surviving Adult
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed for 140 Days... 2 Dee ee ee

Accumulation of Organochlorine Pesticides by Marine
Fishes... ee ee ee ee ee ee

Percentage Mortality of Sheepshead Minnows (Cyprinodon
variegatus) Exposed to Malathion in Flowing, Natural
Seawater for 140 Days. ..........24.268-.

Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 140 Days to Malathion in Flowing, Nat-
ural Seawater. 2. 2. 6 1 ee ee ee ee ee ee ee

Number of Eggs Spawned by Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Malathion in
Flowing, Natural Seawater during Two 10-Day Spawn-
ing Periods. . 2. 1 1 ee eee ew ee ee ee ee

Number of Eggs Spawned per Day per Female Sheeps-
head Minnow (Cyprinodon variegatus) Exposed to
Malathion in Flowing, Natural Seawater during
Two 10-Day Spawning Periods. ............

Hatching Success of Fry from the Eggs Spawned by
Sheepshead Minnows (Cyprinodon variegatus) Ex-
posed to Malathion in Flowing, Natural Seawater. ..

Percentage Mortality, Average Standard Length, and
Weight (Determined In Water) of 28-Day Old Sheeps-
head Minnow (Cyprinodon variegatus) Fry Hatched
from Eggs Produced by Fish Exposed to Malathion
for 87-96 DayS - 2-6 ee ee ee ee eee ee

Percentage Mortality of Sheepshead Minnows (Cyprinodon
variegatus) Exposed to Carbofuran in Flowing, Syn-
thetic Seawater. . 2 6 6 ee eee et te ee

vii

23

23

24

24

25

25

26

26

27

27

28


SECTION 5

RESULTS AND DISCUSSION

CHEMICAL ANALYSES

Mean measured concentrations of methoxychlor in seawater
were from 57-109% of nominal during the 96-hour test and from
45-55% of nominal during the chronic test (TABLE 1). Because
this chlorinated hydrocarbon pesticide is "essentially insoluble
in water (0.10 mg/g @ 25°C)" (Anonymous, 1972) and because ex-
posure to light and the addition of particulate matter and micro-
organisms hastens its degradation (Gardner and Bailey; 1975),
these mean measured concentrations were within an expected and
acceptable range.

TABLE 1. NOMINAL AND MEASURED CONCENTRATIONS OF METHOXYCHLOR
DURING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MIN-
NOWS (CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.

Concentration (yg/2)

Test Nominal Measured
QO hour 96 hour Mean % of nom.

Acute Control <0.01 <0.01 -

22 13 21 17 77
30 lL 29 20 67
40 25 26 26 65
53 33 35 34 64
70 26 54 40 57
93 67 86 78 84
125 62 209 136 109
Mean S.D. Range % of nom. #4 samples
Chronic Control 0.2 20.5 0-2 - 18
Sol. control -? - - - -
6 3 +2 1-7 50 15
11 5 +4 2-18 45 15
22 12 +7 4-30 55 15
45 23 +19 9-85 51 15
90 48 +10 34-53 53 4

4anot analyzed.

14


Ecological Research Series

XICITY OF METHOXYCHLOR,
ATHION, AND CARBOFURAN TO
"SHEEPSHEAD MINNOWS

(Cyprinodon variegatus}

DS
ee
& &a
SZ
tz
%

Vp
74. pao?

WN agenc

Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561


TECHNICAL RCPORT DATA

(icase read lustruc tons on Uie reverse before comple tines

1, REPORT NO. 2. ° 3 RECIPIENT'S ACCESSION NO,
ERL-GB-0010
a TITLE ANDSUBTITLE—OCOC~C~“—~SSS~S~S~S:S:SCS~SCSCSCS~Ss~CSstCst‘“sSOSSSSCSSCSdS ROA OTE OOOOCOSOSOSOCS

_April 1977 (Issuing Date)

6. PEA FOAMING ORGANIZATION CODE

Chronic Toxicity of Methoxychlor, Malathion,

and Carbofuran to Sheepshead Minnows
Cyprinodon variegatus

T AUTHOR - ; la PEAFOAMING ONGANIZATION REPORT NO
Patrick R. Parrish, Elizabeth E. Dyar, Mark

A. Lindberg, Chiara M. Shanika, and Joanna M.

lg. PERFORMING ONGANIZATION NAME AND AOORESS 10. PROGRAM LLEMENT NO,

EG&G, Bionomics 1EA615

Marine Research Laboratory TY, CONTRACT/GHANT NO

Route 6%, Box 1002

Pensacola, Florida 32507 68-03-0264

12.,SPONSORING AGENCY NAME AND ADDRESS 43. TYPE OF REPORT AND PERIOD COVERED
Environmental Research Laboratory Final

Office of Research and Development 14, SPONSORING AGENCY CODE

U,S. Environmental Protection Agency

Gulf Breeze, Florida 32561 EPA~ORD

15. SUPPLEMENTARY NOTES

76. ABSTRACT

Sheepshead minnows (Cyprinodon variegatus) were exposed to each of
three pesticides--methoxychlor, malathion, and carbofuran--in flowing seay
water to determine the acute and chronic (partial life-cycle) effects.

Mortality of adult fish exposed to concentrations of methoxychlor
223 wg/2 and hatching success of fry from eggs spawned by fish exposed to
23 ug/2 were significantly different from the control. The maximum ac-
ceptable toxicant concentration (MATC) was estimated to be >12<23 ug/2;
application factor (AF) limits were 0.24-0.47.

Mortality of adult fish exposed to concentrations of malathion ‘218
ug/& and mortality of fry hatched from eggs spawned by fish exposed to 9
and 18 yg/2 were significantly different from the control. The MATC was
estimated to >4<9 ug/k; AF limits were 0.08-0.18.

Mortality of adult fish exposed to concentrations of carbofuran 249
ug/2, hatching success of fry from eggs spawned by fish exposed to 49
ug/2, and mortality of fry hatched from eggs spawned by fish exposed to
23 and 49 ug/2 were significantly different from the control. The MATC
was estimated to >15<23 ug/k; AF limits were 0.04-0.06.

17, KEY WOROS ANO DOCUMENT ANALYSIS

a. DESCRIPTORS bIOENTIFIERS/OPEN ENDED TERMS fe. COSATI Fick

Toxicity Chronic toxicity

Fish Flowing seawater

Saltwater Application factor

Pesticides

Methoxychlor

Malathion

Carbofuran

13. DISTRIBUTION STATEMENT : 15. SUGURITY CLASS (Thine hkeports 71_NO, OF PAGS.
Unclassified 36

Release unlimited 20 SECURITY CLASS Whit pace) «22. PRICE
Unclassified L |



SECTION I

INTRODUCTION

These studies were undertaken to gain information about the
effects of three pesticides--methoxychlor, malathion, and carbo-
furan--on sheepshead minnows (Cyprinodon variegatus), a saltwater
fish. Data from these studies can be used to derive application
factors, first described by Mount and Stephan (1967). An appli-
cation factor is obtained by dividing the concentration consid-
ered to be "safe" over a long period by a short-term toxicity
value. The application factor may then be used to establish wa-
ter quality criteria for each pesticide. The use of sheepshead
minnows for long-term, life-cycle tests was proposed by Schimmel
and Hansen (1975) and a tentative method was described by Hansen
and Schimmel (1975). In our short term studies, data on 96-hour
LC50's (the concentration of each pesticide estimated to be lethal
to 50% of the test animals after 96 hours of exposure) were ob-
tained. In our long-term studies, parental sheepshead minnows
were exposed for 28 days or more and then effects on spawning
and progeny were determined. "Safe" concentrations were esti-
mated after evaluation of data on number of eggs spawned by pa-
rental fish, hatching success of embryos, mortality of fry, and
growth of fry.

Methoxychlor is a stable, chlorinated hydrocarbon compound
used to control a wide variety of insects which attack fruits,
vegetables, field and forage crops, and livestock. It is also
used to control certain household and industrial insects. Me-
thoxychlor is a replacement for DDT in many applications (Anon-
ymous, 1972). Methoxychlor is ostensibly insoluble in water but
soluble in lipids (Gardner and Bailey, 1975) and thus might be
expected to accumulate in fish and to be cumulatively toxic dur-
ing long-term exposure.

A recent publication (Gardner and Bailey, op. cit.) provides
an excellent overview of the effects of methoxychlor on environ-
mental quality and states that although there are variances in
estimates of the acute toxicity of methoxychlor to fishes, all
reported LC50's ranged from 5-80 micrograms per liter (ug/2), ex-
cepting three.

We know of only one chronic (partial life-cycle) toxicity
study that has been conducted with methoxychlor and fish (Merna
and Eisele, 1973). Data from that study were insufficient for

a


ACKNOWLEDGMENTS

We thank the Project Officer, Mr. David J. Hansen, for his
guidance and patience during these studies. Thanks to Mr. Terry
A. Hollister, EG&G, Bionomics Marine Research Laboratory, for
his help with statistical analyses, and thanks to Ms. Susan
Walker for typing the manuscript. The assistance of Mr. Kenneth
S. Buxton, EG&G, Bionomics Analytical Chemistry Laboratory, -is
appreciated, as is the review of the manuscript by Kenneth J.
Macek, Ph.D., and Sam R. Petrocelli, Ph.D., EG&G, Bionomics
Aquatic Toxicology Laboratory.

ix


REFERENCES

Anonymous .— 1971. Furadan® Insecticide--Namaticide. FMC Cor-
poration, Niagara Chemical Division, Middleport, New York.
22 p.

Anonymous. 1972. Methoxychlor technical data sheet. E. I. du
Pont de Nemours & Company, Biochemicals Department, Wilming-
ton, Delaware. 4 p.

APHA, AWWA, and WPCF. 1976. Standard methods for the examina-
tion of water and wastewater. (Fourteenth edition.)
American Public Health Association, Washington, D.C. 874 p.

Cook, G.H., and J.C. Moore. 1976. Determination of malathion,
malaoxon, and mono- and dicarboxylic acids of malathion in
fish, oyster, and shrimp tissue. Agricultural and Food
Chemistry, 24(3) :631-634.

Coppage, D.L. 1972. Organophosphate pesticides: specific level
of brain AChE inhibition related to death in sheepshead min-
nows. Transactions of the American Fisheries Society, 101
(3) 534-536.

Coppage, D.L., and E. Matthews. 1974. Short-term effects of
organophosphate pesticides on cholinesterases of estuarine
fishes and pink shrimp. Bulletin of Environmental Con-
tamination and Toxicology, 11(5):483-488.

Eaton, J.G. 1970. Chronic malathion toxicity to the bluegill
(Lepomis macrochirus). Water Research, 4(1):673-684,

Eaton, J.G. 1973. Recent developments in the use of laboratory
bioassays to determine "safe" levels of toxicants for fish.
Bioassay Techniques and Environmental Chemistry. p. 107-
115.

Eisler, R. 1970. Factors affecting pesticide-induced toxicity
in an estuarine fish. Technical Papers for the Bureau of
Sport Fisheries and Wildlife. 20 p.

Finney, D.J. 1971. Probit Analysis. Cambridge University
Press, London. 333 p.

33


T@

TABLE 8.

NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)

EXPOSED TO METHOXYCHLOR IN FLOWING, NATURAL SEAWATER DURING THREE

10-DAY SPAWNING PERIODS.

FIVE UNSPAWNED FISH (2 MALES AND 3 FEMALES)

WERE PLACED IN A SPAWNING CHAMBER IN EACH DUPLICATE AQUARIUM, A AND B.
Concentration (ng/2)
Solvent

Day Control Control 3 12 23
Day A B A B A B A B A B A B
54
to 443 2532 244% 809 746 921 49 575 162 399 91 5
63
101
to 7718 514 867 1,183 3222 991 476 2772, 94 384189 570
110
116
to 3429 413% 3674 934 528 793 563% 829 517 163% 38 1,085
125
OPAL 1,563 1,180 697 2,926 1,596 2,705 1,088 1,681 773 946 318 1,660
TOTAL 2,743 3,623 4,301 2,769 1,719 1,978

8Deaths occurred in spawning chamber.


TABLE 7. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 140 DAYS TO METHOXYCHLOR IN FLOWING, NAT-
URAL SEAWATER. MEAN STANDARD LENGTH AND STANDARD DE-
VIATION ARE GIVEN IN CENTIMETERS AND WERE DETERMINED
PHOTOGRAPHICALLY. AVERAGE WEIGHT IS GIVEN IN GRAMS
AND WAS DETERMINED IN WATER.

Day 0 Day 58 Day 140

Concentration Length Wt. Length wt. Length Wt.
(ug/2) (cm) (g) (cm) (g) (cm) (g).
Control 1,440.2 0.1 2.94+0.2 0.6 3.740.5 1.3
Sol. control 1.440.2 O.1 2.84+0.3 0.6 3.640.4 1.2
3 1.440.2 0.1 2.840.3 0.3 3.540.5 1.2
5 1,440.2 0.1 2.740.3 0.5 3.540.4 1.0

12 1.5+0.2 0.1 2.840.3 0.6 3.540.4 1.2

23 1,540.3 0.1 2.840.4 0.7 3,740.4 1.5

48 1.5+0.2 0.1 -a - - -

4a11 fish had died.

Fecundity (total eggs spawned) of exposed fish was not sig-
nificantly different from that of control fish (TABLE 8). Be-
cause female fish were killed by male fish in spawning chambers
in all treatments except 23 ug/L, we calculated eggs per female
spawning day- These values were obtained by dividing the num-
ber of eggs obtained from a spawning chamber during a 24-hour
period by the number of live female fish in the spawning chamber
during the same period. There was no significant difference be-
tween eggs per female spawning day in any treatment because of
variability within the treatments, but fewer eggs were spawned
per female spawning day by fish exposed to 5, 12, and 23 ug/2%
than were spawned by control fish (TABLE 9).

No female fish were killed by male fish in spawning chambers
in 23 ug/2, although 1 to 6 females were killed in spawning cham-
bers in lower concentrations and controls. Based on observations
of fish in the spawning chambers, a probable reason is that ex-
posure to 23 ug/’ of methoxychlor decreased aggressive spawning
activity of male fish.

Hatching success of fry from eggs spawned by fish exposed
to 23 ug/2t of methoxychlor was significantly less than hatching
success of control fry (TABLE 10).

Neither fry mortality to 28 days posthatch nor growth of

the fry was significantly affected by exposure to methoxychlor
(TABLE 11).

20


TABLES

Number Page
1 Nominal and Measured Concentrations of Methoxychlor
. during Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing Sea-
water. 2. 6 ee ee ee ee ee ee ee ee ee eee (C1

2 Nominal and Measured Concentrations of Malathion
during Acute and Chronic Exposures of Sheeps-
head Minnows (Cyprinodon variegatus) in Flowing
Seawater . 6 6 ee ee ee ee ee ee ee ee eee

3 Nominal and Measured Concentrations of Carbofuran
during Acute and Chronic Exposures of Sheepshead
Minnows (Cyprinodon variegatus) in Flowing Sea-
water. 2 6 6 ee ee ee ee eee eee ee ee ee (16

4 Analysis of Parent and Hydrolyzed Carbofuran in Sea-
water Samples Collected from the Nominal Concen-
tration of 500 ywg/k during a 131-Day Exposure of
Sheepshead Minnows (Cyprinodon variegatus) ..... 16

5 Acute Toxicity of Three Pesticides to Sheepshead
Minnows (Cyprinodon variegatus) Exposed in Flow-
ing Seawater . . 2. 2 1 ew ee ew ee ee ee eee C18

6 Percentage Mortality of Parental Sheepshead Minnows
(Cyprinodon variegatus) Exposed to Methoxychlor
in Flowing Seawater. ....... + ee eee eee 19

7 Growth of Sheepshead Minnows (Cyprinodon variegatus)
Exposed for 140 Days to Methoxychlor in Flowing,

Natural Seawater... 1. 1 ee ee ee ee eee . 20
8 Number of Eggs Spawned by Sheepshead Minnows

(Cyprinodon variegatus) Exposed to Methoxychlor

in Flowing, Natural Seawater during Three 10-Day

Spawning Periods . .... 2... ee eee eee ee 621
9 Number of Eggs Spawned per Day per Female Sheeps-

head Minnow (Cyprinodon variegatus) Exposed to

Methoxychlor in Flowing, Natural Seawater during

Three 10-Day Spawning Periods. .........4.. 22

vi


Response--30 ng of carbofuran and 80 ng of 3-hydroxycarbofuran
gave half-scale response with retention times of 3.4
and 6.4 min., respectively

Extraction efficiency and mean recovery for the analytical
methods were 89.9+9.8% for methoxychlor, 84.5411.7% for mala-
thion, and 79.0+7.3% for carbofuran. Data in this report are
corrected for recovery.

Fish Tissue

Fish were collected for residue analyses as follows:
a. adults alive at the end of the respective exposure;
b. fry alive at the end of the 28-day growth period; and
c. eggs randomly collected during the spawning periods.

Methoxychlor--

Tissues were analyzed by an adaptation of the methods of
U. S. Environmental Protection Agency (1971 and 1974). Fish
tissue was weighed to the nearest 0.01 g in a beaker. The tis-
sue was transferred to a 100-mg graduated cylinder, which had
been cut at the 80-mg mark to reduce the height of the cylinder;
the beaker was rinsed with dichloromethane, which was then added
to the cylinder. The tissue was homogenized with approximately
30 mg of dichloromethane for 20 seconds by using a Brinkman
Polytron Homogenizer, Model PT 10/20.

The homogenate was filtered through No. 3 Whatman paper in-
to a clean beaker, approximately 30 mz of dichloromethane was
added to the graduated cylinder, and the Polytron probe was
rinsed for approximately 10 seconds in the solvent while the
homogenizer was operating at low speed. The probe rinse was
added to the filter and finally the filter was washed with di-
chloromethane,.

The solvent was evaporated to approximately 5 mg over a
steam bath, cooled, and evaporated to dryness at room tempera-
ture by using a gentle stream of clean air. At this point the
extract was cleaned by florisil column chromatography according
to U. S. Environmental Protection Agency (1974). Methoxychlor
eluted quantitatively from the 6% ether-in~petroleum ether frac-
tion and was sufficiently free of interfering substances to
permit analysis by electron capture detection. The 6% ethyl-
petroleum ether fraction containing methoxychlor was evaporated
to approximately 3 mt in a Kuderna-Danish evaporator equipped
with a three-ball Snyder column, and the unit was cooled to room
temperature. The receiver was disconnected, the remainder of
the solvent was evaporated to dryness at room temperature by using
a gentle air flow, and a known volume of hexane was added to the

11


100% of the fish in one duplicate and 95% in the other after 14
days. Mortality of fish exposed to 49 ppb was significantly
greater than mortality of control fish after 30 days of exposure
(TABLE 20).

TABLE 20. PERCENTAGE MORTALITY OF SHEEPSHEAD MINNOWS (CYPRINODON
VARIEGATUS) EXPOSED TO CARBOFURAN IN FLOWING, SYNTHE-
TIC SEAWATER. MORTALITY IS THE AVERAGE FROM DUPLICATE
AQUARIA AND DOES NOT INCLUDE DEATHS FROM SPAWNING

ACTIVITY.
Concentration (yg/2)

Day Control 6 15 23 49 100

1-30 0 5 2 5 40 100
31-60 5 5 8 10 10 -
61-90 2 0 5 0 0 -
91-131 0 0 0 0 0 -
Total 7 zo 15 15 504 1003

Asignificantly different from the control.

Carbofuran did not significantly affect growth of parental
fish or number of eggs spawned in any concentration (TABLES 21-
22).

TABLE 21. GROWTH OF SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS)
EXPOSED FOR 131 DAYS TO CARBOFURAN IN FLOWING, SYN-
THETIC SEAWATER. MEAN STANDARD LENGTH AND STANDARD
DEVIATION ARE GIVEN IN CENTIMETERS AND WERE DETER-
MINED PHOTOGRAPHICALLY. .AVERAGE WEIGHT IS GIVEN IN
GRAMS AND WAS DETERMINED IN WATER.

Concentration Day 2 Day 60 Day 131
(ug/k) Length Length Length Weight

>
Control 2.940.4 3.840.3 4,140.3 1.15
6 3,840.5 3.6+0.4 4.0+0.2 1.10
15 3.140.4 3.640.3 3.940.3 1.16
23 2.840.4 3.2+0.4 3.840.3 0.92
49 2.840.5 3.340.3 3.740.5 1.05

100 3.040.5 -2 - -

#a11 fish had died.

28


TABLE 22. NUMBER OF EGGS SPAWNED BY SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) EXPOSED TO CARBOFURAN IN
FLOWING, SYNTHETIC SEAWATER. ALL POSSIBLE PAIRS
OF FISH IN EACH DUPLICATE AQUARIUM WERE SPAWNED.

Concentration Number of eggs
(ug/2) Replicate A Replicate B Total
Control 650 449 1,099
6 854 852 1,706
15 848 1,295 2,143
23 475 820 1,295
49 248 154 4024

4pifty percent mortality had occurred.

Hatching success of fry from eggs spawned by fish exposed
to 49 wg/k was significantly less than hatching success of con-
trol fry (TABLE 23).

TABLE 23. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-
HEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO CAR~
BOFURAN IN FLOWING, SYNTHETIC SEAWATER. MEAN PER-
CENTAGE HATCH AND STANDARD DEVIATION REPRESENTS
POOLED DATA FROM SPAWNING PAIRS IN DUPLICATE AQUARIA.

Concentration Percentage Number of eggs
(ug/k) hatch examined
Control 9843 450

6 9943 700
15 96+10 937
23 98+2 609
49 864104 233

asignificantly different from the control.

Mortality of fry hatched from eggs spawned by fish exposed
to 23 and 49 ug/2 was significantly greater than mortality of
control fry. Growth of surviving fry in all concentrations was
not affected, however (TABLE 24).

29


Our justification for and methodology of determining the
hydrolyzed products of carbofuran are as follow:

Carbofuran and 3-hydroxycarbofuran were completly hydro-
lyzed within 24 hours when saturated solutions were made pH 9.2
and stored at 25°C (FMC, 1969). Similiar to the base-catalyzed
degradation of Sevin® to l-hydroxynaphthol, carbofuran was ex-
pected to eliminate the methylcarbamate group to form an hydroxy-
sub-stituent at the benzyl-oxygen. Therefore, an analytical pro-
cedure featuring derivatization of l-naphthol was utilized to
verify any degradation products of carbofuran which possess ac-
tive hydroxy-substituents. Parent carbofuran is not detected by
this analyses since it does not contain the reactive hydroxy sub-
stituent.

Approximately 2 g of carbofuran were added to 300 m% of nor-
mal sodium hydroxide and heated to 60°C for six hours. The solu-
tion was cooled, hydrochloric acid was added to adjust the solu-
tion to pH 4.0, and the hydrolyzed carbofuran was extracted into
methylene chloride. The solvent was evaporated and a portion of
the hydrolyzed carbofuran was weighed and dissolved in benzene
to produce the hydrolyzed carbofuran working standard solution.

An analytical procedure (U. S. Environmental Protection
Agency, 1974) designed for the gas chromatographic determination
of l-naphthol in urine, following derivatization with chloroace-
tic anhydride, was utilized to verify the hydrolysis of carbo-
furan. Working standards of hydrolyzed carbofuran were deriva-
tized and chromatographed under operating conditions previously
described (except by using electron capture detection) with the
following results:

Weight of hydrolyzed Peak height response (mm)
Carbofuran/7 mg benzene Retention _ 3 jl Retention — 4.2 mi
(ug) time = 3.0 min. “time = 4.2 min.
0 4 3
1 5 9.5
2 6 18
5 15 4l
10 31 116

An aliquot of the seawater sample extracts (see TABLE 4) was
derivatized and the gas chromatograms were examined for the pre-
sence of quantity of hydrolyzed carbofuran. A graph of peak
height versus weight of hydrolyzed carbofuran was constructed,

17


DISCLAIMER

This report has been reviewed by the Gulf Breeze Environmen-
tal Research Laboratory, 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.


Weiss, C.M. 1961. Physiological effect of organic phosphorus
insecticides on several species of fish. Transactions of
the American Fisheries Society, 9(2):143-152.

Wellborn, T.L., Jr. 1971. Toxicity of some compounds to striped
bass fingerlings. The Progressive Fish Culturist, 33(1) :32-
36.

Westman, J.R., and R. Compton. 1960. Responses of salt marsh
killifishes to certain environmental changes and to mala-
thion. Proceedings of the New Jersey Mosquito Extermination
Association, 47:116-123.

36


FMC. 1969. Personal communication. FMC Corporation, Agricul-
tural Chemical Division, Middleport, New York.

FMC. 1976. Personal communication. FMC Corporation, Agricul-
tural Chemical Division," Middleport, New York.

Gardner, D.R., and J.R. Bailey. 1975. Methoxychlor: Its Ef-
fects on Environmental Quality- NRCC/CNRC Ottawa, Canada.
164 p.

Hansen, D.J., and A.J. Wilson. 1970. Significance of DDT resi-
dues from the estuary near Pensacola, Fla. Pesticides
Monitoring Journal, 4(2):51-56.

Hansen, D.J., E. Matthews, S.L. Nall, and D.P. Dumas. 1972.
Avoidance of pesticides by untrained mosquitofish, Gambusia
affinis. Bulletin of Environmental Contamination and Toxi-
cology, 8(1):46-51.

Hansen, D.J., and S.C. Schimmel. 1975. An entire life-cycle
bioassay using sheepshead minnows (Cyprinodon variegatus).
Federal Register 40(123), part II:26904-26905.

Holland, H.T., and J.I. Lowe. 1966. Malathion: chronic effects
on estuarine fish. Mosquito News, 26(3) :383-385.

Keuls, M. 1952. The use of the studentized range in connection
with an analysis of variance. Euphytica, 1:112-122.

LaRoche, G., R. Eisler, and C.M. Tarzwell. 1970. Bioassay pro-
cedures for oil and dispersant toxicity evaluation. Journal
Water Pollution Control Federation, 42(11):1,982-1,989.

Lewallen, L.L., and W.A. Wilder. 1962. Toxicity of certain or-
ganophosphorus and carbamate insecticides to rainbow trout.
Mosquito News, 22(4) :369-372.

McKim, J.M., and D.A. Benoit. 1971. Effects of long term ex-
posure to copper on survival, growth, and reproduction of
brook trout, Salvelinus fontinalis. Journal Fisheries Re-
search Board of Canada, 28(5) :655-662.

Merna, J.W., and P.J. Eisele. 1973. The effects of methoxychlor
3 aquatic biota. Ecological Research Series EPA-R3-73-046.
io

Mount, D.I., and W.A. Brungs. 1967. A simplified dosing appara-
tus for fish toxicological studies. Water Research, 1:21-29.

Mount, D.I., and C.E. Stephan. 1967. A method for establishing
acceptable toxicant limits for fish - malathion and the bu-
toxyethanol ester of 2,4-D. Transactions of the American
Fisheries Society, 96(2) :185-193.

34


SECTION 3

RECOMMENDATIONS

Spawning groups comprising five sheepshead minnows in the
ratio of 3 female fish:2 male fish are satisfactory to determine
spawning success.

A 10-day spawning period for a spawning group is sufficient
to monitor spawning success.

Studies should be conducted with sheepshead minnows and
other toxicants in chronic (full life-cycle) tests becausé tests
with this saltwater fish appear to provide accurate estimates of
MATC's in a shorter time and with less effort than do tests with
most freshwater fishes.


laboratory. The pump intake was 85 meters (m) offshore at a
depth of approximately 3 m. Water was pumped by a #316 stain-
less steel pump through hard polyvinylchloride (PVC) pipes into
an elevated fiberglass reservoir. En route, the water passed
through a fiberglass, sand filter and a 10-micrometer (ym) poly-
propylene bag filter. From the reservoir, in which the water was
continuously and vigorously aerated, water flowed by gravity
through PVC pipes to the diluters.

No attempt was made to alter the salinity of the water, but
temperature was maintained at 30+1 degrees Celsius (°C) by heat-
ing the incoming seawater in small fiberglass-coated plywood
boxes above the diluters with electric quartz heaters and by
placing test aquaria in constant-temperature water baths.

Carbofuran

All water used for holding, acclimation, and testing was
synthetic seawater, formulated according to the methods of
LaRoche et al. (1970). Freshwater was pumped from a 120-m deep
bed-rock well at the Wareham, Massachusetts, laboratory into two
1,500-2 fiberglass tanks. Ingredients were added, mixed with
freshwater, and aerated. Water was drained from one tank at the
rate of approximately 1,200 2 per day until it was empty. Then,
water was drained from the second tank while fresh synthetic sea~-
water was being prepared in the first tank.

All water flowed by gravity through PVC pipes to the diluter.
Temperature was maintained at 30+1°C by placing the test aquaria
in constant-temperature water baths.

TEST ANIMALS

Methoxychlor and Malathion

All sheepshead minnows used in these studies were collected
from Big Lagoon, near Bionomics Marine Research Laboratory. They
were held in sand-banked ponds on the laboratory grounds and in
fiberglass tanks in the laboratory. All fish were acclimated to
test conditions for 14 days before testing according to the con-
ditions of U. S. Environmental Protection Agency (1975). Mor-
tality was <3% during acclimation. During holding and acclima-
tion, fish were fed frozen or live Artemia salina (San Francisco
Bay Brand) which contained <0.1 ug/g of chlorinated hydrocarbon
pesticides or polychlorinated biphenyls as determined by our
electron-capture gas chromatograph analyses.

Size of fish was: methoxychlor--acute test, 1.7-3.8 centi-
meters (cm) standard length (SL) and chronic test, 1.0-1.9 cm SL
and 0.15 g mean weight (determined in water); malathion-~-acute
test, 0.8-1.8 cm SL and chronic test, 1.0-1.8 cm SL and 0.llg
mean weight (determined in water).


CHRONIC TOXICITY OF METHOXYCHLOR, MALATHION, AND
CARBOFURAN TO SHEEPSHEAD MINNOWS (Cyprinodon variegatus)

by

Patrick R. Parrish, Elizabeth E. Dyar, Mark A. Lindberg,
Chiara M. Shanika, and Joanna M. Enos
EG&G, Bionomics
Marine Research Laboratory
Pensacola, Florida 32507

Contract No. 68-03-0264

Project Officer

David J. Hansen
Environmental Research Laboratory
Gulf Breeze, Florida 32561

ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561


spawned (TABLE 18) but did affect the fry. Mortality
hatched from eggs spawned by fish exposed to 9 and 18
malathion was significantly greater than mortality of
fry. Growth of surviving fry was not affected (TABLE

of fry
ug/k of
control
19).

TABLE 18. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-

HEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO MALA-
THION IN FLOWING, NATURAL SEAWATER. MEAN PERCENTAGE
HATCH AND STANDARD DEVIATION REPRESENTS POOLED DATA
FROM DUPLICATE AQUARIA DURING TWO 10-DAY SPAWNING

PERIODS.

Concentration Percentage Number of eggs
(ug/k) hatch examined
Control 9745 850

Sol. control 9942 700

4 9743 1,000
9 9742 900
18 9646 350

TABLE 19. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND

WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) FRY HATCHED FROM EGGS
PRODUCED BY FISH EXPOSED TO MALATHION FOR 87-96 DAYS.

Concentration Number Mortality Length Weight
(ug/2) of fry (3) (cm) (g)
Control 160 7 1,040.2 0.04

Sol. control 160 10 4 0.03

4 160 9 1.1+0.2 0.04

9 160 14b 1,240.2 0.04

18 120 isb 1.2#0.1 0.03
aNo data.

Significantly different from the control.

Malathion was not detectable (<0.1 ug/g) in fish sampled at

the end of the 140-day study. This is not surprising

because

fish readily convert malathion to the mono- and dicarboxylic

acids of malathion (Cook and Moore, 1976).

Carbofuran

Carbofuran affected survival of parental fish exposed to

the pesticide for 131 days. Exposure to 100 ug/2 was

27

lethal to


Malathion is readily soluble in water and mean measured
concentrations during both the acute and chronic tests reflected
this characteristic; concentrations were 72-143% of nominal
(TABLE 2).

TABLE 2. NOMINAL AND MEASURED CONCENTRATIONS OF MALATHION DUR-
ING ACUTE AND CHRONIC EXPOSURES OF SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) IN FLOWING SEAWATER.

Concentration (ug/2)
Test Nominal Measured
QO hour 96 hour Mean 3 of nom.

Acute Control <O.1 <0.1 -

22 16 16 16 72
30 26 27 26 87
40 25 37 31 78
53 43 50 46 89
70 62 67 64 91
94 78 99 88 95
125 108 lll 109 87
Mean S.D. Range % of nom. # samples
Chronic Control <0.1 - - - 19
Sol. control a - - - -
4 4 +2 1-6 100 20
8 9 +4 4-17 112 20
15 18 +6 8-28 120 20
30 37 +12 20-57 123 10
60 86 +15 70-101 143 3

Anot analyzed.

Mean measured concentrations of parent carbofuran were from
44-62% of nominal during the acute test and from 18-24% of nomi-
nal during the chronic test (TABLE 3). Evaluation of the analy-
ses of water samples collected during days 1-30 of the chronic
test shows that concentrations of parent carbofuran were approxi-
mately 40% of nominal. Thereafter, despite the use of a flowing-
water exposure system, measured concentrations of parent material
decreased to approximately 10% of nominal. Concurrently, however,
we observed a pattern of increasing concentrations of hydrolyzed
carbofuran derivitives (TABLE 4). The rapid decline of measured
concentrations of parent material in the 4-day acute test and the
stability of parent carbofuran in a stock solution over a 12-day
period (confirmed by chemical analyses) is further evidence that
carbofuran was degraded in seawater.

15


RESEARCH REPORTING SERIES

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

Environmental Health Effects Research

Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)
Interagency Energy-Environment Research and Development
“Special” Reports

9. Miscellaneous Reports

ONOTDSwON=

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

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


TABLE 10. HATCHING SUCCESS OF FRY FROM EGGS SPAWNED BY SHEEPS-
HEAD MINNOWS (CYPRINODON VARIEGATUS) EXPOSED TO
METHOXYCHLOR IN FLOWING, NATURAL SEAWATER. MEAN PER-
CENTAGE HATCH AND STANDARD DEVIATION REPRESENTS POOLED
DATA FROM DUPLICATE AQUARIA DURING THREE 10-DAY SPAWN-
ING PERIODS.

Concentration Mean percentage Numbers of eggs
(ug/k) hatch and S.D. examined
Control 9843 1,200

Sol. control 98+4 2,040

3 9843 2,400
5 9546 1,450
12 9742 700
23 732187 1,055

asignificantly different from the control.

TABLE 11. PERCENTAGE MORTALITY, AVERAGE STANDARD LENGTH, AND
WEIGHT (DETERMINED IN WATER) OF 28-DAY OLD SHEEPSHEAD
MINNOW (CYPRINODON VARIEGATUS) FRY HATCHED FROM EGGS
SPAWNED BY FISH =XPOSED TO METHOXYCHLOR FOR 54-63
DAYS.

Concentration Number Mortality Length Weight

(ug/2) of fry (8) (cm) (g)
Control 160 0 1.4+0.1 0.07
Sol. control 160 0 1.440.1 0.05
3 160 1 1,440.1 0.07

5 80 0 1,340.1 0.06

12 120 4 1.4+0.1 0.06

23 59 4 1.540.2 0.06

Methoxychlor was accumulated by adult fish exposed continu-
ously for 140 days. The pesticide was also accumulated in eggs
spawned by these fish. Accumulation was dependent upon water
concentration during exposure (TABLE 12). Concentration factors
(based on measured water concentrations) ranged from 113-264.
These values are much lower than concentration factors for other
chlorinated hydrocarbon pesticides and marine fishes (TABLE 13).
A maximum concentration of 1.1 ug/g was detected in eggs spawned
by fish exposed to 12 ug/2.

23


TABLE 27. SUMMARY OF SIGNIFICANT EFFECTS OF METHOXYCHLOR,
MALATHION, AND CARBOFURAN ON SHEEPSHEAD MINNOWS
(CYPRINODON VARIEGATUS) DURING CHRONIC EXPOSURES
IN FLOWING SEAWATER.

Measured.
concentration
Generation Life stage Effect (ug/2)
Methoxychlor Parental Adult Death 223
Fi Embryo Decreased 23
hatch

Malathion Parental Adult Death 218

Fy Fry Increased 9 and 18
mortality

Carbofuran Parental Adult Death 249

Fy Embryo Decreased 49
hatch
Fry Increased 23
mortality

32


by using the peak eluting in 4.2 minutes, and any hydrolyzed
carbofuran found was determined with the graph. The data are
presented as relative concentrations found in the seawater since
the extraction efficiency of the hydrolyzed carbofuran moiety
from the seawater was unknown.

The concentrations of hydrolyzed carbofuran in seawater
continued to increase during the test period. It is important
to note that sample extracts were not treated with an aqueous
base, but were derivatized directly. Therefore, any hydrolysis
product of carbofuran detected was extracted from the seawater
sample. Additionally, parent carbofuran working standards sur-
vived the derivatization procedure virtually unchanged and the
peaks at 3.0 and 4.2 minutes due to hydrolyzed carbofuran were
not observed.

ACUTE TOXICITY

The acute toxicity of methoxychlor and malathion to sheeps-
head minnows was similar; carbofuran was one order of magnitude
less toxic (TABLE 5).

TABLE 5. ACUTE TOXICITY OF THREE PESTICIDES TO SHEEPSHEAD MIN-
NOWS (CYPRINODON VARIEGATUS) EXPOSED IN FLOWING SEA-
WATER. SEAWATER FOR THE METHOXYCHLOR AND MALATHION
TESTS WAS NATURAL; THAT FOR THE CARBOFURAN TEST WAS
SYNTHETIC. CALCULATIONS WERE BASED ON MEASURED CON-
CENTRATIONS OF EACH PESTICIDE.

Compound 96-hour 95% confidence Salinity Temperature
LC50, ug/k limits, wg/2 (2/00) (°C)
Methoxychlor 49 37-65 23 30
Malathion 51 41-63 20 29
Carbofuran 386 311-480 21 22

The acute toxicity of methoxychlor to sheepshead minnows
tested under dynamic conditions was within the range reported
for other estuarine fishes under static conditions, where esti-
mated 96-hour LC50's ranged from 12-150 ug/%. In static tests
with two cyprinodontid fishes, Eisler (1970) estimated 96-hour
LC50 values of 30 and 36 ug/k for the striped killifish
(Fundulus majalis) and mummichog (F. heteroclitus), respectively.

In flowing water tests, malathion was more acutely toxic to

sheepshead minnows than to the freshwater fathead minnow (96-
hour LC50 9,000 ug/2) (Mount and Stephan, 1967) or bluegill

18


deriving an application factor.

Malathion is an organophosphate pesticide used throughout
the U. S. to control a variety of pests. A major use of mala-
thion is for mosquito control in both freshwater and estuarine
areas. Several studies (Parkhurst and Johnson, 1955; Westman
and Compton, 1960; Weiss, 1961; Lewallen and Wilder, 1962;
Holland and Lowe, 1966; Wellborn, 1971; and Post and Schroeder,
1971) have shown that malathion is acutely lethal to a variety
of freshwater and saltwater fishes under both field and labora~
tory test conditions. Sublethal effects (avoidance and inhibi-
tion of brain acetylcholinesterase) have also been observed in
fishes exposed to malathion (Hansen et al., 1972; Coppage, 1972;
and Coppage and Matthews, 1972).

Chronic studies have been conducted with malathion and two
freshwater fishes, fathead minnows (Pimephales promelas) (Mount
and Stephan, 1967), and bluegill (Lepomis macrochirus) (Eaton,
1970). The studies showed the application factor limits for the
two fishes to be very similar, ranging from 0.02-0.06.

Carbofuran is a carbamate pesticide utilized both as a con-
tact poison or as a soil-applied systemic poison. It is regis-
tered for soil-applied use on a variety of crops, including rice,
and for direct use on several insects, including mosquitoes
(Anonymous, 1971). Little research has been performed on the
effects of carbofuran on aquatic organisms.

Carbofuran was the first compound tested. Tests were con-
ducted from March-July 1974 in the EG&G, Bionomics Aquatic Toxi-
cology Laboratory, Wareham, Massachusetts. Our new laboratory
was opened in January 1975, and tests with methoxychlor and mala-
thion were conducted from June-October 1975, at EG&G, Bionomics
Marine Research Laboratory, Pensacola, Florida.


SECTION 4

MATERIALS AND METHODS

TEST MATERIALS

Methoxychlor used in this study was obtained from E. I. du
Pont de Nemours & Company, Biochemicals Department, Wilmington,
Delaware. It was contained in a plastic jar labeled "Methoxy-

chlor Technical, 1 Kg." Although active ingredient was not
listed on the label, a technical data sheet dated April 1972
which accompanied the chemical stated that "...methoxychlor tech-

nical...contains 88% (minimum) 2,2-bis-(p-methoxyphenyl)-1, 1,
1-trichloroethane and 12% (maximum) other isomers and reaction
products.

Malathion was obtained from the American Cyanamid Company,
Agricultural Division, Princeton, New Jersey. It was contained
in a metal bottle, apparently aluminum, with a transluscent cap.
The bottle was refrigerated at all times. The material was la-
beled "MALATHION Technical, Active Ingredient: Malathion* 953%;
Inert Ingredients 5%. *0,0-dimethyl phosphorodithioate of di-
ethyl mercaptosuccinate; (1 Gallon contains 9.7 1b of malathion)."

Carbofuran was obtained from FMC Corporation, Agricultural
Chemical Division, Middleport, New York. It was contained in a
plastic bag labeled "Carbofuran Technical (99%), FURADAN® In-
secticide, Mr L514, 2Kg. C4717-54-A, 9/14/73."

Concentrations of each pesticide are reported here as micro-
grams (ug) of the technical material described above per liter
(%) of seawater.

Stock solutions of all three pesticides were prepared on a
weight:volume basis by dissolving them in reagent grade acetone.
These 1-2 stock solutions were placed in amber glass bottles and
stored in the laboratory. New stock solutions were prepared as
required.

TEST WATER

Methoxychlor and Malathion

All water used for holding, acclimation, and testing was
natural seawater which was pumped from Big Lagoon into the

5