SCIENTIFIC DATA | TECHNICAL INFORMATION
To Enable
THE ENVIRONMENTAL PROTECTION AGENCY
TO DEVELOP REGISTRATION GUIDELINES FOR
INSECT GROWTH REGULATORS § PHEROMONES
INSECT GROWTH REGULATOR SUPPLEMENT
CRUSTACEAN CHRONIC HAZARD ASSESSMENT
Contract 68-01-2444
January 1976
Prepared by
ZOECON CORPORATION
975 California Avenue
Palo Alto, CA 94304
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INDEX TO PART VII-B
HAZARD EVALUATION SUPPLEMENT
Page
INTRODUCTION . VII-64B
BIOASSAY OF INSECT GROWTH REGULATORS WITH
DAPBIIIA MAGNA VII-67B
Abstract of Results , . VII-67B
Report of Bionomics Experiment ....... VII-70B
Report of Zoecon Morphological Observations on
Treated Daphnia magna ............ VII-103B
BIOASSAY OF INSECT GROWTH REGULATORS WITH
PALASHONETES PUG10 VII-125B
Abstract of Results VII-125B
Report of Bionomics Experiments VII-127B
Report of Zoecon Morphological Observations on
Treated Palaemonetes pugio ......... VII-165B
RECOMMENDATIONS FOR FUTURE EPA WORK IN THE AREA OF
CRUSTACEAN TOXICOLOGY- .............. VII-172B
RECOMMENDATIONS FOR REGISTRATION GUIDELINES FOR
INSECT GROWTH REGULATORS WITH RESPECT TO CRUSTACEAN
HAZARD ASSESSMENT VII-173B
VII-63B
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PART VII - HAZARD EVALUATION SUPPLEMENT
INTRODUCTION: Crustacean Chronic Hazard Assessment of
Insect Growth Regulators
Insect growth regulators by design have indirect
lethal effects. By definition the effects are delayed
and dependent upon the developmental state of the
insect. Owing to some expectation for similarity of
biochemical systems of Insecta and Crustacea, it seemed
prudent to portend the effects of insect growth regulators
on crustacean species of importance in the wild with
studies of model Crustacea. To provide a practicable
bioassay as a predictive tool which could be established
as a registration requirement, it was desirable, if
possible, (as suggested by EPA scientists) to elucidate
a "rapid safety screen". To this end the approach of
looking only at that development period in crustacean
organisms which corresponded to the sensitive stage
in various insects was considered. This approach was
eventually discarded when Zoecon entomologists and
insect physiologists met with aquatic biologists from
contract laboratories and agreed upon the impossibility
of correlating development stages with any confidence.
Despite a practical desire for rapid safety screens,
it appeared necessary initially to design rigorous
chronic studies to review not only gross, albeit delayed,
lethal effects but also to detect subtle changes in
morphology, rate of growth, reproductive success and
the like. It was desirable to gain the capability to
uncover any subtle effect which would affect crustacean
viability in the wild.
Several alternative organisms as indicated in Table
I were considered for the study. The criteria used
included availability on a year round basis, availability
of published techniques and general practical experience
for maintenance of specimens in the laboratory, relevance
to important food web or commercial organisms and
diversification. The brine shrimp Artemia salinus and
the water flea Dap'nnia magna were immediately culled as
likely choices based on experience in maintaining labo-
ratory cultures. The brine shrimp was eventually
rejected as not suitable as a general model but the
cladoceran D. magna was chosen for a first attempt at
chronic exposure.
Of the remaining organisms proposed the Penaeid
shrimp were most desirable by virtue of being not a
model but an example of an important marine resource.
The unknowns in laboratory culture were, however,
VII-64B
-------
Table I
Organisms Considered as Model Decapod Crustaceans
for Insect Growth Regulator Nontarget Hazard Evaluation
Common Name
Scientific Name
Experience with
Rearing
Availability
Value as a
Model Decapod
Crustacean
Brine Shrimp
Artemia salinus
Excellent
Excellent
Low, primitive
crustacean found
only in high
salinity water
Water Flea
Daphnia magna . , -
, Excellent
Excellent
Good
Marine grass
shrimp
Palaemonetes pugio
Fragmented,stimulation
of molt in the lab is
capricious
Seasonal
Good
Pink and brown
shrimp
Penaeus setiferus
Fragmented/poor
Seasonal
Not a model, a
most important
marine resource
Blue Crab
Callinectes sapidus
Fragmented
Seasonal
Good
Crayfish
Procambarus or
Astacus spp.
Fragmented
Seasonal
Good
Mudcrab
Rhithropanopeus
spp.
Fragmented
Seasonal
Good
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PART VII - HAZARD EVALUATION SUPPLEMENT
considered too great a risk that no useful data would
be gained after lengthy experimentation. Furthermore,
the cost of performance of bioassays with Penaeid shrimp
was not readily forecast and therefore did not fit with
the conception of this contract. The crab and crayfish
proposals suffered for similar reasons. Detailed
literature exists for raising each through various life
stages but few laboratories can claim that egg to egg
rearing details are secure. Use of these organisms
could result in data on portions of developmental cycles
but by chance miss sensitive stages.
A second model crustacean decided upon was the
marine grass shrimp. Experience with the egg to egg
rearing of this creature, Palaemonetes pugio, has grown
over the past years and the eventual subcontractee EG&G
Bionomics Incorporated included on its staff several
scientists skilled in laboratory maintenance of P. pugio.
Thus data, on a fresh water cladoceran with considerable
historical background would be extended by data on a
marine shrimp model of somewhat greater uncertainty and
rearing but a significant step closer to the important
marine resource which EPA has the mission to protect
from damage by pesticides.
Because of insufficient lead time to initiate this
project, verbal bidding was solicited from Stanford
Research Institute in Menlo Park, California, and
Bionomics Aquatic Toxicology Laboratory in Wareham,
Massachusetts. Bidding was concomitant with refinement
of a plan of action. Because of significant price
differentiation, written price quotation for the study
was obtained only from Bionomics, After discussion of
various proposals with Zoecon and EPA, chronic study of
the water flea Daphnia magna and the grass shrimp
Palaemonetes pugio were initiated.
The first of these studies involved a 42-day chronic
exposure? of Daphnia magna looking at survivability and
reproductive potential. The study was run with the two
insect juvenile hormone analogs ALTOSID and R-20458 as
well as the chitin inhibiting insect growth regulator
TH-6040. Assessment of toxic hazard and effect on
reproductive capacity was made by survivor counts and
counts of adult versus young. Daphnids were randomly
sampled and preserved in alcohol for shipment to Zoecon
where control and highest dose surviving daphnids were
examined microscopically. Neither unusual morphology
nor effects on rate of growth were evident as compared
to controls with any of the insect growth regulator
treatments. The three insect growth regulators showed
VII-66B
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PART ¥11 - HAZARD EVALUATION SUPPLEMENT
quite a spread of the levels of toxicant necessary to
evoke* response and response was not qualitatively
unlike that expected for a standard toxicant.
The purpose of performing a 42-day chronic assay,
namely, to provide indication that a standard assay of
shorter duration (rapid safety screen?) or perhaps
longer duration would be a necessary requirement for
insect growth regulator registration was largely met.
In the case of Daphnia magna a single life history
bioassay is probably sufficient to assess the hazard
potential to crustaceans. Furthermore, solid evidence
is provided that simple acute bioassay is insufficient.
The study of Palaemonetes pugio was designed to
expose grass shrimp from hatch through reproductive age
with the insect growth regulators. As with Daphnia
magna the purpose was to determine if the grass shrimp
was a suitable crustacean model and to further refine
protocol design in the event that grass shrimp became
the standard for decapod crustacean toxicity required
for registration of insect growth regulators. One
problem surfaced immediately. The assay, if made a
requirement for registration, will apparently have to
be run on a seasonal basis. The stimulation of spawning
of grass shrimp in the laboratory was not possible with
present technology. It appears necessary to obtain
gravid females in the wild. As with D. magna, the
results showed that subchronic bioassay gave a more
realistic picture of potential for hazard to crustaceans
than did acute studies. The following sections provide
details of studies performed under EPA Contract 68-01-2444.
BIOASSAY OF INSECT GROWTH REGULATORS WITH DAPHNIA MAGMA
X. Abstract of Results
After 48 hour acute bioassay, three insect
growth regulators were subjected to chronic, two
generation dynamic bioassay with Daphnia magna.
Altosid, R-20458 and TH-6040 were delivered by
proportional diluters to four replicate flow
through aquaria for each dose level. At the
start daphnids were between 0 and 12 hours old,
Survival was assessed by counts each week and
the reproductive capacity was gauged by the
ratio of young to females. The ratio of sex of
offspring was noted. A gross summary of the
observations is found in Table II. Daphnids
were more sensitive to TH-6040 than the two
juvenile hormone analogs by more than a factor
VII-67B
-------
Table II
Summary of Chronic Bioassay
Data with Daphnia magna
Insect
Growth
Regulator
Static
48 Hour
LC n (95%
Conf. Interval)
ygA
Lowest Chronic
Concentration'
Producing Effect
on Survivability
pg/Ju
Lowest Chronic
Concentration3
Producing Effect
on Reproduction
vg/L
1st Gen.
2nd Gen,
1st Gen.
2nd Gen.
Altosid
89
(68-130)
97
97
51
51
TH-6040
1.5
(0.8 - 2.9)
0.61
0.61
0.61
_b
R-20458
330
(270 - 410)
221
221
24
24C
a Mean measured concentration during test; except for TH-6040 -
see text,
¦ Reproduction abnormal in treated (all levels) md controls,
c Total absence of females, see text.
VII-68B
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PART VII - HAZARD EVALUATION SUPPLEMENT
of a hundred. On the other hand TH-6040 does not
show an effect on reproduction at a lower level
than that at which survivability is affected. The
ratio of sex of offspring appeared to have been
altered by R-20458; however, this also happens
in nature and even the natural mechanism of action
or cause remains a mystery.
Analytical uncertainties in the case of TH-604 0
cast a shadow of doubt on the definition of TH-6040
levels as compared to the other two insect growth
regulators. The nonsynchrony of the second
generation makes reproductive data qualitative
rather than quantitative but the lack of any
observation of significance peculiar to the
second generation makes the value to be gained
by the necessary effort to accomplish a truly
quantitative two generation study open to question.
In addition to the comments above, the
following set of conclusions summarize the
experimental results.
a. In terms of survivability to chronic exposure
R-20458 offers least hazard followed closely
by Altosid; however, both are followed at a
significant distance by TH-6040.
b. In terms of hazard to reproductive capacity
the order of R-20458 and Altosid are reversed
and, as in the survivability portion, TH-6040
appears to offer hazard'to reproduction at
significantly lower levels.
c. Acute bioassay is not a reliable indicator
of chronic effect levels.
d. Analytical indications raise the question of
biological availability of TH-6040 when
applied to natural Daphnia habitat.
e. Insect growth regulators offer as broad a
spectrum of hazardous levels as do standard
toxicants.
f. No unusual or unique effects on Daphnia magna
growth or development are evoked by chronic
exposure to the three chosen insect growth
regulators.
VI1-69B
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PART VII - HAZARD EVALUATION SUPPLEMENT
Report of Bionomics Experiment
On the following pages is found the final report
on chronic toxicity of Altosid, TH-6040 and R-20458
to Daphnia magna as performed by EG&G, Bionomics
Aquatic Toxicology Laboratory, Wareham, Massachusetts.
VII-70B
-------
THE CHRONIC TOXICITY OP ALTOSIDR,
TH-604 0, AND R-20458 TO Daphnia
magna.
FINAL REPORT
SUBMITTED TO:
ZOECON CORPOPATI ON
PALO ALTO, CALIFORNIA 94304
BY;
EG & G, BIONOMICS
AQUATIC TOXICOLOGY LABORATORY
7 90 MAIN STREET
WAREIIAM, MASSACHUSETTS 02571
VII-71B
-------
INTRODUCTION
The continuing growth in the introduction of new chemical
pesticides, as well as the recent introduction of juvenile
hormone type pesticides, into the environment suggest a
significant requirement for adequate methods to screen and
identify those pesticides which may constitute a potential
threat to non-target organisms. For assessing such potential
threats in the aquatic ecosystems. Mount and Stephan (1967)
introduced the Laboratory Fish Production Index (LFPI) for
evaluating the toxic effects of a compound on survival,
growth, reproduction, and spawning behavior of fish during
long term exposures. This method, however, is also of value
when used with aquatic species other than fish. Arthur
(197.0) has utilized long term exposure methods successfully
with the aquatic invertebrate species Garmnarus pseudolinmaeus,
Campalona decisum, and Physa Integra. Macek et al. (1975a)
have utilized similar methods to evaluate the chronic toxicity
of lindane to a number of aquatic organisms including the
invertebrates Daphnia magna and Chironomus tentans. In this
study, v;e were primarily concerned with the survival and
reproductive capacity of Daphnia magna to estimate the maximum
acceptable toxicant concentration (MATC) of three insect
growth regulators (Altos .id1*, TH-6040, K-2 04 58) for this
zooplankton species. The MATC is the highest concentration
of the toxicant, during continuous aqueous exposure, which
has no adverse effects on the parameters mentioned,
R
The insect growth regulators, Altosid , l'H-6040, and R-20458
'serve to inhibit the successful maturation and survival of
insect larvae or pupae. One of the compounds, (TH-6040)
is known to act by inhibiting chit in synthesis during ecdysis
(Miura and Takahash, 1974). In view of this mode of action,
a greater understanding of the effects of these compounds
on other organisms in which ecdysis occurs should be obtained
before any large scale usuage of the pesticides occurs.
The water flea, Daphnia magna, was chosen as the test species
because ecdysis generally occurs anywhere from 12 to 28 times
during the development of this organism (Pennak, 1953).
Daphnia magna was selected also, because the susceptibility
of this organism to toxic substances appears to be comparable
to the general suscept i.bility of the predominant zooplankters.
Since the MATC will not indicate if or when the insect
growth regulators affect ecdysis or development of Daphnia
magna, samples of the exposed Daphnia. were taken and preserved
periodically and submitted to Zoecon, for future morphological
examination.
1 VII-72B
-------
Daphnia magna were chronically exposed to the growth regulators
for 42 days in order to determine which concentrations of the
pesticides affected survival and production of young. The
48 hour LC5 0's for Daphnia magna were established from acute
static bioassays and used to calculate application factors
(MATC limits/LC5Q)
MATERIALS AND METHODS
ACUTE TOXOCITY BIOASSAYS
The methodology for conducting the acute bioassays closely
fallowed the Methods for Acute Toxicity Test with Fish,
Macroinvertebrates, and Amphibians, issued by the Committee
on Methods for Toxicity Tests with Aquatic Organisms,
Environmental Protection Agency, (1975). The tests were
conducted under static conditions at a temperature of 21 ^
1°C. Five daphnids, 0 to 48 hours old, were used per
replicate. Three replicates were used per concentration.
The test aquaria consisted of 250 ml beakers with 200 ml of
aged {>1 week), well water. Observations of survival
were made after 24 and 48 hours of exposure.
Analysis of the results consisted of converting the test
concentrations and percent mortality to logs and probits
respectively, and the calculation of a linear regression
equation. Prom this, the 48 hour LC50 value was determined,
along with the 95% confidence interval.
CHRONIC EXPOSURE SYSTEMS
The water chemical delivery system consisted of a proportional
diluter (Mount and Brungs, 1967) with a 0.5 dilution factor.
The toxicant, dissolved in acetone, was delivered to the
chemical cells from a 10 ml glass syringe thru a stainless steel
needle. Prom the chemical cells, the toxicant was adequately
diluted to 5 concentrations in mixing chambers and delivered
to each of the four replicates per treatment. Diluent water
was delivered to each of the four replicate control aquaria.
Water was delivered to each replicate container by individual,
glass delivery tubes originating from the respective mixing
chamber. Fifty ml of toxicant solution or diluent water
were delivered to each test aquarium every thirty minutes.
Cylindrical glass battery jars were used as test aquaria.
These aquaria were 18 cm high and 13,5 cm wide. A 3 x 8 cm
notch was cut in the tops of the aquaria to allow fox-
drainage. The notches were covered with 4 0 mesh Nytex screening
to prevent the escape of Daphnia. The water depth was 15
cm and volume was 1.75 liters.
2
VII-73B
-------
The dilution water was pumped from a 400 ft. subterranean
well to a cement holding tank. Prom the holding tank the
water was delivered, by aged PVC piping, to a stainless steel,
heated headbox. The water was then gravity fed, via tygon
tubing, to the water cells of the diluter. The headbox
temperature was maintained with immersion fused quartz heaters
and thornio-regula tors.
Lighting was provided by a combination of Durotest {Optima
PS) cind wide spectrum Grow Lux fluorescent lights. The
entire test units were enclosed with black polyethylene
curtains to inhibit outside light filtration and to
minimi zo disturbance of the daphnids. The photoperiod was
controlled by an automatic timer to provide 13 hours of light
during each 24 hour period.
To initiate the first generation exposure, Daphnia magna
(<12 ho ;rs old) were obtained from laboratory stock cultures.
Thirty-eight (38) daphnids were randomly distributed to each of
the twenty-four (24) experimental units. Of this number, 18
were removed periodically during the first seven days of exposure
and preserved in 7 0£'for morphological examination (Tables 1,
2 and 3}, Observations of the survival and production of
young per female of the remaining organisms in each aquarium
were made every 7 days. When progeny were present {days
14 and 21), they were counted and discarded or utilized to
initiate the second generation exposure. At the initiation
of the second generation exposure, precise estimates of the
age and developmental stage of selected daphnids were not
possible since those young animals remaining in the aquaria at
the termination of the first generation exposure could have
been from <1 - <168 hours old. An effort was made, at this time,
to randomly select from the young forms available, an equal
number of organisms to initiate the second generation exposure.
Daphnids were also sampled during the second generation
exposure for morphological examination.
During the initial 3 days of both the first and second generation
of the chronic exposures, the test containers were kept under
static conditions in total darkness. These precautionary
measures were taken because Daphnia are phototactic and an
attraction to the surface, during this early stage of development,
would result in entrapment and death at the air/water interface
due to surface tension.
Dissolved oxygen and temperature were measured in a least .
one replicate of each treatment daily with a YSI dissolved
oxygen mater, using a combined oxygen and temperature probe.
3
VII-74B
-------
TABLE 1. SAMPLING SCHEDULE FOR Daphnia magna EXPOSED TO
ALTOSIDr FOR FUTURE EXAMINATION. THREE Daphnia WERE
TAKEN FROM EACH REPLICATE AT THE DESIGNATED SAMPLING
TIMES
Daphnia
DeveXopmen t
Date
Day
Time
age (hrs)
Sample #
Stage
GENERATION
I
11/21/74
0
4:00
P.M.
8-20
1
Juvenile
11/22/74
1
8 : 00
A.M.
24-36
2
Juvenile
11/22/74
1
4:00
P.M.
32-44
' 3
Juvenile
11/23/74
2
4:00
P.M.
56-68
4
Juvenile
11/25/74
4
8:00
A.M.
96-108
5
Juvenile/Adult
11/28/74
7
8:00
A.M.
168-180
6
Adult
GENERATION
II
12/12/74 '
" 21
4:00
P.M.
0-168
1
1
12/13/74
22
8:00
A.M.
16-184
2
12/13/74
22
4:00
P.M.
24-192
3
12/14/74
23
4:00
P.M.
48-216
4
12/16/74
• 25
8:00
P.M.
84-252
5
_—„
^Stage of development is unknown due to variation in age
VII-7SB
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TABLE 2. SAMPLING SCHEDULE FOR Daphnia magna EXPOSED TO
TH-6040 FOR FUTURE EX AMI RATIO?!. THREE Daphnia
WERE TAKEN FROM EACH REPLICATE AT THE DESIGNATED
SAMPLING TIMES
Daphnia ' Development
Date
Day
Time
ago (hrs)
Sample
# Stage
GENE POTION
I
2/28/75
0
4:00 P.M.
8-20
1
Juvenile
3/1/7 5
1
8:00 A.M.
24-36
2
Juvenile
3/1/75
1
4:00 P.M.
32-44
3
Juvenile
3/2/7 5
2
4 : 00 P.M.
56-68
4
Juvenile
3/6/75
6
8:00 A.M.
144-156
5
Juvenile/Adult
3/7/7 5
7
8:00 A.M.
168-180
6
Adult
GENERATION
II
3/20/75
21
4:00 P.M.
0-168
7
1
3/21/75
22
4:00 P.M.
24-192
8
3/22/7 5
23
4:00 P.M.
48-216
9
3/26/75
27
4:00 P.M.
144-312
10
Adult
3/27/75
28
4:00 P.M.
168-336
11
Adult
1
Stage of
development
is unknown
due to variation
in age
VII-76B
-------
TABLE 3. SAMPLING SCHEDULE FOR Dap hit i a magna EXPOSE!) TO R-20458
FOR FUTURE E XAMI NAT ION ~ T H REK" Dap'lVn ia WERE TAKEN
FROM EACH REPLICATE AT THE DESIGNATED SAMPLING TIMES
Date
Day
Time
Daphnia
age (hrs)
Sample 1
Development
Stage
GENERATION I
2/27/75
0
o
o
P.M.
8-20
1
Juvenile
2/28/75
1
CO
o
o
A.M.
24-36
2
Juvenile
2/28/75
1
o
o
P.M.
32-44
3
Juvenile
3/1/75
2
O
o
*4)
P.M.
56-68
4
Juvenile
3/5/75
6
8:00
A.M.
144-156
5
Juvenile/Adult
3/6/7 5
7
o
o
CO
A.M.
168-180
6
Adult
GENERATION II
3/20/7 5
21
©
o
*3«
P.M.
0-168
7
1
3/21/7 5
22
O
O
¦cr
P.M.
24-192
8
3/22/7 5
23
«{>
o
o
P.M.
48-216
9
3/26/75
27
4:00
P.M.
144-312
10
Adult
3/27/75
28
4:00
P.M.
168-336
11
Adult
Stage of development is unknown due to variation in age
VII-77B
-------
The Daphnia were fed a combination of trout starter food and
cerophyll in a 20:1 ratio respectively. The combination was
blended in water and the suspension was filtered through a
102 mesh stainlacs ste^L screen before use. A 0,2 ml aliquot
of the supernatent of this food solution (35 mg/ml) was
pipetted into each aquarium 3 times daily.
y
The mean weekly survival and reproductive data of each replicate
were subjected to analysis of variance according to Steele
and Torrie (I960). If significant differences (P =.05) due
to treatment were indicated, the means of the replicates were
analyzed utilizing Duncan's Multiple Range Test to determine
which treatments were statistically different CP =,05} from
the controls,
CHEMICAL METHODS
The chemical stocks of Altosid5, TH-604 0, and R-20458 were made
using nanograde acetone. No solvent was delivered to the
controls. The highest concentration of acetone ever
present in one aquarium never exceeded 12 ul/1. Determinations
of the aqueous concentrations of toxicant were made by periodic
sampling of water from an aquarium to provide a composite sample
for chemical analyses. Withdrawal of equal volumes of water
from control aquaria was performed utilizing the appropriate
procedure.
Water samples were provided for Altosid1* analysis by withdrawing
100 ml of test solution from the appropriate aquarium hourly
over a 4-8 hour period. Analysis of Altosid in the composite
water sample was performed by adding 10-20 g of reagent grade
sodium chloride to 4 00-800 ml of the sampled test water in
a 1 liter separatory funnel to yield a salt concentration of
25/000 rug/1, The sodium chloride was dissolved and the water
was extracted once with 100 ml of nanograde petroleum ether
by shaking the funnel for two minutes. After phase separation
the water was drained from the funnel and the solvent was
added to a 500 ml Kuderna-Danish evaporator, equipped with a
three-ball Snyder column. The solvent was evaporated to ca 3 ml
over a water bath at 7 0°C. The Kuderna-Danish evaporator was
cooled to room temperature and dismantled. The remaining solvent
was evaporated at ambient temperature to less than 1 ml, using
a gentle stream of clean, dry air.
The extract was then diluted to 1.0 ml using nanograde hexane
and an aliquot was removed for analysis by gas/liquid
chromatography.
Aliquots of the water extract were analyzed using the following
instrumental operating conditions;
Instrument; Tracor Model MT550 gas chromatograph
Detector: Electron capture with 15 mCi Ni^3
VII-78B
7
-------
Column: 4* x 1/4" glass, packed with 5% DC-200 on 80/100
mesh Supelcoport
Carrier gas: 70 cc/min of nitrogen
Purge gas: 20 cc/min of nitrogen
Temperatures: \
Inlet: 23 0°C Outlet: 24 0°C
Column: 205°C Detector: 28 5°C
Recorder: Corning Model 841 strip chart, response 0-1
m¥ full scale
R
Response; 100 ng of Altosid (active) gave half-scale recorder
pen deflection using electrometer attenuation of
1.6 x 10 amperes. AltosidR eluted in 8.0 minutes
under the above conditions
Prior to the analysis of a series of Altosid1* water extracts,
the gas chromatograph was equilibrated by injecting 3-5
mg of Altosid11 into the column , and allowing 2-3 hours for
the recorder pen to establish a stable baseline. Varying
weights of known AltosidR standards were analyzed and a
graph of response (peak height} versus nanograms of Altor.idR
chromatographod was constructed. All sample peak heights
were converted to Altosid1* weights using the standard graph.
Six quality control samples were manufactured by adding 1.5 ml
of an acetone solution containing 100 (jg/ml of Altosid® to
800 ml of distilled water to produce concentrations of 0.188
mg/1 of Altosid in each sample. The samples were analyzed
according to the above procedure with 84 ^ 10% recovery of
AltosidR. All reported Alto:;.idR concentrations have been
corrected for the percentage recovery of the analytical
method.
Analysis for the presence of TH-6040 in the test aquaria
required the removal of 1000 ml water samples from the
nominal concentrations of 0.25, 0.50, and 1.00 pg/1. Two
thousand ml water samples were taken from the nominal
concentrations of 0,062 and 0.125 jag/1. These composite water
samples were generated by removing 125 ml of test solution hourly
over an eight hour period for the former (1 liter} samples,
and 125 ml hourly during two successive 8 hour days for the
latter (2 liter) samples.
Each 1 liter sample, or each of two 1 liter portions of a 2 liter
sample were placed in a separatory funnel and extracted 3 times
with 30 ml portions of nanograde methylene chloride. The
total extracts of each sample were combined in a Kuderna-
Danisn evaporator, equipped with a three-ball Snyder column,
and evaporated to ca 5 ml over a water bath at 70°C. The
remaining solvent was transferred to a 15 ml centrifuge tube
and evaporated to complete dryness at ambient temperature using
VII-79B
-------
a gentle stream of clean, dry air. The residues were stored
and shipped under dry ice to Dr. Donald W. Fuhlhage,
Thompson-Hayward Chemical Co., 5200 Speaker Road, Kansa
City, Kansas.
Prior to liquid/liquid chromatographic analysis, the
extract:; were dissolved in a known volume of acatonitrile.
The minimum detectable concentration of TH-604 0 in the
acetonitrile solution was 1 yug/ml (personal communication,
Dr. P. Fuhlhage, Thompson-Hayward Chemical Co.)* The 2
liter water samples from the nominal test concentration 0,062
and 0.125 jug/1 contained 0.124 and 0.250 p.g of TH-6040. When
dissolved in 100 pi of acetonitrile prior to analysis, the solution
contained 1.24 and 2.5 0 jug/ml of TH-604 0, thus the
concentrations necessary for detection were present.
The operating conditions of the liquid/liquid chromatograph
were as follows:
Column; Bondapak CIS/corasil column, 2 ft* by 1/8 in. I.D.
Phases; 40-45% acetonitrile and 60-55% water
Detector; UV detection at 254 nm
Response: With a 5.5 ml retention volume at 0.44 ml/min,
a peak height of 33 cm/ppm TH-6040 was obtained
with a setting of 0.01 (ODP8). The reference
standard was 1 ppm TH-604 0 in acetonitrile.
Five quality control samples containing 0.5 and 0.1 jug/l
of TH-6040 (1 liter and 2 liter volumes, respectively)
were analyzed by the above methodology. The percentage
recoveries were 88 - 27 and 76 - 16% respectively.
Water samples were provided for R-20458 analysis withdrawing
4 00 ml of test solution from the appropriate aquarium over a
4 hour period. Analysis of R-20458 in the test water was conducted
in the following manner: a 400 ml portion of water was measured
volumetrically, added to a 1 liter separatory funnel, and
10 g of reagent grade sodium chloride was added. The sodium
chloride was dissolved and the water was extracted once with
50 ml of nanograde petroleum ether, by shaking the funnel
for two minutes. After phase separation, the water was
drained from the funnel and the solvent was added to a 250
ml Griffin beaker. The solvent was evaporated to ca 3 ml
over a water bath at 70°C. The remaining solvent was evaporated
at ambient temperature to less than 1 ml, using a gentle
stream of clean, dry air. The extract was then diluted to
1.0 ml using nanograde hexane and an aliquot was removed
for analysis by gas/liquid chromatography.
Aliquots of the water extracts were analyzed using the following
instrumental operating conditions;
Instrument; Tracor Model MT550 gas ehromatograph
VII-80B
9
-------
63 *
Detector: Electron capture with 15mCi Ni
Column: 4' x 1/4" glass, packed with 5% DC-200 on 80/100
mesh Supelcoport
Carrier gas: 60 cc/min of ntirogen
Purge gas: 45 cc/min of nitrogen
Temperatures:
Inlet: 23 0°C Outlet: 235°C
Column: 160°C Detector: 29 5°C
Recorder: Corning Model 841 strip chart, response 0-1 mV
full-scale
Response: 50 ng of R-204 58 (active) gave half-scale recorder
pen deflection using an electrometer attenuation
of 1.6 x 10-9 amperes, R-20458 eluted in 4.0
minutes under the above conditions.
Prior to the analysis of a series of R-20458 water extracts,
the gas chromatograph was equilibrated by injecting approximately
3 nig of R-20453 into the column and allowing sufficient time
for the recorder pen to establish a stable baseline. Varying
weights of known R-204 58 standards were analyzed and a
graph was constructed to relate response (peak height) versus
weight of R-20458 injected. All sample peak heights were
converted to R-20458 using the standard graph,
Five quality control samples were made by adding 1.5 ml of
an acetone solution containing 10 pg/ml of R-20458 to 300 ml
of distilled water to produce concentrations of 0.050 rag/1
of R-20458 in each sample. The samples were analyzed
according to the above procedure with 54 - 5% recovery of
R-20458. All reported R-20458 concentrations have been
corrected for the percentage recovery of the analytical
method.
RESULTS
ACUTE BIOASSAY
R
The analysis of the results of the acute bioassay for Altosid
indicated a 48-hour LC50 value of 8 9 pg/1, with a 35%
confidence interval of 68-130 jug/1 (Table 4). The lowest
concentration causing a 100% mortality was 140 pg/1. Based
upon this evaluation the measured concentrations selected for
the chronic exposure of Daphnia magna to AltosidR ranged from
10-140 pg/1.
10
VII-81B
-------
Based on previous experience with the behavior of similar
compounds in diluter systems, we anticipated a discrepancy
betv/een nominal concentrations and measured concentrations
in the aquaria. Thus, we prepared stock solutions calculated
to yield nominal concentrations higher than the desired
measured concentrations.
%
The acute bioassay conducted with TH-6040 indicated a 4 8-hour
LC50 value of 1.5 pg/1, with a 951. confidence interval of
0.8 to 2.9 pg/1 (Table 4). Based on these data, the highest
nominal concentration of TH-604 0 selected for the chronic exposure
was 1.0 jug/l. Analysis of the acute bioassay conducted with
R-20458 indicated a 48-hour LC50 value of 330 pg/1 with a 95%
confidence interval of 270-410 pg/1 {Table 4). Based on these
data, the highest nominal concentration of R-204 58 chronically
exposed to Daphnia magna was 250 pg/1.
CHEMICAL ANALYSIS ,
Daily measurements of dissolved oxygen concentration and
temperature in the test containers indicate that these
parameters did not significantly fluctuate between treatments
within each exposure. The mean, standard deviation, and
range for these parameters are: AltosidR, temperature 18.7
± 0.6 (18 - 0-20.0)°C, DO 4.9 ± 0.8 (4.0-6-3) mg/1, TH-6040,
temperature 16.6 ± 0.6 (15.0-17.0)°c, DO 6-6 - 1.8 (2.8-8.7)
mg/1. R-20458, temperature 16.7 - 0.4 (16 . 0-17.5}°c, DO
6.7 - 1-2 (4.1-8.3) rag/1. Due to mechanical problems with the
temperature control system, mean temperature during most of
the exposure to TH-6040 and R-20458 deviated significantly
(>2.0°C) from the nominal temperature (19°C).
Results of the analysis of the diluent water taken previous
to the initiation of the chronic exposures are summarized
(Table 5). Results of the analysis of the composite water
samples taken throughout the chronic exposures of daphnids
to AltosidR, TH604 0 and R-20458 are also presented (Tables
6, 7 and 8} .
Water samples taken after 7 days of normal diluter functions
in the chronic exposure system previous to the introduction
of Daphnia contained concentrations of TH-6040 close to the
desired nominal concentration. However, samples taken following
the introduction of Daphnia and food to the system were
consistently lower than the desired concentration. Because
the diluter functioned normally throughout the exposure and
the desired amount of TH-604 0 stock was being delivered to the
system, we suggest that the Daphnia or the bacterial flora
within the exposure system were accumulating and/or degrading
the TH-6040. Therefore, measured concentrations of TH-6040
for day 0 water samples were used as an indicator of the amount
of TH-604 0 being introduced into the experimental aquaria.
1.1
VII-82B
-------
TABLE 4. ACUTE TOXICITY OF ALTOSIDR, T1I-G040, AMD R-20458 TO
Daphnia magna DURING STATIC BIOASSAYS
Toxicant
Concentration
(pa/1)
Observed % Mortality
24 hour 48 hour
48 hr. I,C 50
I y 5-i Conf.
Interval)
pg/i
Altosid
R
320
210
140
100
65
42
28
Control
100
93
100
20
0
6
0
0
100
93
100
40
0
13
0
0
89
(68-130)
TH-604 0
3.2
2.8
1.6
1.2
0.6
0.3
0.1
Control
53
46
53
0
0
0
0
0
100
73
53
20
23
6
0
0
1.5
(0.8-2.9)
R-20458
1,000
650
490
420
320
240
180
Control
100
66
73
60
40
13
0
0
100
93
100
66
66
13
0
0
330
(270-410)
1-2
VII-83B
-------
TABLE 5.
CHEMICAL ANALYSIS OF THE DILUENT WATER UTILIZED
DURING CHRONIC AQUEOUS EXPOSURE OP Paphnia magna
TO ALTOSIDR, TI1-G040, AMD R-204 58
Parameter
mg/liter
Parameter
mg/liter
Calcium
8-4
Chloride
17 . 6
Magnesium
2.8
Fluoride
0.5
Potassium
1.1
Cyanide
<0 .005
Sulfate
11.6
Iron*
<0.01
Nitrate
<0.05
Copper
0.004
Nitrite
<0.05
Zinc
0.01
Ammonia
0.1
Cadmium
<0.0 01
Phenol
<0 .001
Chromium
<0,001
Chlorine
<0 .01
Lead
<0.01
13
f-
VII-84B
-------
TABLE 6. MEAN MEASURED AQUEOUS CONCENTRATION OF ALTOSID
(pg/1} DURING CHRONIC BIOASSAY WITH Daphnia magna
CONTINUOUSLY EXPOSED FOR 4 2 DAYS
%
AltosidR
(pg/l)
Nomina" 17
Replicate
Mean
Days of
Exposure
1
. 3
7
14
21 28
35
42
50
12 i 3a
A
17
11
7
10
B
20
8
8
c
15
10
9
14
D
18
11
10
100
27-6
A
29
39
22
24
B
33
25
16
C
24
41
20
30
D
27
20
28
200
51 - 13
A
56
69
40
43
B
57
42
55
C
65
68
45
50
D
47
39
39
400
97 - 29
h
83
90
78
118
B
91
76
86
C
120
99
82
110
D
140
70
115
800
487 - 152
A
580
412
352
465
B
600
390
526
C
610
436
388
417
D
7 02
520
420
aStandard Deviation
14
f
VII-85B
-------
TABLE 7. MEAN MEASURED AQUEOUS CONCENTRATION OF TH-604 0 (pg/1)
DURING CHRONIC BIOASSAY WITH Daphnia magna CONTINUOUSLY
EXPOSED FOR 42 DAYS.
TH-6 04 0. (pg/1)
Nominal/
Replicate
Mean3 ,
Days of
Exposu
re
0
4
11
18
25
32
39
0.06
0.09
- 0.02b
A
0.12
N.D.
N.D.
B
0.07
N.D.
N.D.
C
0. 07
N. D.
N.D.
D
0.11
N.D.
N.D.
0.13
0.12
- 0.03
A
0.11
N.D.
N.D.
B
0.16
N.D.
C
0.12
N.D.
N.D.
D
0.09
N.D.
N.D.
0.25
0.25
- 0.07
A
0.16
0. 07
N.D.
B
0. 35
N.D.
N.D.
c
0.27
0.05
0.09
D
|
0.23
N.D.
N.D.
0,50
0. 61
- 0.14
A
—,
0. 05
0. 05
B
0.45
N.D.
N.D.
C
0.72
0.05
N.D.
D
0.66
N.D.
1.00
1.27
- 0.13
A
1.40
0.13
0.08
B
1.30
0.09
N.D.
C
1.10
0.13
0.10
D
1.30
N.D.
0.07
o,
Mean measured concentration prior to introduction of
Daphnia magna.
v
Standard Deviation
CBelow minimum detectable limit (0.05 pg/1)
ci
Sample apparently contaminated prior to completion of analysis
15
*
VII-86B
-------
TABLE 8. .MEAN MEASURED CONCENTRATION OF R-20458 (jig/1) DURING
CHRONIC BIOASSAY WITH Daphnia magna CONTINUOUSLY
EXPOSED FOR 4 2 DAYS.
R-
20458
(pq/D
NomrnaX/"
Replicate
Mean
Davs of Exposure
4
11
18 25 32
39
15.6
24 ± 5a
A
23
22
31
B
29
19
26
C
17
32
D
22
18
31.2
31 ± 7
A
34v,
28
19
n
D
^ JD
29
33
C
31
44
D
28
30
W £t • tail
62 -12
A
49
72
53
B
65
67
64
C
82
72
D
56
41
125.0
112 -24
A
93
160
110
B
130
_
100
C
12 0
115
D
99
78
250.0
221 ±71
A
-
200
65
B
280
_
200
C
260
290
D
250
220
ct
Standard Deviation
h
Sample lost
16
VII-87B
-------
CHRONIC EXPOSURE - ALTOSIDR
R
Continuous exposure to various concentrations of Altosid
for 21 days significantly (P-.05) reduced survival and
reproduction of Daphnia magna. Analysis of survival data
showed that continuous exposure to a measured mean concentration
of 4 87 /ig/1 for 7 dp,ys resulted in a 1001 mortality. The
toxic effect of Altosid- on survival is cumulative to day 14.
Exposure to 97 pg/1 for 14 and 21 days significantly {P=.05)
reduced survival of daphnids when compared to controls and
all other treatments (Table 9).
R
Altosid affected reproduction at concentrations lower than
those which reduced survival. During the 14 days of
reproduction, those first generation daphnids exposed to measured
concentrations of 97 and 51 jug/1 AltosidR experienced a
significant (P-.Q5) reproductive impairment when compared
to controls (Table 10). Because of the 100% mortality
observed in those replicate test aquaria exposed to 487
jug/1 and the inadequate young production at 97 jig/1
AltosidR, control daphnids were introduced in these treatments
for initiation of the 2nd generation in order to reconfirm
observations made during the first generation exposure,
R
The 21 day exposure of the second generation Daphnia to Altosid
did not indicate an increased cumulative effect on survival or
the production of young greater than that observed during the
first generation (Table 9 and 10). Observations of survival
after 23 and 35 days (1st and 2nd weeks of generation II)
revealed a significant {P=.05) decrease in survival in all
replicates exposed to 97 and 487 p.g/1 Altosid^, Survival data
from day 42 was statistically meaningless because of the
increase in natural mortalities in all treatments.
Analysis of young production, during the second generation
exposure apparently indicated, once again, that reduction of
young production due to AltosidR is not cumulative beyond
what was observed in the first generation {Table 10). During
the 5th and 6th weeks of, production of young was significantly
reduced by exposure to 97 and 51 p.g/1 AltosidR. Not only
were fewer young produced at these concentrations, but many
young daphnids died shortly after release from the females.
As mentioned with respect to survival data previously, statistical
evaluation of the reproduction data obtained on day 42 was of
no value due to variability observed in all treatment levels.
K
Based on the observed effects of Altosid on the production
of young for Daphnia magna continuously exposed to the
toxicant during 2 generations (42 days) of development;
the estimated maximum acceptable toxicant concentration for
this species is >27<51 /jg/1 AltosidR.
17
VII-88B
-------
TABLE 9. PERCENT SURVIVAL OF Daphnia magna CONTINUOUSLY
EXPOSED TO ALTOS .I. DR FOR 42 DAYS . ~
Altosid
(wq/1) *
5
Survival
on Day
Nominal/
Replicate
Mean
Measured
Generation I
Generation
II
7
• 14
21
28
35
42
Control
—
A
100
90
90
90
85
80
B
100
95
95
100
90
75
C
85
75
75
95
55
50
D
80
80
75
100
60
50
X
91
85
84
96
73
64
50
12
A
100
85
30
85
75
0
B
95
95
70
50
50
0
C
95
85
80
85
60
0
D
100
100
65
75
65
0
X
98
91
61
74
62
0
100
27
A
100
90
65
55
50
50
B
60
60
55
90
55
50
C
100
95
95
65
45
45
D
90
85
75
95
75
70
X
88
83
73
76
56
54
200
51
A
100
95
95
90
60
15
B
90
90
90
90
55
25
C
95
95
80
65
65
50
D
90
85
75
95
70
15
X
94
91
85
85
62
"26
4 00
97
A
95
15
15
70a
45
0
B
90
10
10
75a
35
5
C
85
85
80
15 a
0
_
D
55
55
55
25a
10
0
X
81
41
40
46
23
1
800
487
A
0
—
0a
—
B
0
_
Qa
—
_
C
0
_
0a
—
D
0
-
0a
-
_
X
0
0
Survival of young daphnids introduced from first generation
control groups due to high mortality and low reproductive
activity of first generation daphnids
VII-89B
-------
TABLE 10.
PRODUCTION OF YOUNG PER PARTHENOGENETIC FEMALE
Daphnia magna CONTINUOUSLY EXPOSED TO ALTOSIDR
FOR 4 2 DAYS
Altos id v (jug/1)
Young/Parthenogenetic Fentitle.
GENERATION I GENKltATION II
Replicate
Measured
cl
TY
14
Y/?b
21
TY
Y/2
35
TY
i
Y/?
42
TY
Y/9
Control
—
A
5 05
28
686
38
457
27
209
13
B
56
3
300
16
414
23
120
8
C
313
21
319
21
251
23
443
44
D
179
12
554
37
348
29
309
31
X
16
28
25
24
50
12
A
271
16
149
25
463
31
0
0
B
288
15
451
32
208
21
0
0
C
377
22
370
23
339
28
0
0
D
3 23
16
314
24
3 61
28
0 _
0
X
17
26
27
0
100
27
A
363
20
275
22
266
26
179
18
B
.164
14
459
42
263
24
271
28
C
271
14
363
19
219
24 .
196
31
D
401
24
506
34
620
42
259
19
X
18
29
29
24
200
51
A
52
3
137
7
140
12
9
3
B
103
6
226
12
93
9
125
26
C
71
4
91
6
3
0
13
1
D
79
5
171
11
170
12
22
6
X
4
9
8
9
400
97
A
4
1
3
1
28c
3
0
0
B
3
1
2
1
°c
0
0
0
C
10
1
0
0
°c
0
0
0
D
45
4
4
0
3
1
0
0
X
2
~T5
1
0
800
487
A
0
0
0
0
0C
0
0
0
B
0
0
0
0
0e
0
0
0
C
0
0
0
0
0C
0
0
0
D
0
0
0
0
"0C
0
0
0
X
0
0
0
0
Total Young Young/Parthonogenetic Female
i*
'Young produced by control daphnids introduced to initiate
second generation exposure
lg VII-90B
-------
CHROMIC EXPOSURE ~ TH-604 0
Exposure of young Kaphnia magna to mean measured TH-604 0
concentrations (at time 0) of 1.27 and 0.61 pg/1 significantly
(P-.05) decreased survival when compared to controls and all
other treatments (Table 11). Exposure of daphnids bo 1.27
fig/1 TH-6040 for only 2 days resulted in 100% mortality.
During this period, considerable mortality also was observed
among daphnids exposed to 0.61 pg/1 TH-6040 and by day 7 of
the exposure only 2 daphnids.(2,5% of the population) remained.
These animals survived the remainder of the first generation
exposure (21 days) , "No significant effects on survival of
daphnids were observed during 21 days exposure to mean
measured concentrations of TH-604 0 (at time 0) as high as
0.25 pig/1.
The variability in the number of young produced/parthenogenetic
female during the first generation precluded sensitive
evaluation of these data. Empirical observation of the data
(Table 12) clearly indicates that reproduction among daphnids
exposed to a mean measured concentration (at time 0} of 0.25
jug/1 TH-604 0 was comparable to controls. During this
first generation exposure of daphnids to TH-6040, we observed
the development of sexual Daphnia (males). The incidence of
males (20-30%) in the population appeared to be random
including that observed among controls.
Due apparently to the uptake and/or degradation of TH-6040 by
the bionass within the experimental systonus, concentrations of
TH-604 0 in all experimental units were sufficiently low
that no significant effects on survival of daphnids during
the second generation of exposure were observed. Additionally,
although the incidence of males observed during the second
generation was lower (5-10%) than that observed during the
first generation, reproductive activity was depressed an no
indications of toxicant related effects on reproduction during
the second generation exposure were evident.
Based on the effects on survival of young daphnids exposed
to initially mean measured concentrations of 1,27 and 0.61
jag/1 TH-6040, clearly the MATC of TH-6040 for Daphnia
magna is estimated to be <0.61 pg/1.
CHRONIC EXPOSURE - R-204 58
Exposure of young Daphnia magna to 221 pg/1 R-204 58 resulted
in complete mortality of the population within 7 days. During
the first generation exposure, survival of daphnids exposed
to concentrations of R-20458 as high as 112 jug/1 was -not
significantly different from controls {Table i3).
20
VII-91B
-------
TABLE 11. PERCENT SURVIVAL OF Daphnia maqna CONTINUOUSLY
EXPOSED TO TI1-6040 FOR 42 DAYS.
TH-6040
fpg/D t
t Survival
Nominal/
Mean
Generation
. I
Generation
II
Replicate
Measured
a ?
14
21
28
35
42
Control
A
85
85
85
75
40
40
B
100
95
90
85
50
45
C
100
100
100
70
55
50
D
100
100
100
90
90
75
X
96
95
94
80
59
52
0.06
0.09
A
100
100
95
85
70
50
B
95
90
90
95
30
25
C
100
95
80
60
35
20
D
100
100
100
90
80
80
X
99
96
91
82
54
44
0.13
0.12
A
85
85
85
100
100
100
B
100
100
100
75
60
55
C
100
100
100
100
90
80
D
100
100
100
80
50
0
X
9F"
w ~
89
7 5
0.25
0.25
A
100
100
90
0
•«
—
B
100
85
60
100
90
65
C
100
100
100
95
95
95
D
100
100
100
85
85
85
X
100
96
88
70
67
61
0.50
0.61
A
5
5
5
95
90
90
B
0
_
_fc»
—
—
C
0
D
5
5
5
-
_
X
2
2
2
95
90
90
1.00
1.27
*
A
0
......
.. .
—
B
0
-
_
C
0
_
_
_b
—
—
D
0
»-
-
1
0
3Mean measured concentrations at initiation of 42 day
exposure period
No F, generation produced for initiation of 2nd goneration
exposure
#•
21 VII-92B
-------
TABLE 12. PRODUCTION OF YOUNG/PART1IENOGENETIC FEMALE Daphnia
magna CONTINUOUSLY EXPOSED TO TH-604 0 FOR 4 2 DAYS
TH-604 0 (jug/l} Young Produced/ParthenQganet i c Female
Nominal/ Mean GENERATION £ GEMIGRATION -t.I
Replicate Measured 14 21 3 3 4 2
v TY Y/Sc TY Y/¥ TY Y/? TY Y/V
Control
A
88
15
465
79
0
0
37
5
B
151
12
747
50
0
0
69
8
C
168
13
529
44
0
0
31
4
D
174
12
687
49
0
0
58
6
X
13
55
0
6
06
0.09
A
198
10
625
35
4
1
76
8
B
143
8
463
27
0
0
26
5
C
136
8
218
17
4
0
0
0
D
87
5
571
32
158
9
218
12
X
8
28
2
6
13
0.12
A
151
11
445
32
50
2
228
11
B
140
7
606
30
0
0
26
C
154 "
8
603
33
0
0
64
7
D
284
15
678
36
0
0
X
10
33
.5
7
25
0.25
A
188
14
620
56
__u
-
-
B
118
13
185
46
49
3
133
11
C
220
13
661
39
15
• 1
130
7
D
233
15
756
47
37
2
124
7
X
14
47
" "I
8
50
0. 61
A
°d
0
36
36
296
16
157
9
B
d
—
p-
—
—
—
—
—
C
D
_d
X
36
16
9
00
1.27
d
A
~d
""
—*¦
—*
B
d
C
D
~d
"
X
3
Mean measured concentrations at initiation of 42 day exposure
period
Total Young
,Youn9/Parthenogenetic Female
No young due to 100% mortality of adults
VII-93B
22
-------
TABLE 13. SURVIVAL OF Daphnia magna CONTINUOUSLY EXPOSED TO
STAUFFER R-20458 FOR 4 2 DAYS.
R-20453
{jug/1)
%
Survival
. on Day
Nominal/
Mean
Generation I
Generation
11
Replicate
Measured
7
14
21
28
35
42
Control
A
100
100
40
100
90
85
B
100
95
95
100
90
90
C
100
100
55
85
80
80
D
100
100
45
75
20
5
X
100
99
59
90
70
65
15.6
24
A
100
100
80
95
75
75
B
100
100
85
100
100
100
C
100
100
100
75
7 0
70
D
95
35
95
100
100
95
- X
99
90
92
86
85
31.2
31
h
100
100
55
100
100
10 0
B
100
100
100
100
100
100
C
30
30
50
85
85
85
D
100
95
90
95
95 ¦
95
X
97
96
74
95
95
95
62.5
82
A
80
80
75
100
100
100
B
70
70
65
85
65
65
C
100
100
95
100
70
70
D
95
95
95
80
55
55
X
86
86
82
86
72
72
125.0
112
A
100
100
80
95
10
10
B
80
75
70
100
100
75
C
90
90
70
100
90
90
D
95
95
85
90
15
10
X
91
¦ 90
76-
96
54
46
250.0
221
A
0
—
—
_ a
B
0
__
_ a
_
_
C
0
—
_ a
wm
D
0
—
_ a
X
0
'No generation produced for initiation of 2nd generation
exposure
23
VII-94B
-------
During the first week of reproductive activity (days 7-14
of exposure), the production of young when compared to controls
was reduced significantly (p - . 0 5) by exposure to 2 4 , and 31,
jug/1 R-204S8 and completely inhibited by exposure to 62, and
112, jig/1 R-20458 {Table 14). These are particularly significant
observations since during this period the reproduction among
control groups was comparable to that observed after 14 days
among control groups during the AltosidR and TH-604 0 Daphnia
chronic toxicity studies. Also, during the first generation
exposure of daphnids to R-20458, incidence of adult males in
the population was less than'1%. We conclude that the
observed effects on reproductive activity during this period
are toxicant induced. As indicated by an analysis of data
obtained at the end of the first generation exposure, the
production of young was apparently delayed between 1 and
7 days by exposure to 24, 31, 62 and 112 pg/1 R-2 04 58.
However, production of young daphnids during the last week
of exposure was significantly {P~.05) reduced, when compared
to controls, only among daphnids exposed to 112 jug/1 R-20458.
Exposure of a second generation daphnids to all concentrations
tested except 221 ytg/1. R-204 58 was conducted. No statistically
significant effects of exposure to R-20458 on survival of
daphnids were observed during the second generation exposure
(Table 13)- During the first week of normal reproductive
activity {days 7-14 of the second generation exposure), production
of young essentially did not occur except for one control
replicate), and only controls produced young daphnids during
the second generation exposure. Upon examination at the end
of the exposure it became evident all (1005) daphnids among
the R-20458 exposed populations were males rather than
parthenogenetic females. Among the control populations however,
the incidence of males was only 17%. Clearly all of the offspring
from R-204 58 exposed first generation daphnids resulted from
the production of male eggs during the first generation. Only
the first generation controls continued to produce mostly
parthenogenetic female eggs.
DISCUSSION
The 48-hour LC50 value of 1.5 pg/1 obtained for TH-6040 was the
same as determined,by Miura and Takahaski (1974}, despite the
fact that these investigators used a mixed age population of
daphnids. Comparable information on R-20458 and Altos idR
is not readily available.. Based" on the data obtained from the
chronic exposures of Daphnia magna to AltosidR, TH-604 0 and
R-20458, TH-6040 was considerably more toxic than the other growth
regulators to this species. Both AltosidR and R-2 04 5S
inhibited reproduction at concentrations lower than those at
which it decreased survival.
Under "normal" conditions, Daphnia can have from 2 to 40
eggs per clutch (Pcnnak, 1953") , It it due to this wide
24
VII-95B
-------
TABLE 14. PRODUCTION OF YOUNG/PARTIIENQGENTIC FEMALE Daphnia
tnacina CONTINUOUSLY EXPOSED TO STAUFFER R-20458' FOR
4 2 DAYS
Younq Produced/Parthcnocfcnotic Female
Nominal/
Moan
GENERATION
1
GENERATION
II
Replicate
Measured
14
21
35
42
TY
Y/?D
TY
Y/f
TY
Y/V
TY Y/f
Control
A
24 2
12
412
51
o ¦
0
71 6
D
18 6
10
659
35
0
0
53 3
C
197
10
455
41
0
0
113 8
D
250
12
402
45
8
8
9 9
X
~TT
43
2
6
15. 6
24
A
152
8
435
27
_ _
B
47
2
613
36
_
C
66
4
605
30
c
-
_
D
25
1
762
40
c
_
X
4
33
31,2
31
A
-
140
7
813
74
-
B
0
0
704
35
c
-
C
55
4
537
54
-
_
D
14
1
779
43
G
-
-
X
3
51
62. 5
62
A
0
0
540
36
c
-
_ _
B
0
0
426
33
_ _
C
0
0
528
28
c
-
D
0
0
616
33
X
0
0
32
125.0
112
A
0
0
570
36
o
_
_ _
B
0
0
392
28
-
_ _
C
0
0
338
24
- -
D
0
0
497
29
_
_ _
X
—
29
250.0
A
221
d
—
—
—
B
C
D
~d
d
-
-
-
_
X
_
_
j^Total Young
'Jouiii.;/par Ihonoyeni11ic Fcma 1 e
^No young due to absence of females in generation
No young due to 100% mortality of first generation adults
25
VII-96B
-------
range of egg production that statistical comparisons of
reproduction between treatments are riot always precise. During
the first week of reproductive activity in the chronic exposure
to 'I'll — G040, the daphnid populations exposed to 0.09 and 0.12
jjg/1 produced an average of 8 arid 10 young per female
respectively. Statistical analysis indicated these values
were significantly^lower than the controls which produced 13
young per female, 'However, we believe that only among daphnids
exposed to 61 pg/1 (measured-at time 0) where no young were
produced, was there significant effects due to treatment.
This assumption is supported by the fact that young production
among daphnids exposed to 0.25 pg/1 TH-60 4 0 (measured at time
0} was comparable to the controls.
As was reported, the development of male daphnids occurred during
the TH-6040 and R-20458 chronic studies, in both treated and
control groups. Males were anatomically differentiated based
on the basis of smaller sixe, larger attenules, and the
presence on the first legs of a stout hook used in clasping.
Pennak (1953) reports that the factors responsible for the
appearance of males in cladoceran populations are not completely
understood. He reports however, the production of male eggs
seems to be induced mostly by: (1) crowding of females and
accumulation of excretory products; (2) a decrease in available
food; and (3) a water temperature of 14° to 17°C. Apparently
the production of male eggs during the chronic exposure of
daphnids to TH-604 0 and R-204 58 were temperature related,
although this is a naturally occurring phenomenon, it appears
that exposure to R-20458 affected the extent to which it-
occurred during the Daphnia chronic.
Table 15 presents the results of previous chronic exposure
studies with Daphnia magna to several pesticides. TH-604 0
is considerably more toxic to Daphnia magna then the other
pesticides listed, with the exception of toxaphene. It is
difficult to assess the toxicity of R-204 58 compared with the
other pesticides listed because a true safe level was not
determined for this compound. It appears that no generalizations
can be made concerning the toxicity of third generation
pesticides (i.e. growth regulators) compared to the more
classically used pesticides (i.e. organo-phosphates, chlorinated
hydrocarbons) to organisms, such as Daphn.1 a, in which ecdysis
occurs regularly. Just as the more conventional pesticides
display a wide range of acute and chronic toxicity to Daphnia,
so do the growth regulators encompass a wide toxicity range.
26
VI1-9 713
-------
TABLE 15. 48-HOUR LC5 0, MATC, AND APPLICATION FACTORS FOR
SEVERAL PESTICIDES TO Jiaohniil magnet
Compound 48-hour LC5 0 MATC Application
(m/i)
Ciig/1)
Factor
AltosidR
89
>25
<50
>0.
28
<0.
56
Tii-604 0
1.5
<0.
61
<0.
40
R-20458
330
<24
<0.
07
Lindane3
485
>11
<19
>0.
02
<0.
04
. b
Atrazme
6900
>140 <250
>0.
02
<0.
04
, . c
Acrolein
57
>17
<34
>0.
30
<0.
60
c
Heptachlor
78
>12
.5 <25
V
o
»
16
<0 .
32
c
Endosulfan
166
>3
<7
>0.
02
<0.
04
Trifluralinc
193
>2
<7
>0.
01
<0.
04
Toxaphened
10e
>0.
07 <0.12
>0.
007
<0
i, 012
aMacek et. al., 1975a
Id
Macek et. al. , 1975b,
CMacek et. al., 1975c
dKPA, 1975
e
48-hour EC50 value
27
VII-98B
-------
CONCLUSIONS
None of the three insect growth regulators displayed a cumulative
toxic effect during a second generation exposure greater than
that observed during the first generation exposure. Based on
these observations it appears that chronic toxicity of this
class of compounds"*-to crustaceans such as Daphnia magna could
be accurately estimated based upon the results of exposure
during one complete life history . Clearly, there exists
significant differences between acutely toxic and chronically
toxic concentrations of two of these chemicals (AltosidR and
R-20453), and evaluation of hazard based solely on acute
toxicity data from static bioassays may be inadequate.
TH-6040 was the only compound, of the three tested, which did
not reduce reproductive activity of Daphnia at concentrations
lower than those which reduced survival. This observation may
be due to the absence of continuous exposure to measurable
concentrations of TH-604 0. R-2 04 58 postponed the release
of juvenile daphnids fit the lowest treatment level (24 jjg/1) .
This level was 9X less than the concentration of R-20458,
in water, which reduced survival, MtosiuR affected production
of young at a concentration approximately 2X less than that
which significantly affected survival. These observations
suggest that consideration of effects of these types of
chenica]s on the reproductive process are required to completely
evaluate hazard to aquatic organisms.
It is possible that R-20458 nay stimulate the production of
rnalG Daphnia magna. Developing an understanding of the ru chanisn
by which this occurs might aid in elucidating the mechanism
of this inadequately understood phenomenon, as it occurs in
nature.
TH-604 0 is considerably toxic to Daphnia magna when compared
to some of the more conventionally used pesticides. The
toxicity of AltosidR to Daphnia is more comparable with some
of the less toxic pesticides. It appears that no generalizations
can be rcade regarding the toxicity of the insect growth
regulators to Daphnia magna.
28
VII-99B
-------
SUBMITTED BY:
PREPARED BY:
APPROVED BY:
Bionomics, E G & G
Aquatic Toxicology Laboratory
7 90 Main street
Wareham, Massachusetts 02571
Gerald LeBlanc
invertebrate Biologist
Bevier llasbrouek Sleight, III
Director, Aquatic 1'ox.ioology
Laboratory
VII-100B
-------
LITERATURE CITED
American Public Health Association 1971. Standard Methods
for the Examination of Water and Wastewater, 13th ed.
Am. Public Health Assoc,, New York.
Arthur, J.W. 1970. Chronic effects of linear alkylate
sulfonate detergent on Garomarus pseudolinnaeus, Carnpoloma
decisun and Physa Integra, Water Research 4: 251-257.
Biesinger, K,E», R, W. Andrew, J.W. Arthur 1974, Chronic
Toxicity of NTA (Nitrilotriacetate) and Mctal-NTA
complexes to Daphnia magna. J. Fish, Res. Bd. Canada
31(4): 486-490.
Committee on Methods for Toxicity Tost with Aquatic Organisms
1974. Methods for Acute Toxicity with Pish, Macro-
invertebrates and Amphibians. U. S. Environmental Protection
Agency, Ecological Research Scries, EPA-660/3-75-009, 61pp.
Drummond, R.A. and W. F, Dawson 1970. An inexpensive method
for simulating diel patterns of lighting in the
laboratory, Tran . Am, Fish. Soc., 99 {2) : 434-435,
Frear, D, and J, Boyd 1967. Use of Daphnia magna for the
Microbioassay of Pesticides. I. Development of Standardized
Techniques for Rearing Daphnia and Preparation of Dosacje-
Mortality curves for Pesticides. Journal of Economic
Entomology, 60(5): 1228-2136.
Macek, K.J., K.S. Buxton, S.K. Derr, J.W. Dean, S. Sautcr
1975a. Chronic Toxicity of Lindane to Selected Aquatic
Invertebrates and Fishes. U.S. Environmental Protection
Agency, Ecological Research Series {In Press} .
Macek, K.J., K.S. Buxton, S, Sauter, S. Gni.lka and J.W. Dean
1975b. Chronic Toxicity of Atrazi.ne to selected Aquatic
invertebrates and fishes. U.S. Environmental Protection
Agency, Ecological Research Series (In Press) .
Macek, K.J., K.S. Buxton, M.A. Lingberg, S. Sauter, and P.A.
Costa 1975c. Chronic Toxicity of Acrolein, Keptachlor,
endosulfan and trifluralin to Daphnia magna and the
fathead minnow (Fimephalcs prorietas) . U.S. Environmental
Protection Agency, Ecological Research Series {In Press).
¥11-10IB
-------
• Miura, Takeshi and Takahanhi, R.M, 1974. Insect Developmental
Inhibitors. Effects of Candidate Mosquito Control Agents
on Non-target Aquatic Organisms. Environmental
Entomology 3(4): 631-836.
%
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 C.E. Stephan 3,967 . A method for establishing
acceptable toxicant limits for fish-malathion and the
butoxyethano1 ester of 2,4-0. Trans, Am. Fish. Soc.
9 6: 185-193.
Perm a >, R. W. 1953. Fresh Water Invertebrates of the United
States. The Ronald Press Company, Sew "York," p. 350-364.
Steele, R.G.D. and Torrie I960, Principles and Procedures of
Statistics. MoGraw Hill, New York. 481 p.
U.S. Environmental Protection Agency Quarterly Research
Report. March 31, 197 5.
VII-102B
-------
PART VII - HAZARD EVALUATION SUPPLEMENT
3. Report of Zoecon Morphological Observations on
Treated Daphnia magna
In an effort to detect morphogenetic changes
in Daphnia magna corresponding to those which
would be produced by the insect growth regulators
on insects, sample organisms from the bioassays
were randomly withdrawn at regular intervals
for microscopic examination. Examination was
performed by an insect physiologist at Zoecon
under a stereo microscope at 25X. Because no
particular response could be predicted, general
inspection of treated daphnids was performed
in comparison to untreated controls. A record
of the rate of growth of the daphnia took the
form of measurements of the anterio-posterior
axis by means of an ocular micrometer. This
carapace length was plotted against time to compare
growth rate with untreated controls. Neither
unusual morphology nor effects on rate of growth
were evident as compared to controls with any
of the insect growth regulator treatments.
Figures 1 through 10 present the graphical
representation of the data as recorded numerically
in Tables 1 through 10. As the first bioassay
completed, every dose level of the ALTOSID treatment
was analyzed; however, only control and highest
surviving dose was analyzed for the compounds
R-20458 and TH-6040. In several cases a combination
of highest surviving treatment levels was made to
provide sufficient data. The measurements, while
not elucidating rate of growth differences of
sign!figance or revealing any morphogenetic action,
did point out the difficulty of visual selection
of young for a second generation.
The synchrony of the so-called second generation
was sufficiently lost so as to obscure the rate
of growth trend in treated as well as control
daphnids. This loss of synchrony is apparently
owing to the potential for a 168 hour spread in
the ages of daphnids used to start the second
generation. It appears that aquatic biologists
are unable to distinguish the age (size) with
confidence. Bionomics biologists propose two
general solutions to this problem for future work.
VII-103B
-------
PART ¥11 - HAZARD EVALUATION SUPPLEMENT
One solution would be to increase the number
of organisms in the first generation to such an
extent that a synchronous second generation of
young could be selected. Sufficient females would
have to be used to assure that a certain number
of young daphnids would be produced in a narrow
time range, say twelve hours. The difficulty
in this approach is that the census for survival
and reproductive capacity in the first generation
would be a task of monumental proportions. All
daphnia counting is presently done by hand as
solutions are transferred. Moreover distinctions
between adult and juvenile daphnids is performed
visually on the basis of size at this time, A
Zoecon proposal for practical variation on this
theme is to have a large treated population in
a separate aquarium at each toxicant level in
addition to the four replicate aquaria which would
be counted at each time interval. The purpose
of this large single group of organisms would
be only to supply the second generation daphnids
and not to provide first generation survivability
and reproducab.ility information.
The second proposal was to employ some sort
of automatic counter/sizer to enable the aquatic
biologists to handle large populations in each
replicate. At the start of the second generation
the young could possibly be sorted by using a
sizing mesh with the phototropic or other natural
response used to encourage passage through the
sizing screen.
The former proposal as modified seems most
immediately practicable. On the following pages
are found the tables and figures indicating rate
of growth of Daphnia magna in the presence of
the three insect growth regulators.
VIJ-104B
-------
(!) (lib
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s t*
2 tti
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VII-105D
-------
qowth & development of daphxea warn in toe presence of insect growth regulators as hdicheed by camPACE lejgeh
71.Ely
E5.E27
t
i
EB.ESj
4
f
S.E2t
FIGURE 2 - ALTQSID - 800 ug/L ICKEXRL DOSE LEVEL
-j- = first generation
— = second generation
Relative ler.cth units'
times 39.2 = absolute
length in irdcrons
SI.SEi
HS.E2-
H2.2Z-
~*,r—
a.Zz
23. EH
2B.E2-
ia.EZ-
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+
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tsa
tc
r«a
¦>v.
*
n
SSI
tm ->
•
L.1
in
aa
!S
L"?
DAYS OF EXPOSURE 10 l
-------
G50WIH & DEVELOPMENT OF DlgHXZA MACXA IN THE PRESEKCE OF INSECT GSDWIH REGULATORS AS INDICATED BY CARAPACE L5KGIH
73.EDt
EUSf
E.23-
EH.iZ-
HS.EE
H2.SZ-
iS.Sf--
^57 ^rr7_
2S.E2--
22.22-
FIGURE 3 - ALTCSID - 400 ug/L ICMIWi DOSE LEVEL
+
/*
i
-j- = first generation
= second generation
Relative length units
tiiras 39,2 = absolute
length in microns
1S.E*
IS.E.^r
££1
£2
EM
S3,
p.3.
r<
IS4
m
r.i
r- ;
trt
r.:
13
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a"
ta
m
til
rji
rvi
J:..
», -
m
LI
HAYS OF EXPGSDBE TO IGE
-------
GROWTH fi DEVELOPMENT OF DAPHNES KCR IN THE PRESENCE OF INSECT GROWTH REGUXATORS AS UDIGMED BY CARAPACE XESGTH
72.02-
B.E2<
£3.EE-
££.E
SH.SK
*"il. iZj
3H.S2-!
FIGURE 4 - ALTOSID - 200 Ug/L NOMINAL DOSE I£VEL
32 .^r
~Vr T*
= first generation
^ = second generation
Relative length units ^
tines 39,2 = absolute trr
, length in mesons
© Sm
t_ *L_ J >
. C-L±;
^ &
Ia.£34
£zl
sa
ry
m
Irt
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A
ru
r^j
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QMS OP EXPOSURE TO IGR
-------
GROWTH & DEVELDPMS2fT OP DaPtgSR. fflGXA. EH THE PRESENCE OF INSECT G33WTH REGULATORS AS INDICATED BY CMMXE ISN'GTH
?2.22t
FIGURE 5 - ALTOSID - 100 ug/L NOMINAL DOSE LLVEL
ES,E?r
EZ.ZZr
S3.B3-J
13.22-
H3 3>
2J.22-
33.E2t
2£" .zZt
~n. i?nl
—A*- « Lb ,
-j- = first generation
^ = second generation
Relative length units
times 39.2 = absolute
length in microns
6)
, T\ "
/*P\
<37
0
®
©
0
e
i
i
IC.L'rJr
:s
rs
"slf
fM
r/j
c^;
m
hi
tA
L3
Eu
ra
A
m
S3
£53.
R
fU
ES
S3
sr
rM
DAYS OF EXPOSURE TO IGR
-------
GRCWTH 6 DEVELOPMENT OF DftPHNEft. mom IN THE PEESEICE OF INSECT GROWTH REGULATORS PS INDICATED BY CARAPACE LENGEH
* FIGURE 6 - ALTOSID - 50 ug/L NOMINAL DOSE LEVEL
73.E57 ,
+
<
M
M
O
ffi
Ea. 22
EE. EE
S£.E2~
EZ.EZ
HZ.ZZ
H3.E2
2K.Z2
23.02
-%-y
is, m~
= first generation
(J) = second generation
Relative length units
tines 39.2 = absolute
length, in microns
Et:
1 b
-CL.
/
¦^r
_r
-f7
n_
^ i
rr\
¦/#* % •
Lr-rr-
/V.
\u
—
tB
rv
fi
-jg- |-
K
rave rw? i?vTy%erTi>t7 mr* tvt>
-------
Gacwas & mmmrntn of msmm mem in the presence of insbct growth begumtoss as indicated by carapace lensih
?2.S3t
ES.2Z
ES.iZj
SS.E2-
E2.K-
FIGURE 7 - TO 6040 - CONTROL
—j- = first generation
= second generation
Relative length units
tines 39.2 = absolute
length in mcrons >
+
-------
GSDWni & DEVBD0PMB3T OF DaPHMEft. MflGSR. IN THE PBESBCE OF INSECT GHCWIH REGLILKTORS AS BCiICOTED BY CARAPACE LESEZH
FIGURE 8 - TH 6040 - 1.000, 0.500, 0.250 ug/L SOEfflL DOSS LEVEL
-4-" first generation (1.000 ug/L)
EE. EE'
E5.2Z
55. E* ¦
Sa.ES--
4S.05
m.m
3S.SS
33. EE-
H.z:.:
2B.H2-
IS.EB-
!EI. ~;'J :
+
23 —
r<
S
pJ
E2
at
m
Lrt
13
second generation (0.500 ug/L}
PH= second generation(0.250 tsg/L)
Relative length units
tirres 39.3 = absolute
length units in microns _
BJ
KJ
tn
V
2S3
S3
ffl
JS
BB
rn
I
rn
DJ
ZXJ
I pum|i |""-l !¦! mi ill
tPPj ^
(j)
PM
n>j
rw
csa
R
-------
•G3CWIH & DEVELOPMENT OF DAPKNEA. Mft3& IN THE PRESENCE OF BJSBCT G30WTH REGIEATOSS AS INDICATED BY CARAPACE LENGTH
72JZt
E.Z2-
E2.03-
SS.B3-
sa.Esj _
FIGURE 9 - K20458 - eOSTROL
¦f
= first generation
= second generation
Relative length units
tines 39.2 = absolute
length ir. microns.
' 6
^ ©
£t.
,2?
^ F'
.©
CD
IS
nn
£S
-
m
zr
ca
zsi
c:
lj-j
en
m
FS *
133
m
rm
S3
SI
ru
ru
R
S
ESL
ESI
1/1
P4
LO
DJ
ess
csa
K
~3
PJ
-------
GFOWTH. & DEVELOPMENT OF ESftPHNEA MftGNA IN THE PRBSECE OF INSECT G30WTH REGULATORS AS INDICATED BY CARAPACE LENGTH
FIGURE 10 - R20458 - 250.0, 125.0 ug/L NCKEMAL DOSE LEVEL
n?-*-
IB.ESs
KI
SI
S3
sa
s
rn
n
*> ^
S3
t/1
iS
UJ
K»
aa
is.
is
E3
m
-j-= first generation (250.0 ug/L)
-££)= second generation(125.0 uq/L)
Relative length units
tiiiass 39.2 = absolute
length in irdcrcr.s
0
$
W-
0
$
cm
BBSS
DAYS OP KXPnSJTPP 'no -rco
-------
J
GB0TOH & DEVEX/QEMEMF OP naPHNEft MBGMR IN THE PRESENCE OF INSECT GROWTH RBGULKTQKS AS XtOIC&TED BY CftMPflCE IENGIH
MAXIMUM • TKBI£ 1 : ALTO5ID
AGE/DAYS
SL'-'tPLS (CUMULATIVE STAS OF DOSE GSOUP ACTUAL REPLICATE RELATIVE LENGIH
' ssser exposure devsick-eot (hcmesvl coscesthatioh vq/l) cxxjce^rikw merskrbebs
TIME/DAYS)
r-f Ol
o o
o o
0.833
0.833
Juvenile
II
0QOTR0L
0
" • "
A
B
23,23,24
22,23,23
123)
02 i
026
1.5
1.5
Juvenile
ff
A
B
24,28,29
23,24
(25.25)
049
050
1.83
1.83
Juvenile
»
A
B
28*30
29,30
• (28.75)
073
074
2.8
2,8
Juvenile
'
A
B
31,27
28,30'
(29)
09?
093
4.5
4.5
Adult
If
A
B
27,30,45
31,33,33
• (33.15)
121
122
7.5
7.5
Mult
«
A
B
40,45,50
35,53,60* .
i
1
(51.3)
o o
o o
fO M
7.0
7.0
(21)
Wide Age Range
A
B
21,25,25 '
/
{23.66) j
025
026
7.66
7.86
(21.7)
11
SI
2
26,30,35
36,48,48
(36.45) j
s
3
049
050
8,0
8.0
(22)
If
»
;
I
i
A
B
22,26
46,49,50
1
(36.15)
073
074
9.0
9.0
(235
M
11
I
¦
A
B
27,28,32
25,28,31
!
i
(28.6) |
J
09?
098
10.66
10.66
{24.5}
31
11
i
i
t
A
B
44,46,50*
i 26,26,30
I
!
(36.85) j
I
I
i
¦
J * Samples tearing eggs }
-------
GBOWEH & DEVELOPMENT OF OMR ftRQR IK THE PBESJ2CE' OF BSBCI GROWTH REGULATORS AS BJDICMH3 BY CARAPACE t.fmotw
®BIE 2 : ALTOSID
:.-j>3zr
MAXIMUM
, AGE/DAYS
i{CUMULATIVE
exposure
' TIME/DAYS)
STAGE OF
DEVEUDPMEKT
DOSE GROUP
(NOMINAL CONCEMimTION ug/L)
actual
coxcEcrmrias
REPLICATE
RELATIVE LEHGTH
JEASUREJEfTS
005
0.833
I
Juvenile
800 ug/L
482 + 104 ug/L
A
22,23,23
006
0.833
If
B
23,23,24 {23)
029
1,5
Juvenile
A
25,25,25
030
¦ 1.5
If
B
24,30 (26)
053
1,83
Juvenile
A
24,24,32
054
1.83
IS
B
24,23,23 , (24.8)
077
2.8
No Sample / Died
A
/
078
2,8
B
/
101
4.5
No Sarple / Died
A
/
102
4.5
!!
B
/
125
7,5
No Sample / Died
|
A
/
126
7.5
It
i
1
B
/
No 2nd generation
due to 100% kill
of 2nd generation
-------
".'Z2-3S3.
GHCWEH & nEVELOPMEOT OF DAPtEEA tRGKft IN THE PRESENCE OF INSECT GBCWTd REGULATORS AS INDICATED BY CARAPACE L£2>JGTH
TABLE 3 ; ALTO5ID
MAXIMUM
AGE/DAYS
(CUMULATIVE
EXPOSURE
TIME/DAYS)
STAGE CF
DEVELOPMENT
DOSE GROUP
(NOMINAL OOXCCvTRITicx ugA)
rcrjAL
coxc-^vriVvTiox
REPLICATE
RELATIVE LENGTH
MSASURE5EOTS
009
010
0,833
0.833
Juvenile
It
400 ug/L
l* 97 + 20 ug/L
A
B
i 22,23,24
23,23,24
(23) "
033
034
1.5
1.5
Juvenile
If
i
!
A
B
I
24,24,26
24,26,30
(25.6)
05?
QjS
1,83
1.83
Juvenile
11
|
A
B
28.28.28
24.23.29
(27.5)
031
0fc2
2.8
2.8
Juvenile
11
A
B
27,29,31
33
(31}
105
106
4.5
4.5
Mult
1*
A
B
30,35,46
34,35,36
(36)
129
130
7.5
7.5
Mult
it
1
1
A
B
43,54,5?
52,52,50
,
i
j
'
No 2nd generation
due to 100% kill
of 1st generation
i
i
,
1
[ St
\
1
{
f
J
|
-------
GROWTH & DEVEUXWEOT OF DftFHMEA MAGNA IN THE PRESENCE OF INSECT GROWTH BBGULftTORS AS INDICATED BY CARAPACE LENGTH
TABLE 4 : ALTCSID
MAXIMUM
AGE/DAYS
(CUMULATIVE STAGE OF DOSE GROUP ACTUAL REPLICATE RELATIVE LENGTH -
rC-S2R ' EXPOSURE DEVELOPMENT (NOMINAL CQNKSIOTRATICN uc/L) COXCEN'r^TICN JSEASUREME23TS
TIME/DAYS)
013
014
0.833
0.833
Juvenile
11
200 ug/L
j 51+13 ug/L
A
B
23,23,23
22,23,23
(23)
037
038
1.5
1.5
Juvenile
rt
A
B
23 j 25
23,23,25
(23,8)
061
062
1.83
1.83
Juvenile
if
A
B
25,26,28
28,28,30
(27.45)
085
088
2.8
2.8
Juvenile
11
A
B
30,36,37
29,30,30
(32.15)
109
110
4.5
4.5
Mult
11
A
B
38,43,50
35,38,39
(405
133
' 134
7.5
• 7.5
Mult
tr
A
B
57,57,58
60', 59,58
(57.5)
013
014
7.0 t215
Wide Age Range
M
A
B
37,43
36,38,38'
(38.8)
037
033
' 7*.66 (21"7)
11
M
A
B
32,38,44
27,42,41
(37)
061
062
lfQ (22)
W
I!
A
B
35,40,43
37,41,42
;
(39.65)
085
085
»;j} (235
H
II
A
B
48,50,58
48,49,51
(50,6)
109
110
S:S«24-5>
11
tt
i
i
i
i
1 A
; b
1
l
\
i
33,43,48
42,47,53
,
(45,3)
-------
growth & mvemFma of drphsdsr. magna in hie presence of insect srcwtb regulators as mjickth} by carapace length
TABLE 5 i ALTOSID
c:\p^p
!w3ZR
MAXIMUM
AGE/DAYS
(CUMULATIVE
EXPOSURE
TIME/DAYS)
STAGE CF
DEVEiO?>E>H"
DOSE GROUP
(NOMINAL COSCETOATIOM ug/L)
ACTUAL
CG^CEyrmrxos
REPLICATE
relative nerem -
MEASUREMENTS
01?
018
O.S33
0.833
Juvenile
rf
100 ug/L
' 27+6 ug/L
A
B
23,24,2j
23,23,23
(23.5}
041
042
1.5
1.5
Jwenile
it
A
B
24,30,35
25,34
(29.55)
065
066
1.83
1.83
Juvenile
11
A
B
23,29
24,26,29
(26.15)
0^3
093
2.8
2.8
Juvenile
II
A
B
27,28.28
23,28
(26,75)
113
114
4.5
4.5
Mult
H
A
B
37,45,46
34,38,39
(38.8)
137
138
7.5
7.5
.Mult
n
A
B
57,57,58
58,60,68*
(59,5)
017
018
7.0
7.0 (2D
Wide Age Range
E!
A
B
25,25,34
24,30,30 .
(28)'
041
042
If
rr
A
B
32,32,37
27,30,32
(31.6)
085
066
o*2 <22)
8.0
rr
r»
A
B
30,35,35
28,29,34
(31.5)
089
090
9-0 {235
9.0
n
rr
A
B
38,45
29,29,29
(35.25)
113
114
10.66 ,
10.66(24-5}
rr
ii
1
f
f
s
!
i
A
B
26,30,32
44,45,51
{38}
-------
aomi £ DEVELOPMENT OF PAPHNE& mOR IN THE PHI5BCB GF INSECT GKCWTH REOJLATORS AS INDICKTED BY CARAPACE LENGTH
TABLE 6 : ALTOSID
MAXIMUM
AGE/DAYS
£-;-S?I2 (CUMULATIVE STAGE CF DOSE GROUP ACTUAL REPLICATE . RELATIVE LENGTH T.
>:J!2SR EXPOSURE DEVELOPMENT {NOMINAL CONCENTRATION ug/L) COXCSfESKriOK MEASUREMENTS
TIME/DAYS) ________________
021
0:2
0.833
0.633
Juvenile
If
50 ug/L
12+3 ug/L
A
B
24,24,24
23,23,24
(23.5)
045
046
1.5
1.5
Juvenile
It
A
B
29,33
25,33,33
(30.65)
OSS
070
1,83
1.83
Juvenile
n
A
B
29,29,30
27,30,32
(28.3)
1 3
2.8
2.8
Juvenile
t!
A
B
23,35,36
. 28,29,29
(30.5)
117
118
4.5
4,5
Mult
ii
A
B
39,43,45
38,43,45
(42,15)
141
142
7.5
7.5
Mult
1?
A
B
51,57,62
52,60,66*
(57.95)
021 '
022
(21)
Wide Age Range
rt
A
B
47,47,49
25,34,48
(403*
045
046
^cai.r
tf
n
A
B
30,32,39 ¦
31,31,39
(37)
069
070
H (22)
t*
it
A
B
; 44,47,48
38,42,49
(44.65)
OS 3
0S4
!:S (23^
n
n
A
B
49,51,58*
36,37,40
(44.5)
117
113
ig0:^24-5
If
11
i
i a
! B
43,44,44
41,46,49
(44,65)
i * Samples bearing eggs
-------
2-3E
001
002
025
036
019
0 0
073
074
097
098
121
122
001
002
018
019
035
036
GROWTH & DEVELOPMENT OP OftPHNEA. HROR IN THE PBBSEBCE ,0F INSECT GROWTH BEOJEATORS MS INDICATED BY CARAPACE USJGIH
TABI£ 7 : TO 6040
MAXIMUM
AGE/DAYS
{CUMULATIVE
EXPOSURE
TIME/DAYS)
STAGE OP
devhjopment
DOSE GRGCP
{NCMIKAL O3NCEKIRATI0N uy/L)
ACTUAL
CONCEOTRAXICK
REPLICATE
RELATIVE UENGOH
MEASUREMENTS
22,24
21,22,23
Juvenile
(22.5)
20.21
21.22
22,27,28
22,22
23.8)
26,27,28
27,29
Juvenile
it
(27.5)
39,42
41,41,45
(41.25)
38,39,39
44,52
Mult
(43.5)
37,38,38
17,36
Wide Age Range
7lo (213
36,37,43
21,32,39
8.0 (22)
(34.6)
36,37,37
27,27,42
9.0 (23)
(34.5)
-------
S-:-^LE
mxmz & EEmj&msr of baphnea mom, in toe presence of insect growth lEGamTOBS as indicated by carapace lenxjth
TABLE 8 s TO 6040
MAXIMUM
AGE/DAYS
(CUMULATIVE
EXPOSURE
TIME/DAYS}
STAGS OF
DEVSIDPK2NT
DOSE GSOCJP
(XOlMINAL CCNCENrRATIOM ug/L)
ACTUAL
COXQ-ATRATIGX'
REPLICMS
BELKEOT XfiJGHi
MEASUKEMEETIS
0.833
0.833
1.5
1.5
1.83
1.83
2.8
2.8
6.5
6,5
6.5
6.5
7.5
7.5
Juvenile
Juvenile
«
Juvenile
11
Juvenile
it
Mult
H
II
9*
Adult
1.000 ug/L
1.27 ± 0.13
ug/L
initially
0.500 ug/L
0.93 ± 0.65
ug/L
initially
A
23,23,24
B
23,23
(23.2) *
A
22,22,23
B
19,23
(21.5)
A
23,23
B
23,24
(23.25)
A'
25,26,27
B
24,24,26
(25.3}
A
31,35
B
36,39
C
33,34,35
D
32,35,40
(34.9)
A
36,37
B
33,35,48
{41.85
A
13,51
(34.5)
A
49,52,
(50.5)
A
33,60*,60*
(52.6)
A
26,26
E
27,34
C
25
(28)
A
.30,29
B
26,31,41
C
28,29,30
(31.3)
A
32,42
B
39,45
C
32,32,37
(37)
005
007
003
043
041
042
7,0 (21)
8.0 {22)
9.0, (23)
7.0
7-0 (21)
7.0
8.0
8.0 (22)
8.0
9.0
9.0
9.0
0.500 ug/L
0.250 ug/L
0.93 ± 0.65
ug/L
initially
0.25 + 0.07
ug/L
initially
(23}
-------
S.-.'PLE
®CMTii & OF DfiPHKBl MAGKft. IN THE PRESENCE OF INSECT GROWTH REGULATORS AS XMJICRTED BY CARAPACE IB3GIH
TABLE 9 : R20458
MAXIMUM
AGE/DAYS
(CUMULATIVE
EXPOSURE
TIME/DAYS)
STAGS OF
DEVEtOPMHOT
DOSE GROOP
(NOMINAL CX}SGENimTICS3 ug/L)
ACTUAL
coscE'SmTioH
REPLICATE
RELATIVE LBJGUI
MERSUKEMESTS
001
002
0,833
0.833
Juvenile
ii
I OONTRX
f "" " ' 11 ' n... «...
A
B
21,23,23
20,22,22,23
{22.15)
025
026
1.5
1.5
Juvenile
II
A
B
22,24
22,22
(22.5)
P Q
0j0
1.83
1.83
Juvenile
It
'a
B
22,23,28
21,23,23
(23.3)
3
074
2.8
2.8
Juvenile
If
A
B
26,27,23
25,27,28
(26.8)
09?
038
6.5
6.5
Mult
If
A
B
42,45,46
41,42,46
(43.651
111
122
7.5
7.5
Mult
11
A
B
49,61,81
52,57,57
(56.15)
001
0)2
7.0
7.0 (21)
Wide Age Range
A
B
30
28
(29).
0 I
o ¦>
8.0
8.0 {22}
It
tt
A
B
28,26,26 •
32
(29)
Oil
042
9,0
9.0 (23)
ii
if
i
A
B
31,31
32,37,38
(33.3)
061
062
13,0
13.0 ("5
(2
If
1 A
j B
29,32,35
34,42
(35)
081
032
14-° C28i
14.0 (28)
.
II
It
!
i
j
i
i
f
1 i
!
!
A
B
25,32,35
35,41,47
(35.8)
-------
£?:-3LE
KH-3EH
GKCfilH & DGV110RCOT OF DAPKMEA MBQm IN THE PRESENCE OF INSECT GROWTH REGULATORS AS INDICATED BY CARAPACE LEHSTO
SffiLB 10 s R20458
MAXIMUM
AGE/DAYS
(CUMULATIVE
EXPOSURE
TIME/DAYS)
SKIGE OF
DEVELOPMENT
DOSE GROGP
(MMEKKL CXJNC^TRATION ug/L)
ACTUAL
COXCEvTRKTION
RESLICaiS
RELATIVE LEXGIB
MEASUREMENTS
005
006
0.833
0.833
Juvenile
n
250.0 ug/L
j 221 ± 71
i ug/L
J
A
B
22,22,22,22,23,23
21,23,23 (22*15)
025
030
1.5
1.5
Juvenile
tf
A
B
22.22.22
21.22.23
(22)
053
054
1.83
1.83
Juvenile
fl
A
B
22.22.22 '
22.22.23
(22)
077
076
2.8
2.8
Juvenile
1!
a'
B
23.23.23
23.23.24
(23)
105
106
6.5
6.5
Adult"
fr
125.0 ug/L
112 ± 24
ug/L
A
B
39,40,42
40,42,45
(41.3)
129
130
7.5
7.5
• Mult
tf
A
B
49,52,57
43,51,53
(50.8)
005
006
7.0
7.0
(21)
125.0 ug/1
112 ± 24
ug/L
a
B
27,17
17,24,28
(22).
025
026
8.0
8.0
(22)
A
B
29,32,33
35,39
(33.6)
045
046
9.0
9.0
(23)
A
B
31,32,34
33,34,36
(33.3)
065
066
13.0
13.0
<27)
j
A
B
37,38,40
41,42,43
(40.1)
085
036
14.0
14.0
(23)
A
B
38,38
40,41,43
(40) i
-------
PART VII - HAZARD EVALUATION SUPPLEMENT
BIOASSAY OF INSECT GROWTH REGULATORS WITH PALAEMONETES
PUG 10
1. Abstract of Results
For the initial assessment of the sensitivity
of the marine grass shrimp Palaemonetes pugio
to the three insect growth regulators, a static
acute 9 6 hour LC,-n was performed. It was readily
apparent that grass shrimp were significantly
less sensitive than water fleas to the presence
of the insect growth regulators. Median lethal
concentrations of from 6 to 425X the Daphnia levels
were recorded for Palaemonetes pugio. The Bionomics
scientists report that this appears to be higher
than a range of median toxic levels reported for
similar grass shrimp species exposed to standard
pesticides; however, few directly comparable results
on shrimp of any kind are available.
The subchronic exposure of the grass shrimp
to Altosid, R-20458 and TH-S040 was complicated
by technical problems. Winter acclimated Palaemonetes
pugio resisted attempts to induce spawning. Further-
more a significant natural (but extremely uncommon)
alteration in the salinity of feed stock sea water
occurred. Although the reduction from a normal
of approximately 25 parts per thousand to a low
of 4 parts per thousand did not noticeably affect
the survival of untreated control grass shrimp
the salinity stress might have exaggerated toxic
response to the insect growth regulators. The
grass shrimp unfortunately proved too fragile
to manually transfer and count on a weekly basis
so the timing of the expiration of the larval
shrimp with respect to the salinity change could
not be defined.
The purpose of a subchronic 35 day exposure
was to pinpoint narrow sensitive stages which
might be developed into "rapid safety screens".
Because of the single evaluation point the work
did not elucidate such shorter bioassays of any
meaning. The work did, however, point out the
inadequacy of standard static acute testing to
indicate potential for subchronic or chronic hazard.
There was a thousand fold factor between the lowest
effect levels in a subchronic sense and the median
acute lethal levels (the salinity change might
be a factor, however).
VII-125B
-------
Table III
Summary of Chronic Bioassay
Data with Palaemonetes pugio
Insect
Growth
Regulator
Static
96 Hour
LC n (95%
Conf. Interval)
mg g/LEppm
Lowest Chronic a
Concentration
Producing Effect
on Survivability
yg/L=ppb
Altosid
¦
>10 HDT*
972
TH-6040
0.64
(0.13 - 3.1)
0 .625
R-20458
2.0
(1.4 - 2.7)
261
* HDT = Highest dose tested,
a
Mean measured concentration during test except for
TH-6040 - see text.
VII-126B
-------
PART VII - HAZARD EVALUATION SUPPLEMENT
2. Report of Bionomics Experiments
On the following pages can be found the final
report on chronic toxicity of Altos;id, TH-S040 and
R-204 58 to Palaemonetes puqio as performed by EG&G,
Bionomics Aquatic Toxicology Laboratory, Pensacola,
Florida.
VII-127B
-------
THE ACUTE AND SUBCHRONIC TOXICITY
OF R-20458, ALTOS ID AND TH-6040
TO THE GRASS SHRIMP, Palaemonetes
Rtmo.
Final Report
Submitted to
Zoecon Corporation
Palo Alto, California
Bionomics - EG&G, Inc.
Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 32507
December 1975
VII-128B
-------
INTRODUCTION
1
The determination of the chemical composition of natural
hormones which are critical for the normal growth and development
of;insects has led to the formulation of synthetic analogs for the
control of insect pests. Two generic types are of greatest con-
cern at this time. The juvenile hormone type acts by inhibiting
the normal metamorphosis of insect larvae to adults, killing them
in the pupal stage. Stauffer Chemical Company's R-2G458 and Zoecon
Corporation's ALTOSID are examples of this type of insecticide.
The other type of insect growth regulator is a .chitin inhibitor."
Members of this class of insecticides prevent the normal production
and deposition of chi tin by the insect, thus causing malformed
exoskeletons and abnormally developed larvae which do not survive
to the adult stage. Thouipson-Hayward Cherni cal Company's TH-6040
is an example of this second type of insecticide (Miura and Takahaski ,
1974; Sanders, 1975).
Since insecticides are often applied .in areas and under condi-
tions which impact both freshwater and estuarine ecosystems, it is
necessary to assess the nature and magnitude of this impact on
aquatic species. Due to the phylogenetic relationships including
patterns of growth and development between-insects and crustaceans
and because of the mode of action of these growth-regulating insecti-
cides, it is probable that crustaceans will be the most susceptible of
all non-target aquatic species to these compounds. Obviously, the most
important crustaceans in terms of dollar value and human consumption
are the shrimp. Total U.S. shrimp landings in 1369 were 318.5 million
pounds (heads-on) with a value of $124.5 million. By comparison, total
crab landings were 257,3 million pounds with a value of $43.1 million
-------
2
(U.S. Department of Commerce, 1972). Shrimp are also of great
significance in aquatic food webs.
The grass shrimp. Palaemooetes pugio,was selected for these
studies because of its ecological significance, its developmental
pattern (which includes 10-13 zoeal stages, post!arval and adult
stages wi th a molting" rate known to be affected by various internal
and external factors such as type of food available), the signi-
ficant anatomical changes which occur at each molt> and its amenabi-
lity to rearing under experimental conditions in the laboratory
(Broad» 1 957a,b; Williams, 1985; Little,. 1968; Sandifer, 1973).
The effects of the three growth regulating insecticides of
interest on the survival, growth, and norma1 development of
Palaemonetes pugio were investigated under acute and subchronic
conditions.
Spawning attempts
In order to closely control the age and recent acclimation
history of larval grass shrimp to be used in the subchronic portion
of this study and to provide shrimp on demand even under unfavor-
able environmental conditions, we attempted to induce egg deposi-
tion in the laboratory by winter-acclimated P. pugio collected from
^ nrrw ¦ uBiiiiii 'n*T ml . mi i <
the field. Techniques employed were based on the methods reported
by Little (1968), Sandifer (1973; 1974, personal communication)
and Tyler-Schroeder (1574 and 1975 , personal communications) and
involved the controlled adjustments of water temperature and photo-
period to which the shrimp were exposed, P_. pugio were maintained
in continuously filtered sea water in duplicate 75-liter (£) glass
aquaria which were covered completely with opaque, black plastic
sheeting. Some shrimp were collected by seine from Big Lagoon,
VII-130B
-------
near Bionomics Marine Research Laboratory, and others were kindly
supplied by Ms. Dana Beth Tyler-Schroeder, of the EPA, Gulf Breeze
Laboratory, Gulf Breeze, Florida. Initially, shrimp were placed
in tanks which contained filtered sea water at a temperature and
salinity equivalent to ambient field conditions at the time of
animal collection,. Photoperiod was also adjusted to ambient condi-
tions at that time. After a short acclimation period, water temper
ature and light cycle photoperiod were increased in a stepwise
manner according to the regime reported by Little (1968).
Concurrently with these attempts at egg induction,, we con-
structed a brood chamber to facilitate the hatching and collection
of larvae from gravid shrimp, The chamber is based on a design
by Tyler-Schroeder (1975, personal communication). It is composed
of a 35-z glass aquarium (which serves as a holding tank and a
water bath) with a stand pipe drain and contains four battery jars.
Filtered sea water flows by gravity into a splitter head box which
divides the volume into four equal streams. This flow passes via
glass tubing through each of four large bore glass brood tubes,
suspended one on each side of the water bath. One ovigerous shrimp
is placed into each of these four tubes. Upon hatching, which gen-
erally occurs during the night, zoea are swept into the battery
jars where they are held until removed by the investigator. By
using this apparatus, it is possible to prevent predation of
larvae by adult shrimp and to isolate the progeny of one female
from those of others.
While a significant amount of time and effort were expended
in these attempts at induced, gonadal development and egg deposition
these attempts were unsuccessful and larval shrimp used in this
VII-131B
-------
4
study were obtained from ovi gerous females which were collected
from the field and isolated in separate containers in the labora-
tory.
VII-132B
-------
5
MATERIALS AND METHODS
Test materi als
Compounds for testing were Stauffer Chemical Company's R-20458,
Zoecon Corporation's ALTOSID, and Thompson-Wayward Chemical Company's
TH-6040. The sample R-20458" was a clear, amber-colored liquid with
a viscosity similar to light-weight machine oil and was labeled,
"Lot # 2623-16-2» 75% A.I." ALTOSID was a clear, light yellow-
colored liquid with a viscosity similar to that of R-20458 and
was labeled, "Lot # 011033, 90.7% A.I., Technical." TH-6040 was
a fine white powder with the consistency of flour and was labeled,
"Lot v P P -1 2 4 , 98% A.I. , Technical." Stock solutions of each com-
pound were prepared by dissolving the compound in rea gen t-gr arte
acetone. Concentrations are reported as micrograms (pg) of com-
pound per liter of sea water or parts per billion (ppb) and
milligrams {mg) of compound per a of sea water or parts per million
(ppm).
Test animals
Grass shrimp, Palaemonetes pugio Holthuis, were collected by
seine from Big Lagoon, an estuary adjacent to Bionomics Marine
Research Laboratory, Pensacol a, Florida. In the laboratory, IP.
pugio were separated from other related species according to the
characters described by Williams (1965). Hale and non-gravid female
adult shrimp were used in acute toxicity studies and ovi gerous fe-
males were separated from the others to provide larvae for the
subchronic study.
Test procedures
A. Acute toxicity
Static, acute {96-hour) toxicity tests were conducted according
VTT_1
-------
6
to the protocol described in "Methods for Acute Toxicity Tests
with Fish, Macroinvertebrates, and Amphibians" {U.S. Environ-
mental Protection Agency, 1975). Test containers were 3.8-£
(1- gal 1 on) uncovered glass bottles which contained 2 I of
filtered {5 micrometers), natural sea water. Salinity was
26 parts per thousand (°/oo), temperature, 17+1 degrees Celsius
(°C) , a n d' i n i t i a 1 pH, 8.3+0.5 for all acute tests. Each bottle
contained 5 shrimp and each test concentration and the control
was duplicated. Initial dissolved oxygen {DO) concentrations
were from 92 to 98% of saturation (6.7 to 7.1 ppm) and solu-
tions were not aerated during these tests. Concentrations of
each compound to be used in definitive acute tests were based
on preliminary bioassays. The appropriate amount of each com-
pound, dissolved in reagent-grade acetone, was added directly
to sea water in the test containers to obtain the desired con-
centrations. Controls received a volume of acetone equivalent
to the greatest amount added to any of the test concentrations
in that series.
The 96-hour LC501s (the concentrations of the compounds
which were lethal to 50% of the test populations within 96
hours) and their 95% confidence limits were calculated based
on mortality data. Each test concentration was converted to
its logarithm and each corresponding percentage mortality, to
its probit. The LC501 s were then calculated from the resulting
linear regression equation.
Subchronic toxicity
The main component of* the subchronic toxicity test system was
a 500 milliliter (mi.) proportional diluter (Mount and Brungs, 1 967) ,
VII-134B
-------
7
constructed with a dilution factor of 0.5, Each test compound,
dissolved in acetone, was delivered from a glass syringe to the
chemical mixing chamber and from there, to the chemical cells. Mix-
ing of uncontaminated diluent sea water from the water cells with
test solution in the chemical cells produced the 5 test concentrations
which were distributed to the appropriate test containers. The
flow rate of 25,/h resulted in a 95% replacement of water in test
containers in 20 hours (Sprague, 1969). Natural sea water from
Big Lagoon was filtered (10 micrometers), pumped into a 4,000
gallon reservoir and flowed by gravity into the laboratory.
A solenoids controlled by a timer and float switches, regulated
sea water flow into water cells.
Selection of concentrations to be used in chronic assays was
based on acute toxicity data and the chemical characteristics of
the compounds, Stauffer's R-20458 and Zoecon1s ALT0SID were
tested at nominal concentrations of 62.5, 125, 250, 500 and 1,000
ppb. Thompson-Hayward1s TH-6040 was tested at 0 . 625, 1,25, 2.50,
5.00 and 10.0 ppb.
Test containers were 3-a, uncovered, glass battery jars, the
mouth of which was encircled by a band of nylon monofilament screen
cloth with a mesh opening of 475 micrometers (Nitex HC3-475).
Test solutions flowed into battery jars through a glass tube which
extended to the bottom of the jar and drainage was via overflow
through the screen cloth collar. This system maintained the
water depth in jars at 20 centimeters. There were 4 replicate
jars at each of the 5,test concentrations and 4 replicate control
jars for a total of 24 jars per test. Jars were placed in a water
bath and temperatures controlled to ± 2°C. Shrimp were illuminated
by Durotest fluorescent tubes on a 16-hour day, 8-hour night cycle
-------
8
control 1ed by a timer. • •
Prior to initiating subchronic tests, diluters were allowed to
run for several days to check system operation. Tests were begun
with the introduction of 20 zoea <24 hours old to each of the jars,
80 shrimp per treatment. Generally, shrimp hatched during the
night and were <15' hours old when transferred to test containers.
Shrimp were fed newly-hatched brine shrimp (Arteroi a salina) naupli i -
daily. Each day, water temperature and 00 were measured with
a YSI Oxygen Meter and combination temperature-oxygen probe.
Salinity was determined daily with an AO Optical Refractometer -
Salinity Model. Water samples for chemical analysis and shrimp
for microscopical examination were sampled weekly from replicate
jars. Sampling days were staggered to permit same-day extraction
of samples in order to prevent losses of test materials through
storage. The analysis of water samples is described below. Shrimp,
4 individuals from each treatment, were preserved in formalin and
sent to Mr, Don Emlay, Zoecon Corporations 975 California Avenue,
Palo Alto, California.
Exposures were terminated with shrimp at the "juvenile" stage
of development having previously completed approximately 10 zoeal
stages and post!arva stage (Broad, 1957a, b; Little, 1968). At termi-
nation, percentage survival and mean size of shrimp were calculated
and comparisons made by Student1 s t-test for significant differences
between controls and any' of the¦treatments at the P<0.05 level.
Analytical chemistry procedures * ;
Stock solutions of R-20458, ALT0SID and TH-6040 were prepared
in reagent-grade acetone. Mater was sampled from test containers
weekly and analyzed for these compounds.
VII-136B
-------
9
R-20458
¦ESS—--— -- j3
Water samples (800 ma) were extracted once with 100 mi of
nanograde petroleum ether in a 1 -1iter separatory funnel. The ex-
tract v/as dried with anhydrous sodium sulfate and then evaporated
to approximately 3 m£ in a 250-ma Griffin beaker over a 70°C
water bath. The remaining extract was transferred to a Kuderna-
Danish evaporator and concentrated to 1 m£ using a stream of nitro-
gen at ambient temperature or diluted to this volume immediately
prior to analysis by electron-capture gas-liquid chromatography.
The instrument used for analyses was a Perkin-Elmer Model
3920 gas chromatograph equipped with a 6 3Ni detector. The
61 X 1/4" ID glass column was packed with 31 OV 101 on 80/100
mesh Gas Chrom Q. Carrier gas was 10% methane-argon at a flow
rate of 35 mm/min. Operating temperatures were; inlet, 250°C;
column, 160°C; detector, 275°C.
Immediately prior to analysis of samples, the column was
equilibrated by injecting 3 mg of R-20458 and allowing 2-3 hours for
establishment of a stable baseline. Standards of varying weights
of R-20458 were analyzed and a graph constructed which related
response in terms of peak height against nanograms of R-20458 in-
jected. Using this graph, peak heights of samples were converted
to weights of R-20458.
Eight quality control samples were prepared by adding appro-
priate volumes of R-20458, dissolved in acetone,to sea water. These
samples contained 62.5 ppb and 1.0 ppm of R-20458 to bracket con-
centrations used in the chronic exposure.. Samples were analyzed
according to the procedures described above. Recoveries were
VII-137B
-------
10
calculated to be 55+2.6%. All reported R-20458 concentrations
have been corrected for the percentage recovery of this analyti-
cal method.
ALIOS!D
Extraction of ALTOSID from water samples was performed in
the same manner as described for R-20458. Analyses were performed
on the same Perkin-Elmer Model 3920 gas chromatograph as described
above. Conditions were also the same, with the exception of a
column temperature of 210°C for ALTOSID analyses. Recoveries of
ALTOSID were 96+9.9% for samples fortified to 62.5 ppb of ALTOSID
and 74±8.1 % for those at 1.0 ppm. Reported ALTOSID concentrations
were not adjusted for recovery.
TH-6040
Hater samples (1-liter) were extracted 3 times with 50-m£
portions of nanograde methylene chloride. Extracts were combined,
dried with anhydrous sodium sulfate and concentrated to approxi-
mately 3 m£ in a 5Q0-m£ Kuderna-Dani sh evaporator equipped with a
3-ball Snyder Column., The extract was transferred to a culture
tube and evaporated to dryness using a gently-flowing stream of
nitrogen at ambient temperature. The tube was closed with a Teflon-
lined screw cap. Samples were frozen, packed in dry ice, and ship-
ped to Dr. Donald W. Fuh1hage, Thompson-Haywa rd Chemical Co., 5200
Speaker Road, Kansas City, Kansas, for analyses by liquid-liquid
chromatography.
I
VII-138B
-------
11
RESULTS
A. Acute toxicity
The calculated 96-hour LC50 for R-20458 and adult P.. pugio
was 2.0 ppm with 95% confidence limits of 1.4-2,7 ppm {Table 1).
ALTOSID was not acutely toxic to grass shrimp at nominal- concen-
trations up to 10 ppm (Table 2). The 96-hour LC50 for TH-6040
was calculated to be 0.64 ppm with 95% confidence limits of 0.13-
3.1 ppm (Table 3).
VII-139B
-------
12
TABLE ]. Acute toxicity of Stauffer Chemical Company's
R-20458 to Palaemonetes p u q i o In static sea
water. Salinity was 26 °/oo and temperature,
17+1°C.
Nominal concentration
(mg/£;ppm)
Mortali t
y (%>
24 h
48 h
72 h
96 h
Control
0
0
0
0
1.0
0
0
0
0
2.0
0
10
20
50
3.0
10
30
70
90
4.0
10
70
100
100
5,0
100
100
100
100
6,0
90
100
100
100
96-h LC50
(mq/f, ppm)
2.0
951 confidence limits
(mg/E;ppm)
1.4 - 2.7
VII-140B
-------
13
TABLE 2, Acute toxicity of Zoecon Corporation's ALTOSID
to Palaeroonetes puqio in static sea water. Sa-
1 inity was 26 u/oo arid temperature, 17±1°C.
Nominal concentration Mortality {%)
(mg/f, ;ppm)
24 h
48 h
72 h
96 h
Control
0
0
0
0
0.010
0
0
0
0
0.10
0
0
0
0
1.0
0
0
0
0
10.0
0
0
0
10
96-h LC50 951 confidence limits
(mg/&;ppm) (mg/fc;pprc)
>10
VII-141B
-------
14
TABLE 3. Acute toxicity of Thompson-Hayward Chemical Company's
TH-6040 to Palacmonetes pugi o in static sea water.
Salinity was 26 °/oo and temperature, 17±1°C.
Nominal concentration. Mortality (%)
iq/Jl ;ppm) i
24 h
48 h
72 h
96 h
Control
0
0
0
0
0.010
0
0
0
0
0.10
0
0
20
20
0.28
0
0
40
40
0.50
0
20
20
40
0.75
0
20
40
40
1.0
0
40
40
60
96-h LC50 95% confidence limits
lHaZlLE-pml (mq/fc;ppm)
0.64 0,13-3,1
VII-142B
-------
15
B. Subchronic toxicity
Analysis of temperature data revealed a range of 24 to 27°C
over the course of the 35 days of exposure. There was no more than
1°C difference in water temperature from any one day to the next
and the mode temperature was 26°C with 25°C occurring nearly as
frequently. Dissolved oxygen varied from 4.5 to 6.6 ppm (58-88%
of saturation) in the R-20458 exposure with a one-time low of 2.9
ppm (37% of saturation). DO in the ALT0S1D exposure varied from
4,2 to 6,9 ppm (56-89% of saturation). DO was from 3.1 to 6.8
ppm (42-851 of saturation) in the TH-6040 exposure. Salinity
during this period ranged from 4 to 29 °/oo (Tables 4, 5 and 6).
Heavy rainfall which occurred during July resulted in the depres-
sion of salinity of exposure sea water beginning between days 7
and 14 and continuing for approximately 2 weeks , after which sa-
linity began to rise to its usual concentrations for the area at
this time of year. This fluctuation in salinity of bay water was
extremely uncommon for this area and time of year. Similar condi-
tions have not been experienced in this area since salinity record-
ing was begun around 1937 (U.S. EPA, Gulf Breeze Estuarine Research
laboratory. Gulf Breeze, Florida) and therefore were entirely un-
predictable based on historical data.
Results of the chemical analyses of water samples indicated
good agreement between nominal and measured concentrations for
R-20458 (Table 7), Measured ALTOSID concentrations varied from
nominal concentrations (Table 8). Analyses of TH-6040 samples
indicated that measured concentrations were higher than nominal
concentrations, initially, but closely approximated desired con-
centrations thereafter (Table 9). In reporting effects, a simple
VII-143B
-------
16
TABLE 4. Measured salinity, temperature, and dissolved oxy-
gen {00) of flowing sea water during a 35-day expo-
sure of Palaemonetes pugio to Stauffer Chemical
Company's R-204581
Day Salinity (°/oo) Temperature (°C) DO (ppm)
1
26
24
5.8
2
26
25
5.7
3
26
25
5.9
4
27
25
6.0
5
28
25
6.0
6
28
24
5.2
7
29
25
5.4
8
29
26
4.8
9
28
27
4,5
10
28
26
4.5
11
28
25
4.8
12
27
25
5.1
13
27
25
5.1
14
24
25
5.3
15
22
26
5.4
16
18
26
5 6
1?
15
26
6.4
18
8
25
6.4
19
6
26
6.4
20
6
26
5 6
El
6
25
5.3
22
6
24
5.9
23
6
24
6.0
24
5
24
6.4
25
5
26
6.1
26
5
26
6.6
27
4
25
6,2
28
5
. 26
5.4
29
5
26
2.9
30
6
26
4.5
31
8
26
5.7
32
12
26
4.7
33
14
26
5.4
34
15
25
5.8
35
16
25
5.7
VII-144B
-------
TABLE 5. Measured salinity, temperature, and dissolved oxy-
gen (DO) of flowing sea water during a 35-day expo-
sure of Palaemonetes pugio to Zoecon 'Corporation 's
ALTOS ID.
Day Salinity (°/oo) Temperature (°C) DO (ppm)
1
28
25
6.0
2
28
24
5.9
3
29
25
5.8
4
29
26
6.2
5
28
27
5.7
6
28
26
5.5
7
28
25
, 5.9
8
27
25
5.6
9
27
25
6.4
10
24
25
5.8
n
22
26
6.1
12
18
26
6.1
13
15
26
6.6
14
8
25
6.1
15
6
26
6.3
16
6
26
5.6
17
6
25
6.3
18
6
24
5.9
19
6
24
6.0
20
5
24
6.1
21
5
26
5.5
22
5
26
6,2
23
4
25
6.0
24
5
26
6.9
25
5
26
6.4
26
6
26
6,5
27
8
26
6.0
28
12
• 26
5.8
29
14
26
6.5
30
15
25
4.2
31
16
25
5.0
32
18
25
5.9
33
15
26
6.2
34
14
25
5.7
35
13
25
4.3
VII-
-------
18
TABLE 6. Measured salinity, temperature, and dissolved oxy-
gen (DO) of flowing sea water during a 35-day expo-
sure of Palaemonetes pugio to Thompson-Hayward
Chemical Corporation's TH-6040.
Day
Salinity (°/oo)
Temperature (°C}
DO {ppm)
1
27
C
25
6.5
2
24
25
6.4
3
22
26
6.0
4
18
26
5 < 9
5
15
26
6.5
6
8
25
6.3
7
6
26
6.4
8
6
26
5.7
9
6
25
6.3
10
6
24
6.6
11
6
24
6.7
12
5
24
6.8
13
5
26
6.0
14
5
26
6.6
15
4
25
6,5
16
5
26
6.5
17
5
26
5.9
18
6
26
5.9
19
8
26
5.4
20 -
12
26
5.7
21
14
26
5.9
22
15
25
5.1
23
16
25
5.1
24
18
25
3.7
25
15
26
3.1
26
14
25
4.6
27
13
n r
c 0
5.4
28
12
o c
b D
5.4
29
12
25
5 4
30
12
£» •
31
12
25
-
32
14
25
-
33
12
24
6.1
34
16
24
6.0
35
16
24
6.1
aD0 meter inoperative.
VII-146B
-------
19
TABLE 7. Measured concentrations of Stauffer Chemical
Company's R-20458 in flowing sea water during a
35-day exposure of Palaemonetes pugio. Salinity
was 4-29 °/oo arid temperature, 24«27°C.
Concentration (v g/a ;ppb)
Nominal
Day 0
7
Measured
14 2]
28
35
62.5
82.. 2
63.8
100 77.9
50.4
47.0
,125
139
130
141 ¦ 142
136
133
250
266
212
281 218
385
205
500
480
430 '
430 524 '
Q3 ga
397
1,000
1 ,074
930
889 777
-
a0bviously aberrant datura, probably due to sampling error.
VII-147B
-------
20
TABLE 8. Measured concnetrations of Zoecon Corporation's
ALTOSID in flowing sea water duri ng a 35-day
exposure of Palaemonetes pugio. Salinity was
4-29 °/oo and temperature, 24-27°C.
Concent ration fun/RlPPb)
Nomi nal
Dav 0
11
Mea s urod
18'
25
32
35
62.5
49,2
140
182
83.0
44.0
28.2
125
82.8
181
394 .
154
83.0
34.'4
250
183
532
849
299
260
201
500
1,437
1 ,368
1 ,368
601
665
395
1,000
3,994
3,716
3,608
„
-
-
The concentrations of ALTOSID on days 0 through 32 as
listed in Table 8 were determined by an analysis of one of
the four replicates. On day 35, all four replicates for each
concentration were analyzed. The figures presented in Table
8 represent an average of these data.
Table 8A provides the data by replicate from which the
averaged figures were derived.
Table 8A {Day 35)
ALTOSID Concentration (yg/£;ppb)
Replicate
nominal 12 3 4
43.3
13,9
25.0
494
62.5 30,7
125 39.0
250 216
500 446
9,1 29,4
55.5 29.0
162 225
294 34?
VII-148B
-------
21
BLE 9, Measured concentrations of Thompson-Hayward Chemical
Corporation's TH-6040 in flowing sea water during
a 35-day exposure of Palaemonetes pugi o. Salinity
was 4-29 °/oo and temperature, 24-27°C.
Concentration (ug/&-,ppb)
Nominal Measured
Day 4
10
"T7
24
31
35
0.625
0.900
0.770
<0.500
0.550
0.590
<0.500
1 .25
2.69
1 .54
0.940
1 .85
1 ,61
<0.500
2.50
5.51
-
_
-
-
-
5.00
6.79
am>
10.0 16,4
The concentrations of TH-6040 on days 4 through 24 as
listed in Table 9, were determined by an analysis of one of
the four replicates. On days 31 and 35, all four replicates
for both concentrations were analyzed. The figures presented
in Table 8 represent an average of these data.
Table 9A provides the data from all four replicates from
which the averaged figures were derived.
Table 9A
TH-6040 Concentration (yg/Jl ;ppb)
Nominal
Replicate
1
2
3
4
Day
31
0.62
0.65
0.64
0.56
0.50
1.25
2.47
1.55
1.28
1.13
Day
35
0.62
<0.50
<0.50
<0.50
<0.50
1.25
<0.50
<0.50
<0.50'
<0.50
VII-149B
-------
22
average of the measured concentration of each compound over the
duration of the study was used. In the case of TH-6040, only
initial measured concentrations corresponding to nominal concen-
trations of 2.50, 5.00 and 10,0 ppb were used since complete mor-
tality of test animals in those concentrations terminated water sam-
pling. For the remaining two TH-6040 concentrations9 measured values
were averaged for all results greater than detection limits.
R^mss
Zoea exposed to a measured concentration of 918 ppb of R-20458
were all dead within 7 days. Continuous exposure of shrimp to
measured concentrations of 70 and 137 ppb for 35 days had no
apparent effect on mean length or Survival of P., pugio from first
zoeal stage to juveniles. However, exposure to concentrations of
261 and 392 ppb resulted in a significant difference (P<0.05)
in both size and percentage survival of exposed shrimp as com-
pared to the control (Tables 10 and 11),
VII-150B
-------
23
TABLE 10, Growth of Pal demonetcs £hi gjj3 continuously exposed
to Stauffer Chemical Com 5) any 1 s R-204S8 in f 1 ov/i ng
sea water for 35 days. Salinity ranged from 4-
29 °/oo and temperature, 24-27°C.
Concentration Rostrum-telson length (mm)'
N 0 m i n a 1
I-' 0 asiir C: d
Day 7
14
28
35
Control
-
4.6+0.8
7 . 0± 0. 0
13. HI .3
1 5. 0± 0.8
62.5
70.2
4. 8± 0.5
6 . 4± 0, 5b
12.4+0.9
14.9+0.8
125
137
5.2+0.3
6 . 0+ 0. 7b
1 2.0± 0. 9
14.5±0.4
250
261
3.9±0.2
6.1±0.6b
1 0. 5±0.6b
1 2 . 0±0 . 8b
500
392
3.8+0.5
5, 5+0, 4b
8.6±1.2b
10.5±0.9b
1,000
918
_
-
_
-
aSize of
shrimp on
day 0 was 3.0+0.0 mm.
bMean is
significantly different
from that
of control at
P<0.05.
VII-151B
-------
24
TABLE 11. Survival of Palaeroonetes pugio conti nuously exposed
to Stauffer Chemical Company's R-20458 in flowing
sea water for 35 days. Salinity ranged from 4-29 °/oo
and temperature, 24-27°C.
Concentrati on
(ng/t;ppb)
Shrimp
survi ving
at 35 days
Nominal
Measured
JL
% relative
to controls
Control
-
77
96
_
62.5
^4
O
¦
72 '
90
94
125
13?
74
92
96
250
261
57
71
74
500
392
29
36
38
1 ,000
918
0
0
0
VII-152B
-------
o c
CD
ALTOSID
There was 1001 mortality within 7 days when shrimp were
exposed to a measured concentration of 3.8 ppra of ALTOSID.
Length and survival of larvae exposed to concentrations of 80,
155 and 387 ppb was not significantly different from that of the
control through day 35. Mean length of shrimp on days 28 and 35
and survival to day 35 of shrimp exposed to 972 ppb of ALTOSID
was significantly less than that of the control (Tables 12 and 13),
XM^6p4 0
Zoea exposed to measured initial concentrations of 5.5, 6,8
and 16.4 ppb of TH-6040 did not survive to day 7, Sizes of shrimp
exposed to concentrations of 0,70 <:nd 1.73 ppb we re riot signifi-
cantly different from that of the control through day 35 (Table
14). As there was only one animal alive in 1.73 ppb on day 35,
a statistical comparison of the size of that shrimp with the control
would not be valid. Survival to day 35 of shrimp exposed to 0.70
and 1.73 ppb was significantly less than that of the control (Table
15),
VII-153B
-------
26
TABLE 12. Growth of Pa 1aemonetes puqio continuously exposed
to Zoecon Corporation's ALTOSID in flowing sea
water for 35 days. Salinity ranged from 4-29 °/oo
and temperature, 2 4 - 2 7 ° C.
Concentration Rostrum-te1 son length (mm)3
Nominal
Measured
Day 7
14
28
35
Control
-
4.5+0.6
6 . 4±0 . 4
12.4+2.1
16.5+1.3
62.5
87.7
4.2± 1 . 3
6.1+0.6
11.9H.3
13.6+2.5
125
155
4, 4± 0,5
6 . 5± 0.6
11.0±0.7
14,8+4.0
250
387
4.910.2
6 . 5± 0.4
n.i±o.3
14.1+1.6
500
972
4 . 8± 0. 5
6.1+0.2
8.8±1.2b
12.2±1.6 b
1,000 3,773
aSize of shrimp on day 0 was 3.0+0.0 ram.
bHean is significantly different from that of control at P<0.05.
VII-154B
-------
27
TABLE 13. Survival of Palaemonetes pugio continuously exposed
to Zoecon Corporation's ALTOS 10 in flowing sea water
for 35 days. Salinity ranged from 4-29 °/oo and
temperature, 24-2?°C,
Concentrate on
(p g/£;ppb)
Nominal Measured
Control
62.5 87.7
125 155
250 387
500 972
1,000 3,773
Shrimp surviving at 35 days
|~TeTatTvi
# % to controls
69 86
69 86
73 91
68 85
36 45
100
106
98
52
VII-155B
-------
28
TABLE 14. Growth of Palaemonetes pugio continuously exposed
to Thompson-Hayward Chemical Company's TH -604 0
in f 1 ov/ing sea water for 35 days. Salinity ranged
from 4-29 °/oo and temperature, 24-27°C.
Concentration Rostrum-telson length (mm)a
(pg/i;ppb) y±s.D. (n=4)
Nominal Measured Day 7 14 28 35
Control - 5.5±0.0 6.2±0.3 11,8+0,9 13.6±0.5
0.625 0.70 5.8+0.3 6.8±0.8 10.6±0.9 12.8± 0.9
1.25 1.73 5.2+0.6 7.5b 10.0±0.0 10.0b
2.50 5.51 -
5.00 6.79 -
10.0 16.4 -
aSize of shrimp on day 0 was 3.0±0.0 mm.
L
Measurement of 1 animal (see text for explanation).
VII-156B
-------
29
TABLE 15. Survival of Pal aomonotcs pugio continuously exposed
to Thompson- Hayw«ird Chemical Company's TH-6040
in f1owi ng sea water for 35 days . Salini ty ranged
from 4-29 °/oo and temperature, 24-27°C.
Concentration
(pg/i;ppb) Shrimp surviving at 35 days
^TeTati ve
Norn i na1
Measured
jL
X
to controls
Control
-
74
92
-
0.625
0.70
51
64
69
1 .25
1 .73
10
12
14
2.50
5.51
0
0
0
5.00
6.79
0
0
0
10.0
16.4
0
0
0
VII-157B
-------
30
DISCUSSION
w Acute toxicity
Results of the stati c,acute toxicity tests Indicated that,
under the conditions described, TH-6040 was the most toxic of the
three compounds tested and ALTOSID was the 1 east toxic to adult grass
shrimp, Palaemonetes pugio. The relative toxicity of these com-
pounds is somewhat different from that determined with water fleas,
Daphni a magna. In the D. magna study, TH-6040 exhibited the
I ¦ * II ¦¦ ¦ ¦ ¦¦¦! I III |,i| nil II |] ' j- || tllf |, , ,| | — V *
greatest toxicity and R-20458 the least, (Bionomics, 1975).
Grass shrimp were more resistant to each of these compounds
than were water fleas. LC50 values (96-hour) for grass shrimp
were from 6 to 425 times greater than corresponding 48-hour values
for water fl eas .
It is difficult to compare the relative toxicity of these
three compounds with that of other pesticides since there are few
grass shrimp toxicity data reported in the literature which are
based on uniformly performed bioassays. However, Eisler (1969)
reports 96-hour LC501s ranging from 1.8 to 440 ppb for Palaemonetes
vulgaris exposed to 7 organochlori ne and 5 organophosphate insecti-
cides (Table 16). Based on these data with a closely related
"species of grass shrimp, it appears that the acute toxicity of
these three insect growth regulators is less than that of any of
the commonly used insecticides,
B. Subchronic toxicity
Larval grass shrimp were significantly more sensitive to these
growth regulators than were the adults. This was especially ob-
vious in the TH-6040 portion of this study in which zoea exposed
VII-158B
-------
31
TABLE 16. Acute toxicities of 12 insecticides to the grass
shrimp Palaemonetes vulgari s.
96-hour LC50
Insect!citie (pg/& ;ppb)
Organochlori nes
Heptachlor 440
Dieldrin 50
Methoxychlor 12
lindane 10
Aldrin 9
p,p1-DDT 2.0
Endrin 1.8
Organophosphates
Delnav R 285
Malath ion * 82
Phosdri n ^ 69
DDVP 15
Methyl parathion 3
(from Ei sier, 1969)
VII-159B
-------
32
to measured concentrations of 5.51, 6.79 and 16.4 ppb were all
killed prior to the day-7 sampling period. For R-20458 and
ALTOSID, those concentrations which resulted in a significantly
decreased survival of exposed shrimp, as compared to controls,
through day 35 also caused"a significant difference in rostrum-
telson length. This effect was observed at measured concentra-
tions of 261 and 392 ppb for R-20458 and 972 ppb for ALTOSID.
TH-6040, however, caused a significant decrease in survival to
35 days at measured concentrations of 0,70 and 1.73 ppb but no
significant decrease in rostrum-telson length at any test concen-
tration. The lack of adequate numbers of survivors at 1.73 ppb
of TH-6040 at 35 days made it impossible to assess any difference
in length of exposed versus control shrimp.
Due to the extreme fluctuations in salinity during this study,
it is possible that the observed effects on shrimp were the result
of an interaction between salinity stress and exposure to test
compounds. Sandifer {1 973} studied the survival and development
of Palaemonetes vulgaris maintained in the laboratory under combina-
tions of salinity-temperature conditions of 5, 10, 15, 20, 25 and
30 °/oo and 20, £5 and 30°C, respectively, and reported significantly
reduced survival and development of larvae at 5 °/oo salinity. How-
ever, he also noted that P.. vol qari $ is less tolerant of low sa-
linities than is £. pugio. Thorp and Hoss (1975) similarly re-
ported significantly reduced survival of adult P_. vul qari s and
pugio maintained in the laboratory at 5 °/oo as compared with those
held at 20 and 30 °/oo. However, they also note that the relative
abundance of P.. p u g io in collections from areas of constant low
salinity or areas experiencing great fluctuations in salinity was
VII-160B
-------
33
greater than that of JP. vul gari s.
Based on these subchronic toxicity data, the maximum accep-
table toxicant concentration, MATC (i.e., the highest concentra-
tion at which no deleterious effects are observed) for R-20458
is >137<261 ppb; for ALTOSIO, >387<972 ppb; and for TH-6040,
>0.70<1. 73 ppb.
A comparison of acute versus subchronic toxicity data reveals
differences of several orders of magnitude between test concen-
trations which were acutely toxic to grass shrimp and those which
produced significant effects on growth. Based on these data and
on those from the Daphnia study, environmental hazard evaluation
for R-20458, ALTOSID and TH-6040 based solely on static, acute
toxicity data is totally inadequate.
VII-161B
-------
34
LITERATURE CITED
Bionomics. 1975. The chronic toxicity of.ALTOSID, TH-6040 and
R-20458 to Daphni a magna. Report to Zoecon Corporation, Palo
Alto, California: 31 pp.
Broad, A.C. 1957b, The relationship between diet and larval
development of Palaemonetes. Biol. Bull, 112: 162-170.
E i s 1 e r, R. 1969. Acute toxicities of insectici des to marine
decapod crustaceans. Crustaceana 16; 302-310.
Little, G. 1968. Induced winter breeding and larval develop-
ment in the. shrimp, Pa 1aemonetes'pugio Holthuis. pp. 19-26.
In: Crustaceana, Supplement No. 2. Studies on Decapod Larval
Development.
Miura, T. and R.M. Takahashi. 1974. Insect developmental in-
hibitors. Effects of candidate mosquito control agents on
nontarget aquatic organisms. Environ, Entomol. 3(4): 631-638.
Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus
for fish toxicologi cal studies. 'Water Res. 1: 21-29.
Sanders, H.J. 1975. New weapons against insects. Chem. Engn.
News 53: 18-31. .
Sandifer, P.A. 1973. Effects of temperature and salinity on
larval development of grass shrimp* Palaemonetes vulgaris
(Decapoda , Cari dea). Fish. Bull. 71: 115-123.
Sprague, J.B. 1969. Measurement of pollutant toxicity to fish.
I. Bi oassay methods for acute toxi ci ty. Water Res. 3 : 793-821 .
Thorp, J.H. and D.E. Moss. 1975. Effects of salinity and cyclic
temperature on survival of two sympatric species of grass
shrimp (Palaemonetes), and their relationship to natural dis-
tributions. J. Exp. Mar. Biol, Ecol. 18: 19-28,
VII-162B
-------
35
U.S. Department of Commerce. 1972. Fishery Statistics of the
United States. Statistical Digest No. 63,National Marine
Fisheries Service; 474 pp.
U.S. Environmental Protection Agency. 197 5. Methods for Acute
toxicity tests with fish, macroinvertebrates, and amphibians.
Ecological Research Series EPA-660/3-7 5-009: 61.
Williams, A.B. 1965. Marine decapod crustaceans of the Carolinas.
Fish. Bull. 65(1); 1-298.
VII-163B
-------
36
SUBMITTED BY: Bionomics - EG&G, Inc.
Marine Research Laboratory
Route 6, Box 1002
Pensacola, Florida 3250?
PREPARED BY: Sam R, Petrocel1i , Ph.D.
Chief Scientist
r~~)
1? ftbiiwdL.
APPROVED BY; Rod Parrish
T> AXi
—s. f\ * I
Director, Marine
Research Laboratory
VII-164B
-------
PART ¥11 - HAZARD EVALUATION SUPPLEMENT
3. Report of Zoecon Morphological Observations on
Treated Palaemonetes pugio
As with Daphnia magna an effort to detect
morphogcnetic changes in Palaomonotos pugio v.":3
undertaken. Changes corresponding to those which
would bo evoked by the insect growth regulators
in insects could not be predicted so sample organisms
from the bioassays were randomly withdrawn at
regular intervals for microscopic examination,
Examination was performed at Zoecon by insect
physiologists under a stereo microscope at 25X.
General inspection was performed in comparison
to untreated controls recording rate of growth
as indicated by measuring the rostrum-telson length
by means of an ocular micrometer. Zoecon data
compares very favorably with similar measurements
performed at Bionomics and was plotted against
time for visual comparison of growth rate with
untreated controls. No unusual morphology was
observed. Minor effects on rate of growth were
evident as compared to controls with all of the
insect growth regulator treatments. Figures 11
to 13 present the graphical presentation of the
data as recorded numerically in Tables 11 to 13.
Time points for each dose level for which samples
were available were plotted and although the closeness
of the family of growth curves for each insect
growth regulator causes some question of the signifi-
cance of the differences, a fairly consistent dose
response is observed in each case. When a synchronous
population of Palaemonetes pugio is observed in
this way it would appear that the rostrum-telson
length is a sensitive measure of rate of growth
and concomitantly a measure of general sublethal
response to aquatic toxicants.
VII-165B
-------
...
; » ,
;. :j;.. |
*r- *r r ~|~
r 'I
>
rtj
. J
s. I i ! ' 1
¦>» rt
CO
y>
m
ST
m
01
n
o
ro
TO
CM
<£»
i
tt5
° Q
00
rH
VD
H
M
O
H
00
' VD
i
CJ
VII-166B
unu -
-------
uiui - i|^ final
VII-167B
-------
-------
Table XI
ZOECON ALTOSID
3l
Concentration Rostrum-Tels^n length (nun)
(pg/1; ppb) X-S. D ¦
Nominal
Measured
Day 7
14
28
35
Control
5. 0-0.5
6.5-0.5
14.1-1.6
16.8-1.7
82,5
87.7
4.8-0.6
6.9-0.3
12.9±1.3
15.4-2.0
125
155
4.75-0.5
7.6-0.5
12.0±0.8
16.1-4.8
250
387
5.4-0.6
7.3-0.6
12 . 4-1.4
15.4-1.4
500
972
5.3-0.4
6.9-0.3
9.9-0.8
13.0-1. 2
1000
3773
_
_
SL +
Size of shrimp 011 day 0 was 3,7-0.0 ram.
VII-169B
-------
Table XII
STAUFFER R-20458 ZOECON MEASUREMENTS
3,
Concentration. Rostrum-Tel son length (mm) *
Quq/i; ppb) X-s, p.
Nominal
Measured
Day
7
14
28
35
Control
4
.75-0.2
7.0-0.4
14.0-2.1
15.7-1.2
62.5
70.2
4
.9-0.3
6.4-0.5
12.6-0.8
15.0-0.9
125
137
4
.9-0.3
6.1-0.5
12 . 3-1.6
14.3-1.3
250
261
3
.8-0.4
5.7-0.7
10.7-0.3
12.7-0.9
500
392
3
.75-0.3
5.6-0.5
9.4-0.8
11.0-0.7
1000
918
-
-
-
aSize of shrimp on day 0 was 3.6^0.1 mm.
VII-170B
-------
Table XIII
THOMPSON-HAYWARD TH-604 0
£L
Concentration Rostrum-Telson length (mm)
(^tg/1; ppb) X*S . D.
Nominal Measured Day 7 14 28 35
Control 5.0*.2 6.6±.l 11.7^1.2 14.0-.8
15.6 10.2 5.0-.5 6.7-.3 11.3±1.4 14.7^,5
31.2 24.3 4.8-.3 7.3 b 10.3±»3 10.3 b
62.5 39.9
125 50.2 -
250 92.7 - -
a Size of shrimp on day 0 was 2.8-.08 mm,
b
Only one animal available for measurement.
VII-171B
-------
PART ¥11 - HAZARD EVALUATION SUPPLEMENT
RECOMMENDATIONS FOR FUTURE EPA WORK IN THE AREA OF
CRUSTACEAN TOXICOLOGY
The research reported herein with Palaemonetes
pugio and Daphnia magna were mainly designed to assess
chronic toxic hazard of representative insect growth
regulators but also to indicate the practicality of such
tests as future regulatory requirements. Rather than
indicate clear cut data requirements for new pesticide
registration, in genera] the crustacean research reported
herein brought attention to the primitive stage of
development of aquatic toxicology as compared to standard
mammalian toxicology. To be sure the insect growth
regulators appear to be most meaningfully studied in the
longer tests. The data do not, however, provide any basis
for a different data requirement for insect growth
regulators as compared to classical pesticides. Thus
the conclusion can be drawn that data on chronic exposure
to crustaceans should eventually be a requirement for
any pesticide with potential for aquatic residues.
If data of decision making quality is to be provided
to the Registration Division of Office of Pesticide
Programs, EPA, crustacean tests must be standardized to
such detail that they arc reproducable. The test must
be available on a year round basis or at least through
a major portion of the year in any laboratory equipped
to perform general bioassays with aquatic organisms.
Most importantly a data base must be generated in the
context of which the results of crustacean chronic bioassays
on new pesticides submitted for initial registration (or
on old pesticides for which presumption about the aquatic
safety will require additional data for reregistration)
can be judged, The following general categories of
research are suggested by this study. Many of the topics
overlap considerably and much of the work has been under
way for years in Environmental Protection Agency research
laboratories and elsewhere. It is believed that this
work should be emphasized and brought to fruition as
expediently as a sensible budget allows. Until the
results of such research work are in hand it is suggested
that chronic crustacean bioassay be required only in
the event of pesticides for aquatic application where
other evidence such as adverse field observations or
mode of action suggests hazard potential.
The following work is recommended.
VII-172B
-------
PART VII - HAZARD EVALUATION SUPPLEMENT
1. That the Environmental Protection Agency support
¦work through in-house, university and contract/commercial
aquatic toxicology laboratories designed to refine
the details of laboratory culturing of various
crustaceans through an entire life cycle from egg
to egg,
2. That the Environmental Protection Agency support
basic research work on devices and/or handling
methods for assessment of toxic response by either
census or detailed examination of very early
crustacean developmental forms (early larvae).
3. That EPA scientists devise and test, both through
in-house laboratories and under contract, various
protocols for chronic bioassay which offer potential
for multi generation assessment of hazard both with
regard to survival and reproductive capacity.
4. That the Environmental Protection Agency support
in-house, and by grants to university research
teams, investigations to relate laboratory bioassay
indications of toxic hazard to crustaceans to
prediction of real potential for disruption of
crustacean development in the wild.
5. That the Environmental Protection Agency support
research to establish a data base on established
pesticides prior to the institution of chronic
crustacean bioassay as a registration requirement
except in cases where there is presumption of
hazard on other grounds,
RECOMMENDATIONS FOR REGISTRATION GUIDELINES FOR
INSECT GROWTH REGULATORS WITH RESPECT TO CRUSTACEAN
HAZARD ASSESSMENT
Based on the small sample of three insect growth
regulators representing the two major present classes;
namely, juvenile hormone analogs and chitin inhibiting
compounds, the following conclusions of the study can
be drawn which are pertinent to formulation of guidelines
for registration of insect growth regulators.
1. Qualitatively insect growth regulators do not offer
different potential for hazard than standard
pesticides.
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PART VII - HAZARD EVALUATION SUPPLEMENT
2. Quantitatively the insect growth regulators appear
to have as broad a spread of response levels as
standard pesticides. The array of toxic levels
for insect growth regulators overlaps with that
of standard pesticides. Thus the descriptor
"insect growth regulator" portends neither greater
safety nor greater hazard.
3. The state of the art in chronic bioassay of
Crustacea remains relatively primitive.
4. Although a multi-generation crustacean study
would have considerable scientific appeal the
state of present technicalogical development is
not sufficient. The information content of data
afforded by a single generation study (21 days)
of Daphnia magna was not appreciably increased by
continuing the study to a second generation given
the attendant problems of loss of population
synchrony.
5. Subchronic bioassay with Palaemonetes pugio indicated
lower sensitivity of this creature to the compounds
tested. More importantly the work indicated that
the test as performed has limits in providing data.
Given present technology intermediate time points
are difficult to obtain and furthermore reproductive
capacity cannot be assessed,
6. The dynamic flow-through chronic bioassay probably
does not realistically approximate the residue
exposure of crustaceans to pesticides in the wild.
In practical use reapplication of pesticides is
made at intervals of several weeks with interim
residue decline. Even in the test reported herein,
analytical component of the experiment indicated
that decline of insect growth regulators may be
quite rapid.
The studies reported herein with Palaemonetes pugio
and Daphnia magna were mainly designed to assess chronic
toxic hazard of representative insect growth regulators
but also to indicate the practicality of such tests as
future registration requirements. The data do not provide
any basis for difference in registration requirements
between insect growth regulators and standard pesticides.
The work also seems superficially to-fn~dXcate that
since chronic hazard cannot be adequately assessed
by acute studies a chronic test should be required
for all pesticides likely to result in aquatic residues.
To be sure the younger life stages of Crustacea appeared
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PART ¥11 - HAZARD EVALUATION SUPPLEMENT
more sensitive and the longer exposure times produced
effects at lower concentration levels. The results are
not really surprising, however. The observation that
chronic insult is often more damaging than acute is well
documented in mammalian toxicology. It appears that the
conclusion of adding a chronic crustacean study as a
requirement for registration of all pesticides, while
rational, is somewhat premature. The requirement of
chronic studies should not be required until the time
when such testing affords reproducable data, is generally
available, and a data base exists in context of which
to interpret results. Until that time the research arm of
the Environmental Protection Agency should support work
to reach that goal and the Registration Division should
consider each pesticide on a case by case basis depending
on data on the persistence of the compound and the use
pattern to provide presumption of hazard.
The following suggestions for guidelines for the
registration of insect growth regulators are the result
of this study of crustacean chronic hazard.
1. Insect growth regulators should meet the same
registration requirements as standard pesticides.
2. A chronic crustacean study should be required of all
aquatic use pesticides having high persistence or
depending upon the frequency of reapplication rates.
Any compound which is likely to find its way into
the aquatic habitat or for which field or laboratory
observations portend adverse effects on crustaceans,
should likewise be inspected,
3. A suitable interim chronic crustacean bioassay
is the 21 day study of Daphnia magna.
It is believed that the above recommendations will,
for the present, afford practical protection of the
crustacean resource. The Environmental Protection Agency
scientists should review on an annual basis the availability
of models which more closely approximate crustaceans of
greater importance in the food web. They should encourage
the study of compounds for which presumptive hazards
exist under more experimental protocols but be willing
to make judgments using such data with some flexibility.
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