ABSTRACT:
In this experiment ! compared the toxicities of Exxon Corexit 7664
9550, 9527, and 9580, SLIK-A-WAY, Nokomis 3-F4, and Nutri-Bio 1000,
with and without 1 gram of crude oil, using the splash pool copepod
Tigriopus californicusas a test organism. Investigating T. californicusas
a test organism revealed that males are more sensitive than females to
change, that the organisms' survival is inversely proportional to the
amount of crude in their environment, and that they survive longer in
filtered water than in tide pool water when no outside air is available
These preliminary studies resulted in the final sample of 5 female 7.
californicus ( with eggs), in 3ml tide pool water, in a 12hr light/dark
cycle, at 15°C, with and without 1 gram crude oil to test the cleaning
reagents.
INTRODUCTION.
Since the Torrey Canyon and Santa Barbara spills in the 60's when
toxic dispersants were used to remove oil from the rocks and in the
process irreversably damaged the environment (Smith 1968), more
"environmentally safe“ cleaning reagents have been developed, but
evidently not perfected. Äfter the 1989 Exxon Valdez spill, public
awareness about the tragic effects of oil in the environment and the
ineffectiveness, costliness, and destructiveness of clean-up procedures
has grown. So has the need to evaluate the ecological effects of the
current cleanup procedures beyond the anecdotal (Foster, Tarpley and
Dearn 1990). As a result, I chose to do a comparative study on the effects
of dispersants, fertilizers, and detergents. I tested Corexit 7664, 9550,
9527, and 9580, SLIK-A-WAY, Nokomis 3-F4, and Nutri-Bio 1000 with and
without crude oil fractions on the marine organism Tigriopus californicus.
I californicus, a harpacticoid copepod, in its natural environment of
high splash pools on rocky coasts must cope with the severe environmental
fluctuations in salinity, oxygen, temperature, availability of food,
population density, and buildup of toxins (Haderlie et al., 1978). 7.
californicus can also be easily maintained in laboratory conditions that
are almost identical to the original habitat, overcoming the problems of a
gap between the experimental and natural system (Foster, Tarrpley, and
Dern 1990). To devise a stable and easily replicable experimental system
for bioassay tests, I reviewed the studies of Kontogiannis and Barnett
(1973 and 1975) and Dorit (1977), and experimentally established
standards for container size, number of test animals, sex, type of medium
and light cycles. I also investigated questions raised about the differential
survival of male and female T. californicus with and without eggs when
exposed to a variety adverse conditions (Dorit 1977).1 evaluated the
effects of different crude oll fractions in 24hr light, 24 hr dark, and
reqular 12hr light and dark cycles, in tide pool and filtered water to
establish the best parameters to create repeatable and significant
experimental conditions to properly test a variety of oil spill cleanup
techniques.
MATERIALS AND METHODS:
Tigriopus californicus used in this study were collected from a high
splash pool west of Loeb lab at Hopkins Marine Station, Pacific Grove,
California. They were kept in a 100ml container filled with tide pool
water at 15° C with alternating 12hr light and dark cycles for at least a
week before being tested. To determine the best experimental density and
sex ratio, 5ml jars were placed within both the laboratory and field
populations overnight. Äfter removal, these samples were sexed and
counted under a dissecting microscope. Samples were also collected with
a sterile pipette from small (3Om1), naturally stressed pools (Vittor
1971) in the field. The T. californicus from the laboratory population were
tested in different container sizes, varying conditions of light, and tide
pool or filtered water. The only food avatlable was the unicellular algae
and bacteria contained in their water. They were placed in in Sml flat
bottomed jars to facilitate counting. The jar samples tested in 24hr
darkness were wrapped in tin foil and placed under soup bowis with
sufficient space for air circulation. They 24hr light samples were placed
under an incandescent light and were subject to slightly higher
temperatures. The final experimental samples used to analyze the cleanup
reagents and crude oil controls consisted of 3 adult females with eggs and
2 adult females without eggs in 3ml of tide pool water (in a Sml jar), kept
in alternating 12hr light and dark cycles (normal indirect sunlight) at
15•c.
The crude oil used in this experiment was provided by the Texaco Inc.
fields in San Ardo, California. This heavy, tar-like oil (sp. gr. 0.9799)
consisted of 75% naphthenes, 17.3% paraf fins, 6.5% aromatics and 2.27
sulphur (Texaco Inc. R1350 Crude 011). Weathered crude was prepared by
placing 60ml of San Ardo crude on 600ml sea water in an evaporat ing pan
exposed to sunlight and no rain. This oil was collected after 24 hours and
72 hours (1 and 3 days), losing 50% of its volume by evaporation in the
first 24hrs and 60% by the 3rd day (Wheeler 1978). Mineral oil was used
to examine the physical effects of crude oil in the absence of toxins since
mineral oil is essentially crude oil without impurities (Stecher, 1968).
When 0.5 ml crude or mineral oil were added to the samples, the oil
naturally positioned itself on the water surface, and blocked gas exchange
between the air and water. Otherwise, only 1 and 0.5 grams (about 0.1
and 0.0Sm1) of crude were weighed then added on the end of a toothpick (to
hold the substance in the water column and to prevent it from rising and
disturbing the surface gas exchange).
Samples of Exxon's (Houston, TX) Corexit 9527, 9550, 9580, and 7664,
MI-DEE's (Hayward, CA) SLIK-A-WAY and NOKOMIS 3-F4, and A & V Inc.'s
(Sussex, WI) Nutri-Bio 100 were generously donated by the companies.
Solutions were prepared to manufacturers reccommendations and added to
the lcm* surface and 3ml of tide pool water in the samples. Since about
five US gallons per acre of Exxon's two open water dispersants Corexit
9527 and 9550 and MAR-LENS SLICK-A-WAY are normally added to spills,
after conversion, O.Iml of the solutions (about 2 drops) were added per
sample. A mixture of O.Iml Corexit 9527 diluted with 1Oml seawater was
also prepared, according to the reccomended use, and 0.Iml of this
solution was tested. 1.24 ml of Corexit 9580, a shoreline cleaner (one
gallon is normally added to 100 square feet in undilute form) was added to
the samples as well as a smaller amount, 0.Iml Corexit 9580. O.3m1
Corexit 7664 (normally added as a 1-3% solut ion to 10 to 15 square feet)
was also tested. 0.05 and 0.001mI NOKOMIS 3-F4 were tested, equivalent
to seawater dilutions of 1:15 and 1:100. Finally, O.0Iml and 0.001ml of
Nutri-Bio 1000 were also evaluated, equivalent to sea H20 dilut ions of
1:10 and 1:100.
RESULTS:
There were about 10 females (with no eggs) and 2 males per 5ml
bottle (about 2 antmals per mi) left in the splash pools overnight. In the
jars from the laboraory populat ion there were about 8 females (one with
egg) and 3 males per bottle (about 2 per ml). In my analyses of stressed
(small) high splash pools 1 found about 33 animals per 12m1 pool, 15 of
which were females with eggs, 15 females without eggs, 2 males, and
some small juvenile females, together averaging about 3 animals per ml.
The test Light, 0-24hrs (Fig. 1) describes the experimental controis
for 5 females (3 with eggs) in tide pool H20. The points where the
population increases are due to the appearance of juveniles. In the
laboratory condition of alternat ing12hr light and dark cycles the organism
survived over 30 days with a 608 reduction in its original population.
Exposed to 24hr light, the entire sample died in 7 days. Exposed to 24hr
darkness, the sample died in 30 days.
The Repetetive Crude Oil Fractions (Fig. 2) show that three
separate experiments under the same experimental condition respond in an
almost identical and repeatable manner. When exposed to 0.5q crude
sample T. californicus lived about 10 days as opposed to its 5 day suryival
when exposed to Ig crude. When exposed to a 0.Sml crude oil cover the
samples died in 4-5 days and died in 6-7 days when covered with the same
amount of mineral oil.
The Repetitive Female Crude (Fig. 3) shows that two sets of 5
females with no eggs in tide pool water, exposed to 1 gram of crude died
in 7 days. Two sets of 5 females (3 with eggs), exposed to the same
conditions died in 6 days. Two sets of 5 females (all with eggs) in the
same conditions died on the 6th and 7th day (although the early mortality
in one sample may be due to the fact that on the fifth day the oil had
broken loose and formed a cover over the surface). Overall, no difference
was seen between the organisms with or without eggs when tested with !
gram of crude oil.
Females w/ Eggs vs. Females & Hales (Fig. 4) reveals that 5
males in tide pool water exposed to 0.5 grams crude oil survived 8 days
and when exposed to 1 gram of crude survived only 3 days. The group of 3
females with eggs and 2 females with no eggs exposed to 0.5 grams of
crude survived10 days. Females with and without eggs survived around 6
days when exposed to 1 gram of crude (weather or not they were in tidal or
filtered water or if the crude had first been weathered 3 days before
addition).
The Male and Female, 24hr Light, Mineral OII Cover (Fig. 5) is a
repetitive experiment in tide pool water that shows two sets of females
living 4 days, twice as long as the two sets of males that lived 2 days in
tide pool water.
In the Crude OiI Cover vs. Hineral Oil Cover in 24 hr Dark (Fig.
6) the life of animals in filtered sea H20 with a mineral oil cover in the
dark is almost triple that of the tide pool H20 with mineral oil samples.
Similarly, in the Crude & Mineral OII, 12hr Light/Dark Cycle (Fig. 7)
both the males and females survive almost twice the time 12-13 days
when covered with mineral oil if they are in filtered seawater.
Cleaning Reagents:
The addition of shoreline cleaners is often performed in con junction
with steam cleaning. Although steam was not tested with all the
solutions, boiling hot water added to a sample caused complete mortality
within 7 days (Fig. 10).
Corexit 7664 (Figs. 8 &9): O.3ml Corexit 7664 by itself was mildly
toxic, killing all the samples within 12 days. When O.Sml crude oil was
added to 0.Jml Corexit 7664, the sample died overnight. Otherwise, O.3m1
Corexit 7664 prolonged the life of the 1g (0.1m1) crude samples from 5 to
15 days and the sample with a 3 gram tar ball from 7 to 11 days. The
sample with ig mineral oil and 0.Jml Corexit 7664 also died on the 15th
day.
Corexit 9550 & 9527 (Fig. 10): The open water dispersants Corexit
9550 and 9527 (Fig. 10) proved to be much more detrimental to 7.
californicus. O.Iml of Corexit 9527 killed the sample within 15 minutes
and 0.00 Iml killed the sample in 4 days. 0.Iml of Corexit 9550 with or
without Ig crude killed the sample in 3 days. 0.1 ml of Corexit 9550
killed the samples within 15 minutes. 0.001 Corexit 9550 also killed the
samples within four days.
Corexit 9580 (Fig. 11): 1.24ml of the shoreline cleaner Corexit 9580
blocked the air/water interface and killed the sample in 2-3 days.
(Absolute mortality due to mineral oil blocking gas exchange occurs in
about 5 days). O.Iml of Corexit 9580 (not enough to block surface gas
exchange) and Ig crude killed the sample in 5 days (the same as ig crude
alone) and just O.Iml of Corexit 9580 killed the sample in 8 days.
SLIK-A-WAY & Nokomis (Figs. 12 & 13): O.ImI SLIK-A-WAY with or
without Ig crude killed the samples within 1Sminutes and 0.001m1
SLIK-A-WAY with or without ig crude killed the samples by the 2nd day.
0.05ml Nokomis with or without Ig crude killed the samples by the 2nd
day. 0.00 Iml Nokomis with ig crude killed the sample in 6 days (one day
beyond just Ig crude) and the same amount without crude killed the
sample in 7 days.
Nutri-Bio 1000 (Fig. 14): O.0Iml of Nutri-Bio 1000 killed the
samples with or without Ig crude in at least 15 minutes. Ig crude and
0.001ml Nutri-Bio 1000 resulted it absolute mortality by the 4th day.
DISCUSSION.
I. californicus' survival is inversely proportional to the amount of
crude it is exposed to (Dorit 1977, Barnett and Kontogtannis 1975).
Figure 2 shows T californicus living about 10 days when exposed to 0.59
of crude and about 5 days when exposed to ig crude. When the oxygen
exchange was blocked by a 0.Sml crude oil cover the sample died within
4-5 days. When mineral oil was used (crude oil without impurities), total
mortality took 6-7 days (Fig. 7). These results are identical to the
findings of Barnett and Kontogiannis (1973) and reillustrate the fact that
I. californicus is affected by the physical as well as the toxic elements
of crude oil and that an experiment performed on S animals in Jml tide
pool water yields the same experimental results as tests on 50 animals in
20ml of tide pool water. The fact that T californicus survived about
twice the time, 12-13 days when covered with mineral ofl when they were
in filtered seawater (Fig. 7) is probably due to the lack of other oxygen
requiring organisms in the filtered water.
From his experiments, Dorit (1977) proposed that females with eggs
survive longer than females without eggs when exposed to crude ofl
fractions. I was unable to find evidence for this in repetitive experiments
using females with and without eggs (Fig. 3). In fact, males showed a
much greater sensitivity to crude oil fractions than females. Exposed to
I gram of crude, the female samples out lived the male samples by twice
the time (Fig 4). Likewise, the mineral oil in 24hr light tests (Fig 5)
showed that out of the organisms subjugated to higher temperatures and
oxygen deprivation, the two sets of females lived 4 days, twice as long as
the two sets of males. It was previously noted that in the juvenile stages,
female T. californicus had a better chance of survival than males (Vittor,
1971). This may be due to the fact that they are copulating with larger
males and thereby avoid being preyed upon by other T. californicus(Burton
1985). Yet, since adult males are the first to die when exposed to adverse
environmental conditions and since females can have 15 to 20 consecutive
broods after a single copulation (enough to last the rest of their 90 day
lifespan), rendering males relatively useless (Vittor, 1971), it is to the
population's advantage to have lesser numbers of males when under stress.
This would explain the sex ratios (1adult male:15 females) in the stressed
splash pools. It is also interesting to note that under the starving and
oxygen deprived conditions in figure 7 the male population crashed, but not
completely. Perhaps males are also more sensitive to changes if their
density is greater, explaining their scarcity but not extinction in the
naturally stressed ecosystems. Nonetheless, since females dominate the
populations under stress, and are the most abundant T. californicus they
were used as the experimental controls for the cleaning reagents.
Cleaning Reagents:
SLIK-A-WAY was the most toxic reagent tested (0.001ml with or
without ig crude killed the entire sample overnight). Next were Corexit
9550, 9527 and Nutri-Bio; 0.001ml of the solutions killed all the samples
by the fourth day (one day sooner than ig crude). Nokomis was slightly
less toxic followed by Corexit 9580 (when it did not disrupt oxygen
exchange) then Corexit 7664.
Corexit 7664 had the lowest toxicity to T. californicus. Past studles
have shown that it has had small negative effects on copepods (Venezia
and Fossato 1977). Verily, O.3ml Corexit 7664 by itself was mildly toxic,
killing all the samples within 12 days. The hypothesis that there is a
positive synergistic effect between Corexit 7664 and crude ofl
(Elgershuizen and De Krui jf, 1976) was false only in the experiment with
O.Sml crude oil and 0.3ml Corexit 7664, since this sample died overnight
and O.Sml crude usually takes 4 days to kill T californicus Otherwise,
O.Jml Corexit 7664 prolonged the life of the Ig crude samples from 5 to
15 days and the sample with a 3 gram tar ball from 7 to 11 days.
Interestingly, the sample with ig mineral ofl and 0.3ml Corexit 7664 also
died on the 15th day, indicating that it may not be the toxins in the crude
that are detrimental when combined with Corexit 7664 but the physical
properties that affect the organisms.
Further tests of cleaning reagents might include generat ional studies
where the organisms' fitness could be measured against the number of
eggs per clutch since when female T californicus are exposed to toxins
the number of eggs per clutch decreases (Vittor 1971 and observations).
An experiment that could analyze this without disturbing the organisms
would be an important measure of toxicity. Generational and
recolonization investigations could also be conducted if even smaller
fractions of the cleaning reagents were used on larger populations and
studied for a longer period of time.
If a spill cannot be prevented from reaching the shore, political haste
must not cause early or unnecessary cleansing since the natural
degradation of the oil and the cleaning of the surf are usually the best
cleaning options, both economically and environmentally (API, 1985).
Highly toxic cleaning reagents should not be used on living ecosystems and
should only be used post mortem if their negative effects can be shown not
to inhibit habitat restoration.
ACKNOWLEDGEMENTS:
would like to express infinite thanks to Alan Baldridge and Chuck
Baxter. I would also like to thank the companies who generously donated
their products; Texaco Inc, Exxon, Hydro-Tech, and A & V Inc. Thanks also
to Terri Shaw and the students of Bio 175H for their support and to the
copepods who gave their lives for my experiment.
BIBLIOGRAPHY
American Petroleum Institute. (1985). Oil soill cleanuo: Options for
minimizing adverse ecological impacts A. P. I., (Washington, D.C.) pp.
191-222.
Barnett, C., Kontogiannis, J. E. (1973). The effect of oil pollution on
survival of the tidepool copepod, Tigriopus Californicus
Environmental Pollution 4, 69-79.
Barnett, C., Kontogiannis, J. E. (1975). The effect of crude oil fractions
on the survival of a tidepool copepod, Tigriopus Californicus
Environmental Pollution 8, 45-54.
Burton, R. S. (1985). Mating system of the intertidal copepod Tigriopus
californicus. Marine Biology 86, 247-252.
Cairns, J., Buikema, A. L. (1984). Restoration of Habitats Impacted by
Oil Spills Butterworth Publishers, (Boston).
Crothers, J. H. (1983). Field experiments on the effects of crude oil and
dispersant on the common animals and plants of rocky sea shores.
Marine Environmental Research 8, 215-239.
Eljershuizen, J. H. and Nuwayhid, (1976). Effects of crude oil and
dispersants on bivalves. Mar. Pollut. Bull 7, 22-25.
Foster, M. S., Tarpley, J. A., Dearn, S. L. (1990). To clean or not to clean:
the rationale, methods, and consequences of removing ofl from
temperate shores. The Northwest Environmental Journal 6,105-120.
Haderlie, C., Abbott, D. P., Caldwell, R. L. (1978). Between Pacific Lide
Stanford University Press, (Stanford).
Jordan, R. E., Payne, J.R (1980). Eate and Weathering of Petroleum
Spills in the Marine Environment. Ann Arbor Science, (Ann Arbor).
Stecher, P. (1968). The Merck Index: an encyclopedia of chemiçals and
drugs. Merck & Co., (New Jersey).
Venezia, L. D., Fossato, V. U. (1977). Characteristics of suspensions of
Kuwait oil and Corexit 7664 and their short- and long-term effects
on Tisbe bulbisetosa (Copepoda: Harpacticoida). Marine Bioloqy 42,
233-237.
Vittor, B. A, (1971). Effects of the Environment on Eitness-Related Life
History Characters in Larioous californicus. University Microf ilms,
(High Wycomb).
Wheeler, R. B. (1978). The fate of petroleum in the marine environment.
Exxon Production Research Company Special Report.
FIGURE LEGENDS:
Fig. 1: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool and filtered seawater in no light, 12hr light/dark
cycles, and 24hr light.
Fig. 2: Number of T califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater containing 0.5 m1 (5 grams) crude oil on
the water surface and 0.1 ml (I gram) crude in the suspension.
Fig. 3: Number of T califoricus surviving over time for 5 females
with 3 eggs, 5 females with no eggs. and 5 females with 5 eggs, in tide
pool and filtered seawater exposed to one gram crude.
Fig. 4: Number of T. califoricus surviving over time for 5 females
with 3 eggs, 5 females with no eggs. and 5 females with 5 eggs and 5
males in tide pool and filtered seawater, exposed to 1 or 0.5 grams crude.
Fig. 5: Number of T. califoricus surviving over time for 5 females (3
with eggs) and 5 males in tide pool seawater containing 0.5 ml mineral ofl
on the water surface in 24 hr light.
Fig. 6: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool and filtered seawater with or without 0.5 m1
mineral oil on the water surface in 24 hr dark.
Fig. 7: Number of T. califoricus surviving over time for 5 females
with 5 eggs, 5 females with 3 eggs, and 5 males in tide pool and filtered
seawater with 0.5 ml mineral oil or crude oil cover.
Fig. 8: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater exposed to 0.5 ml crude on the surface or
I gram crude in the suspension with or without Corexit 7664.
Fig. 9: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with a 3 gram tarball or 0.5 ml mineral
ofl on the surface, with and without 0.3 ml Corexit 7664.
Fig. 10: Number of T califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with or with out 1 gram crude and 0.1 ml
Corexit 9550, 0.1 and 0.001 ml Corexit 9527, and one sample tested with
steam.
Fig. 11: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with 0.5 ml mineral oil on the surface,
1.24 ml Corexit 9580 on the surface, or 0.1 ml Corexit 9580 in the
solution with or without 1 gram crude.
Fig. 12: Number of T. califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with 0.1 and 0.001 mI SLIK-A-WAY with
and without I gram crude.
Fig. 13: Number of T califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with 0.05 and 0.001 ml Nokomis with and
without 1 gram crude.
Fig. 14: Number of T califoricus surviving over time for 5 females (3
with eggs) in tide pool seawater with 0.01 and 0.001 ml Nutri-Bio 1000
with and without 1 gram crude.
Light, 0-24hrs
8-
po
se ooo

4

Les * eg bog
lag boo

o
—
Time (days)
—
5 females (3 eggs). Tide Pool Seawater
— 5 fomalos (3 eggs), Filterod Seawatar
5 females (3 eggs). Tioe Pool Seawater. 24 1r dart
5 females (3 eggs). Tioe Pool Seawater. 24 h liont
Figure 1
Figure 2
Repetetive Crude Oil Fractions
3
b
2
L
L
Time (days)
S fomalos (ogs). Too Pool H20. O.Sol Crude Cov
—
5 semalos (Segga). Tioe Pool 120. O.Sml Crude Cover

5 females (Seges). Tide Peol 120. 19 Crude Cover
5 females (o eg0a). Tide Pool H20. 19 Crude Cover

5 females Gegp). Tide Peol 10. 19 Crude Cwer
—0— 5 fomales Cegga). Ie Pool H20. 19 Crude Cover
Repetitive Female Crude

bg
2-

0 +

Time (days)
—0— 5 females (3 eggs), Tide Pool Seawater, Ig Cruge
—— 5 females (3 eggs), Filtered Seawater, lg Crude
5 females (5 eggs). Tide Pool Seawater, la Crude
5 females (3 eggs). Tide Pool Seawater, la Crude
5 females (5 eggs). Tide Pool Seawater, l9 Crude
—
5 females inc egos). Tide Fool Seawater, ' Crude
Figure 3
Figure 4
Females w/ Eggs vs. Females & Hales
0 +
L
L
2
8 10 12
Time (days)
5 females (Zegys), Flltered HO. Ig Crude
—0—
5 females (Jogys), Tldo Pool N20. 1/29 Crudo
5 females (Zeggs), Tide Pool H20, lg Crudo. 24 Dack
5 females (3egys). Ilde Pool H20. lq Crude
5 fomales (Jegos). Tide Pool H20. Ig Crude
5 females (Joggs), Tide Pool H20. Ig Crude Weathered 3 days
—
5 females (Seggs), TIde Pool H20. 19 Crude
—
5 fomales (Sogys), Tido Pool H20. Ig Crudo
.... ....
5 Tomales (no eggs). Tide Pool H0. 19 Crude
—
5 males.  ol H20, 1/29 Crue
—— 5 males. 1 Pool H20, 19 Cus
Figure 5
Hale and Female, 24hr Light, Hineral Oil Cover
6
0 +

—
3
4
Time (days)
femaies (7egs), ae Pool Sewater, O.Sm ineral Oil, 24h Lot

eawte

h

—
Figure 6
Crude Oil Cover vs. Mineral Oil Cover in 24 hr Dark
o
stesstes boood
1


Le g bog
bes boo
1
ol
Time (days)
—O
5 females (3 eogs), Tioe Pool Seawater
—
5 females (3 eggs), Filtered Seawater
5 females (3 eggs), Tide Pool Seawater
0.Sml Mineral Oil, 24hr Dark
5 iemales (3 eogs), Tide Pool Seawater, 24hr Dark
5 females (5 eggs), 1e Pool Seawater
O.5ml Mineral Oil. 24hr Dark
5 females (no egs), Filtered Seawater
0.5ml Mineral Oil, 24hr Dark
Figure 7
Gude & Mineral O11, 12hr Light/oark Cycle
5

boo


T
Time (days)
âO— 5 femalos (Seggs). Te Pool 120. 0.Sml Crude Covor
—
5 females (Segos). Tide Pool HO. OSml Crude Cover
—
5 females (Seogs), Tide Pool H20. O.Sml Hineral Oil Cover
5 fomalos (Jegga). Tide Pool H20, O.Sml Hineral Oil
5 females (Segos). Tide Pool H20, O.5mi Hineral Ol
5 females (Jeogs), Filtered 120, O.Sml Hineral Oil.
5 males, Toe Pool H20. O.Smi Hineral Oil
—— 5 males, Filtered H0, O.5ml Hineral Oil
s— 5 females (no eogs), Tide Pool H20, O.Sml Hineral Oil
Figure 8
Exxon Corexit 7664 and 19 San Ardo Crude
H
—

+


Time (days)
—— 5 females (Jeggs). Tide Poo1 H20. 0 Sml Crude Cover. 0 Jmi Corex:t 7664
—— 5 females (Zeggs). Tide Pool H20. 0 3ml Corexit 7664
—— fems'es (Jeggs). Tice Pool H20. lq Cruce, next day 0 3ml Corexit 7664
—4— 5 females (Jeqgs). Tide Pool H20. lq Cruce. O 3ml Corexit 7664
——— 5 females (Jeggs). Tise Sool H20, lq Crude
Figure 9
Exxon Corexit 7664 with Tar and Hineral 01l
5199


V
2

20
Time (days)
—0— 5 fomales (Jogga). Tido Pool H20. O. 3mi Coroxit 7664
——
5 females (Jeygs), Tide Pool H20, H1S terball
——
5 females (Jeggs). Tide Pool H2O. HS tarball, O Jmi Corexit 7664
—— 5 females (Jeogs), Tide Pool H20, O.Sml Hineral Oil, O.3mi Corexit 7664
4
—
—
Figure 10
Corexit 9550, 9527 and Steam
—
4 5 6
Time (days)
5 females (Jeggs), Tide Pool H20, 19 Crude
5 females (Jeggs). Tide Pool H20, Steam Cleaned
5 females (Jeggs). Tide Pool H20, O. Imi Corexit 9550
5 females (Seggs) Tlde Pool H20, Ig Crude. O. Imi Corexit 9550
5 females (Segga). Tide Pool H20. O. Imi Corexit 9527
5 females (Seggs). Tide Pool H20. O. Imi of Diluted Corexit 9527 with
1Om Ses H20
Figure 11
Exxon Corexit 9580
—
4
b

0 -

0
Time (days)
-------
5 Temales (3 eggs), Tde Pool Seawater, 0.5mi Tiner al Ol
—
5 females (3 eggs). Tide Pool Seawater, lg Crude, 1.24 ml Corexit 9580

5 females (5 eggs), Tide Pool Seawater, 1.24 ml Corexit 9580
—
5 females (3 eggs). Tide Pool Seawater, 0.1 mi Corexit 9580
—
5 females (5 eggs), Tide Pool Sezwater, Ig Crude. 0.1 ml Corexit 9580
— 5 females (no eggs), Filtered Seawater, 1g Cruge
Figure 12
SLIK-A-WAY
—0
O
5
Time (daya)
—
5 females (Jeggs). Tide Pool H20. 1q Crude
5 females (Jeggs)
Tide Pool H20, 19 Crude, 0 Imi Slik-A-Way
5 females (Jeggs). Tide Pool H20, O. Imi Slik-A-Way
5 females (Jeogs), Tioe Pool H20, O. Imi Slik-A-Way+ 1Omi Sea 120
——— 5 females (Joggs). Tide Pool H20. lg Crude. O. Imi Slik-A-Way- 10m H20
Figure 13
Nokomis
6
5
4-
kv v-


4 5 6
0 1 2
Time (days)
—0— 5 females (Jeggs). Tide Pool H20, 19 Crude
5 females (Jeogs). Tide Pool H20, O. Imi of NOKO- I.5ml Sea H20(1:15)
—
5 females (Jeogs). Tide Pool H20. Ig Crude. O.Imi of NOKO15m Sea R
—
— 5 females (Jeggs), Tide Pool H20. O. Iml of NOO- 1Oml Sea H20 min
—s— 5 females (Jeggs). Tide Pool H20, Ig Crude, O Imi of NOKO- 1Oml Sea R0
Figure 14
Nutri-Bio 1000
67

I
0
Time (days)
— . 5 females tes).  fool H20, 1a Cu
les (s).  ol H.0. O.Im Nutri-Bioim Se H20 mix 10)
—  es es) T ol H20. 19Cruoe, O Im Nutri-Bio- Im Ses H20 mu(1.10)
—les (s) Filered H20. lCrue. O ll Nutri-Biom Ses H20 mi(1 10
—  Siemlese) ool H20 19Crue. O.Im Hutri-Plo1Om Set2 m 100