-------
100
90-
_ 80
£
•o
* 7O
(ft ' u
ro
"v
tt 60
c
0)
§ 50
z
CO
40-
n
! 30
CO
2Q-\
Figure 5.7.
TKN
Ammonia
Total Phosphorus
20
40 60
Time (Days)
80
100
Cumulative Release of Ammonia, Total Kjeldahl Nitrogen (TKN), and
Total Phosphorus from MAG AMP Briquettes in Static Flask Experiments.
-------
MAGAMP can be formulated into dense bricks. Bricks weighing
8 and 40 Ibs were field tested as an alternative physical form
for fertilizer application. The usefulness of these bricks in
the field comes from their positional stability on the beach
without an anchoring device. However, these bricks could not be
produced in large quantity and were, therefore, unavailable for
use in any of the fertilization studies. However, because of
their potential promise as an alternative physical form of
fertilizer, separate beach mechanics studies were conducted to
evaluate the nutrient release and distribution characteristics of
these bricks. The very slow release of ammonia from MAGAMP made
it necessary to determine, under controlled field conditions, if
nutrient release could be detected in the field.
Beach pore water sampling stations were placed down-gradient
from MAGAMP bricks as shown in Figure 5.8. Samples for ammonia
analysis were collected 12, 24, and 96 hours after initial
placement of the bricks. The data are shown in Figure 5.9. The
40 Ib brick released up to 138 MM of nitrogen as ammonia, with
the highest concentrations directly down-gradient from the block.
Significant quantities of ammonia were observed up to 2 m from
the bricks at low tide. Ammonia also appears to be well
distributed around the brick. The data suggest that this type of
point source for fertilizer application could be quite useful in
the future.
Sierra Chemical Granules
Two tests were conducted to study the effect of water
exchange rate on nutrient release from this fertilizer. Figure
5.10 shows the cumulative nutrient release pattern with a
variable exchange rate (5 exchanges on the first day, 2 exchanges
per day thereafter through the 10th day, daily thereafter through
the 40th day, and every other day thereafter. The amount of
nitrogen (ammonia and nitrate) released after 80 days was 77% of
the total available nitrogen. When this frequency of water .
exchanges was doubled, 95% of the total nitrogen (approximately
half ammonia and half nitrate) was released after 80 days. The
shape of the release curves were similar. This effect of water
exchanges was probably due to the mechanical agitation of the
system prior to each exchange and to the possible abrasive action
of the fine mesh screen used to drain the granules.
Inipol EAP 22
The results from static tests with this fertilizer are shown
in Figure 5.11. All of the nitrogen (>100%) was released within
the first few water exchanges. The release of more nitrogen than
was theoretically thought to be in the Inipol formulation
suggests that manufacturer's specifications for this batch of
Inipol were incorrect.
43
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Top Transect
Middla Transect
Bottom Transect ( BLH-
Left
Position
Fertilizer
Brick
0.5m
1.0m MMMH 1.0m
-2.0m-
1.0m
-MBMW-
Middle
Position
-2.0m-
Mean High Tide
•WBR]
Mean Low Tide
Right
Position
Figure 5.8. Sampling Point Locations for Magnesium Ammonia
Phosphate Fertilizer Field Test
-------
I I 12 hour* after placeman!
24 hours atler placement
I 96 hours alter placement
140 —
c 2 —
o *
i E
5* 70 —
gz
o ^
r-
1
1
° TM
140 —
it ~
* E
is ro —
• *
g*
O w
°3 _
8
H
G
9
^
|
|
Ml MM MR
140 —
Concentration
(jiM N trom NH3)
-j
> 0
1 1 1
'////////////////////////A
BL BM BR
MAP 1
40- Ib. Brick
4.0 —i
2.0 —
4.0 —i
TM
4.0 —I
2.0 —
ML MM MR
4.0 —|
2.0 —
I
BL BM BR
MAP 2
8- Ib. Brick
2.0 —
4.0 —i
2.0 —
4.0 —i
2.0 —
I
TM
ML MM MR
MAP 5
6- Ib. BrlcH
Figure 5.9. Magnesium Ammonium Phosphate Fertilizer Test: Ammonium Concentration in Beach Pore Water at 12, 24,
and 96 Hours After Placement of Fertilizer (Sampling Locations Given In Figure 5.8)
-------
100
10
Total Phosphorus
I I I I
0 20
I I I
40
I I I
60
I I I
80
I i
100
Time (Days)
Figure 5.10. Cumulative Release of Ammonia and Nitrate from Sierra
Chemical Granules in Static Flask Experiments
-------
120
(A
10
4)
4)
K
e
0)
3
«B
Total Phosphorus
Figure 5.11. Cumulative Release of Ammonia and Total Kjeldahl Nitrogen (TKN)
from Inipol EAR 22 in Static Flask Experiments.
-------
An intermittent submersion test was run on the oleophilic
fertilizer applied to oil-covered Prince William Sound beach
material. The data are shown in Table 5.3. Within 5 minutes
after Inipol-treated oiled rocks were covered with seawater, over
60% of the available nitrogen was released as TKN. However,
following this initial burst, TKN appeared to be released more
slowly, i.e., very little increase in TKN occurred over the next
115 minutes. After decanting the water off the beach material,
allowing it to sit unsubmerged for 6 hours and recovering the
rocks with water, only 8.3% of the available nitrogen was further
released as TKN. Concentrations of ammonia and phosphate
released were quite low, but generally followed the same pattern
as the TKN.
Allowing the fertilizer to remain in contact with the oil
for 6 hours prior to the addition of water did not change the
nutrient release patterns. This suggests that the amounts of
nutrient which sequester with the oil (i.e., not washed off) are
incorporated very soon after fertilizer application.
In addition, mixing the beach material as the Inipol was
applied, or warming the Inipol to 25"C before application, did
not significantly change the amount of nitrogen released in the
first few minutes.
DISCUSSION AND CONCLUSIONS
From these studies it was concluded that bags of "Woodace"
fertilizer briquettes would be used in the initial field
demonstration for slow-release fertilizer. This fertilizer had
good nutrient release characteristics, excellent durability in
the field, and ready availability. Also, given the time
constraints of the bioremediation field demonstration project,
this fertilizer was a reasonable first choice.
Recognizing that bagged briquettes could not be produced in
sufficient quantities for large-scale application, slow-release
fertilizer granules (Sierra Chemicals) were selected for the
second field test, as this material could be easily broadcast
over the beach surface in a large-scale operation. The granules
had good nutrient release characteristics but were not as long
lasting or durable as the briquettes.
Tests with the oleophilic fertilizer, Inipol EAP 22, showed
that it retained nutrients on the surface of oil, although
approximately half of the available nitrogen was lost in the
first minutes following application. Except for the gel point
(ll'C) being high enough to require warming the fertilizer in
cold weather, this liquid fertilizer was potentially very
suitable for large-scale application.
48
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Table 5.3. Release of Ammonia, Total Kjeldahl Nitrogen (TKN),
and Total Phosphorus (TP) from Inipol EAP 22 During Intermittent
Submersion Experiment
Min . from
Start of
Experiment
Ammonia Released (mgN/L)b
5
15
30
60
120
510C
540
600
TP Released (mgP/L)
5
15
30
60
120
510e
540
600
TKN Released (mgN/L)b
5
15
30
60
120
510C
540
600
5 Min.
Contact
Time'
1.1
l.l
1.4
1.3
1.4
0.2
0.1
0.0
1.3
1.2
1.0
1.5
1.0
1.1
0.9
0.9
24.6
26.1
27.2
32.5
29.4
4.6
4.6
4.3
6 Hour
Contact
Time"
0.5
0.4
0.5
0.7
0.7
1.4
1.2
1.1
1.3
1.0
29.8
34.8
35.5
34.3
32.3
a
Time between fertilizer application and initial submersion.
b Initial concentration of nitrogen = 57 mg/L.
c Water drained; beach material remained unsubmerged for 6 hours;
seawater replaced.
49
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SECTION 6
FIELD TEST DESIGN AND METHODS
TEST PLOT SAMPLING DESIGN
Sampling Procedure
The beach sampling design was formulated to generate
scientifically defensible conclusions relative to the success of
bioremediation. Each test site was divided into a series of
plots. The plots were generally 30 m long and 12 m wide running
the length of the beach. Plot size was controlled by the
available beach (i.e., sections of relative uniformity), the
extent of beach covered by the oil, and the prominence of certain
topographical features. Buffer zones of at least 5 m separated
the plots. Larger buffer zones (>20 m) were established between
treated and reference plots to minimize cross contamination.
Cross contamination of nutrients between plots was not expected
because of a small tendency for lateral movement along the
beaches and extensive dilution.
Approximately equal sampling effort was used in three
intertidal zones; high, mid, and low. Zonal sampling was used
to uncover any effect due to length of time of ocean coverage,
rainfall, and freshwater runoff or temperature (exposure to sun,
air, ocean, etc.) that sampling intensity was intended to be
great enough that may have influenced biological and physical
degradation. Sampling intensity was intended to be great enough
that if active biodegradation occurred in only one tidal zone,
then a sufficient number of samples per zone still would be
available for analysis. If degradation occurred in all three
zones, three points could be utilized to discover trend from high
to low tide and to explain changes in biodegradation rates.
From each intertidal zone, blocks were derived by dividing
the beach plot length into seven equal segments, thus creating a
total of 21 blocks. It was recognized that certain sampling
points on the beach were not representative of the entire beach.
For instance, stream runoff flows over one section of the beach
might have been caused by an underlying solid rock outcrop near
the surface of the beach. Having seven samples for each beach
stratum allowed for the existence of a nonrepresentative sample,
or for the possibility of a sample with an obvious gross error
due to a flaw in sampling or analysis. In essence, several
samples were insurance against a host of potential problems. In
addition, several samples were needed to ensure adequate power of
statistical tests.
Each block was divided into 1 m x 1 m sampling grids.
Therefore, although the number of blocks within plots did not
51
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vary with beach size, the number of sampling grids within a block
for a particular plot did vary. Each plot was laid out using
rope secured to rebar stakes to identify the boundaries of the
blocks. Squares of rebar (1 m x 1 m) were used to delineate
sampling grid cells.
For each designated sampling time, a sample was taken from
one grid cell within each block for all analyses. The sampling
grid selection procedure included the following steps:
The sampling crew began at the upper-left-hand corner of
block 1 or 15 and from a random number table picked two
numbers that fell within the confines of the block. That
is, if the block size for the particular plot was 5 m in
length and 3 m in width, the table was used to pick a number
from 1 to 5 to designate the distance along the beach from
the starting point. A second number from 1 to 3 was picked
to designate the distance toward the low-tide mark. Squares
of 1 m X 1 m rebar were used to locate the sampling grids.
The intersection of the two randomly selected points was the
upper-left-hand corner of the selected sampling grid. The
same sampling grid location was used for all blocks in a
single plot during a single sampling event.
• A 1 m x 1 m frame was placed on the beach in the designated
grid cell and samples were collected from the center of the
frame.
• If a sample could not be taken at the center of the grid
cell, a random number between 1 and 12 was chosen. These
numbers represented positions on the face of a clock, in
which 12 pointed to high tide. The sampler then moved away
from the center of the frame toward the indicated clock
position until an appropriate site was found within the
sampling frame. The sampling crew used judgment in many
situations, e.g., if a large boulder was encountered, the
site was discarded and step 3 was repeated.
This procedure was repeated for -each block until completion
of the beach plot sampling. For all analyses of the samples,
except nutrients, site selection was the same.
All sampling was performed at low tide. Two days were
required to sample all plots at each test site. Consequently,
only one-half of the control plots were sampled each day.
For chemical measurements, a 25% error was assumed for the
sampling system, and changes of 30% to 50% were suggested as
significant changes in the measured variables. For testing a
hypothesis at the 95% confidence level with a power of 0.90, 6
replicates are needed to detect a 30% change in the measured
variable and 3 are needed to detect a 50% change. For a power of
52
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0.95, 8 and 3 replicates are needed to detect a 30% and a 50%
change, respectively.
For biological measurements, a change of 50% in population
value was considered as a significant response. Experience
suggested that measurement variability could range from 25% to
75%. For hypothesis testing as described above, sample sizes of
3, 9, or 20 are needed to detect 25, 50, or 75% change in the
measured variable with a power of 0.90, and sample sizes of 3,
11, or 25 are needed to detect 25, 50, or 75% changes with a
power of 0.95.
This analysis assumes the data all follow a normal
distribution. Unfortunately, environmental populations often are
not normally distributed. In the present case, differences in
the length of time sample sites were underwater, inhomogeneous
drainage of freshwater across the beach, drift, and other factors
affected the variability of beach conditions, and therefore
sampling system errors. All these concerns tended to inflate the
number of samples needed to ensure adequate power of statistical
analyses.
The overall design of beach sampling efforts was non-optimal
in a statistical sense. The major limitation arose from the lack
of duplicate beaches for each treatment (and reference).
Measured effects were attributable to both nutrient treatment
effects and beach effects. It could not be determined
statistically whether an increased bioremediation rate at a site
was due to either the treatment or to a fortuitously good
location, since these two variables were confounded. When only
one treated beach was successful, low confidence should be
assigned to the result; however, because two types of beaches and
two types of treatments were used, when one or both treatments
were successful on both types of beaches, confidence in the
results may be high.
Sampling Method
On mixed sand and gravel beaches, samples were taken by
placing a metal pail with the bottom removed onto the beach
surface and working the bucket down into the substratum. As
small rocks were encountered that prevented the pail from going
further into the beach material, the material around the pail was
excavated and the rock removed. If 50% of the rock was inside
the perimeter of the pail, it was added to the pail and included
in the sample. If 50% or more was on the outside, it was
excluded from the sample. All large rocks (approximately 4 cm or
larger in any dimension) were discarded from the sample, since
the amount of oil covering their surface was insignificant
relative to oil in the entire sample, and exclusion of these
rocks reduced variability in substrate characteristics of the
sample.
53
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Once the pail was worked down into the beach material to a
depth of approximately 13 to 14 cm (using marks on the inside of
the pail), all of the beach material down to 10 cm, including
small rocks that protruded more than 50% above that mark, were
included in the sample. Rocks that did not protrude more than
about 50% above the mark were left behind. All beach material
removed from the sampler was placed in new paint cans that had
been previously washed with a detergent solution and thoroughly
rinsed. The contents of the paint can were then thoroughly mixed
with a steel spoon. A subsample of material sufficient to fill a
400 ml wide-mouth jar was taken from the mixed sample. The jar
and its contents were subsampled for microbiology analysis and
then frozen.
Cobblestone beaches were sampled by removing all the rocks
from the sampling area covered by the bottomless pail and placing
them in a paint can. Enough sample of the underlying mixed sand
and gravel to fill a 400 ml wide mouth jar was then collected.
Samples in both the jar and the paint can were frozen for
subsequent analysis.
In each treatment and control plot, in situ jars were
inserted into the beach material. These jars served as a
consistent source of beach material in which the oil
concentration and composition was well defined. The jars were
straight-sided, high-density polyethylene containers 10" high and
8" in diameter with screw cap lids. The jars were perforated
with 1/16" wide and 2" long slits at 2" spacings in the walls,
cap, and bottom to allow adequate percolation of beach
interstitial water through the contained beach material. These
jars contained a known amount of oil-contaminated beach material.
To fill the jars, a large amount of contaminated beach material
was collected and thoroughly mixed in a large plywood box for 30
minutes. Subsamples were used to completely fill each jar, and
the jars were implanted into the beach material 4 inches below
the beach surface. Subsamples were also taken and frozen for T=0
chemical analysis. Duplicate jars were placed in the sediment,
with a spacing of 4 inches horizontally between any two
containers, and the lid placed up-gradient. A total of 40
containers were placed in the plot treated with oleophilic
fertilizer, 42 in the plot with the water-soluble fertilizer, and
18 in the reference plot. The differences in numbers were
functions of the plot size and the availability of containers.
These containers were sacrificed when significant
biodegradation occurred as evidenced by a reduction in the
pristane/phytane ratio from samples taken from the beach material
in the plot. Subsamples were frozen for subsequent analysis of
changes in residue weight and composition of the oil. Enough
jars were available to allow two samplings.
54
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The in situ sampling jars were placed along a transect
between the high-tide and mid-tide zones and between the mid-tide
and low-tide zones. Along these transects, two jars were placed
in each block, equidistant from the corners of the block, except
in control plots, where pairs of jars were placed in alternate
blocks.
FERTILIZER APPLICATION
Slow-Release Water-Soluble Fertilizers
Slow-release fertilizers used in this project were
briquettes that were applied in mesh bags and granules that were
broadcast. The following paragraphs describe the methods used to
place these fertilizers.
Herring-seine net bags filled with slow-release fertilizer
briquettes (Woodace) were placed on the beach in a manner that
was intended to provide complete exposure of the beach material
to nutrients leaching from the bags. Each bag contained
approximately 33 pounds of briquettes. Application of the
briquette bags occurred on June 11, 1989. The total quantity of
briquettes applied to the 35 m x 12 m plot (Otter Beach) was
800 pounds, representing approximately 100 pounds nitrogen and
24 pounds phosphorus (as P2O5). The bags were tethered to 3-foot
sections of 1-1/8 inch diameter steel rods that were buried 6
inches below the surface of the beach. Figure 6.1 indicates the
positioning of the 24 bags in the experimental plot. Three rows
of eight bags were placed at 2 m, 6m, and 10 m from the top of
the plot.
On June 20 and 21, 1989, the bags were repositioned
according to the layout in Figure 6.2, as the bags located at the
2 m row were not being submerged consistently by the high tide
(see below). Additionally, preliminary data indicated that the
nutrients were being channelled vertically down the beach. Four
more bags were added to the previous 24 bags for a total of
28 bags, resulting in 920 pounds of fertilizer (130 pounds N).
The same arrangement and repositioning was used for the bri-
quette bags on Seal Beach. This beach was smaller (28 m wide
rather than 35 m) and, thus, the weight of briquettes applied per
bag was 26 pounds (rather than 33 pounds) for a total of
620 pounds, increasing to 730 pounds after the four new bags were
added.
Figures 6.3 and 6.4 represent the significant tidal fluctua-
tions typical of Snug Harbor. These tidal fluctuations affected
the amount of time each zone was underwater and that nutrients
were being dissolved and transported. For example, in the sand
and gravel plot treated with the fertilizer briquettes, the top
row of fertilizer bags were placed at a relative tidal height of
55
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I I 1 i I i I I
7 ^
7 \
r •>
^ ^
\ \
/
7 \
\ \
r ^
7 \
f 1
7 ^
f 1
7 ^
[ 1
7 \
7 *
Figure 6.1. Placement off the Bags off Fertilizer Briquettes on Otter
and Seal Beaches (See Figure 4.1. for beach locations).
-------
Figure 6.2. Repositioning of the Bags of Fer'^zer Briquettes on Otter
and Seal Beaches (See Figure 4.1. for beach locations).
-------
0)
0)
CD
Date
Figure 6.3. Tidal Fluctuations for High Tides, Snug Harbor, June 6-30, 1989.
-------
-3
Date
Figure 6.4. Tidal Fluctuations for Low Tides Snug Harbor, June 6-30, 1989.
-------
13 feet. As shown in Figure 6.3, the top row of bags were only
underwater approximately one-fourth of the days in June. Conse-
quently, precipitation was a primary factor in controlling the
dissolution and transport of the nutrients in this zone. This
high-tide zone, which was contaminated with oil in Snug Harbor,
was representative of other oil-contaminated zones in the Prince
William Sound.
Slow-release granules were applied to Tern Beach in Passage
Cove using a commercial broadcast fertilizer spreader, at a rate
of approximately 0.0033 lbs/ft2. The total application of
nitrogen and phosphorus by slow-release granules in Passage Cove
was approximately 400 Ibs and 40 Ibs, respectively. The granules
stuck to the oil on the rock surfaces and were therefore not
easily displaced from the beach or redistributed by the tidal
action.
Oleophilic Fertilizer
Oleophilic fertilizer (Inipol EAP 22) was first applied to
Otter Beach in Snug Harbor (mixed sand and gravel) on June 8,
1989. A total of 10 gallons (83 pounds) was applied, which
represented approximately 5% of the estimated weight of the oil
on the treated beach. The following computations were made to
determine the application rate:
Plot was 20 m x 12 m = 240 m2 = 2,600 ft2
Assumptions:
6 inch oil depth: 2,600 ft2 x 0.5 ft = 1,300 ft3
20% void volume: total rock volume = 1,300 ft3x 0.8 =
1,000 ft3
Specific gravity of rock = 2.6 or 160 pounds/ft3
Weight of rock = 160 pound/ft3 x 1,000 ft3 =
160,000 pounds
Oil = 1% of weight of rock = 1,600 pounds
• Specific gravity of Inipol = 1.0
Based on a 5% loading rate of the Inipol/oil, 1,600 pounds
of oil x 0.05 = 83 pounds Inipol or 10 gallons.
A second application 10.5 gallons of Inipol was made on
June 17, 1989, to the Otter Beach plot based on recommendations
from Elf Aguitaine representatives.
60
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The first oleophilic application at Seal Beach in Snug
Harbor (cobble) was on June 9 at a rate of 13 gallons. The
second application of 14 gallons occurred on June 18th.
Oleophilic fertilizer was applied on the plots as the tide
was going out in the evening. Application was initiated
beginning at the top of the beach, an hour after the tide was
past the lowest zone in the plot. The fertilizer was applied
using a backpack sprayer with a capacity of four gallons. The
fertilizer was initially wanned, to ensure uniform application
and to prevent clogging of the spray nozzle.
The weather during the first applications on June 8 and 9
was rainy and cool. During the second application, both days
were clear and sunny, with temperatures around 60*F. Examination
of the plots the day after the second application indicated a
noticeable gelatinous sheen on the surface of the sediment and
rocks where the fertilizer was applied. The sheen lasted for two
days. Wave action was minimal over this period. This sheen was
not seen with the first application.
Sprinkler System
Kittiwake Beach in Passage Cove was used to evaluate the
effectiveness of application of nitrogen and phosphorus via spray
irrigation. Nitrogen and phosphorus fertilizers dissolved in
seawater were sprayed onto the beach daily. The spray irrigation
system used sprinkler heads typical of lawn sprinklers. The
fertilizer solution was pumped by a gasoline-driven well pump to
four sprinkler heads set on each side of the plot. Each
sprinkler swept a 180" arc across the plot during application.
Typical applications were about 0.4 inch of water per day.
Application rates were established to supply 6 M9/1 of nitrogen
and 3 /jg/1 of phosphorus to pore water in the saturated beach
material to a depth of 2 m.
ANALYTICAL PROCEDURES
Detailed information on the standard operating procedures
are given in the Quality Assurance Plan, which is available upon
request from Dan Heggem at the Environmental Monitoring Systems
Laboratory in Las Vegas, NV. Only brief accounts of the
analytical procedures will be given here.
Oil Chemistry
Beach samples consisted of either mixed sand and gravel
frozen in 400 ml I-Chem jars or cobblestones wrapped in aluminum
foil and frozen. The mixed sand and gravel was thawed
immediately prior to the initiation of oil analysis, and the
contents were mixed thoroughly. A weighted 100 gm subsample was
removed and mixed thoroughly with 300 mis of methanol in a
61
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separatory funnel. The slurry was shaken for five minutes, and
the methanol was decanted into a 2 L separatory funnel. The
samples were similarly re-extracted two times with 300 ml of
pesticide - or HPLC grade methylene chloride. The three organic
fractions were combined and back-extracted with 100 ml of 3%
aqueous sodium chloride. The phases were separated and the
aqueous portion was extracted with 50 mL of fresh methylene
chloride. This aqueous extraction in methylene chloride was
added to the combined organic fraction.
The combined organic fraction and 3 or 4 clean boiling chips
were placed into a 1 L round bottom flask fitted with a three-
ball Snyder column. The volume of solvent was reduced until the
color was approximately the color of dilute weathered oil (ca 15
mg/2 ml methylene chloride). The final volume of the extract was
measured with a syringe having an appropriate graduated cylinder,
and an aliquot was transferred to a GC autosampler vial.
All of the cobblestones were extracted using the same
procedure (methanol, followed by methylene chloride), except that
shaking was replaced by gentle swirling to remove oil from the
rock surfaces.
Gas chromatographic (GC) analysis was accomplished with an
instrument capable of reproducible temperature programming with a
flame ionization detector and a reliable autosampler. The GC
conditions were:
Column: DB-5, 30 m X 0.25 mm, film thickness 0.25 urn
Initial Temperature: 45"C, 5 min. hold
Temperature Rate: 3.5*C/min
Final Temperature: 280°C, 60 min. analysis
Injector: splitless, 1 in valve closure
Injector Temperature: 285"C
Injection: 2.0 /il
Detector: FID, 3508C
Those samples that demonstrated significant evidence of
biodegradation were fractionated to allow separate determination
of aliphatics and aromatics. Aliquots of the sediment or oil
extracts selected for fractionation were solvent exchanged to
hexane under a stream of dry nitrogen. A volume of 50 p.1 of
hexamethylbenzene (80 ng//xl) and 25 Ml of n-decyclohexane (1
fig/Hi) was added to each sample extract prior to fractionation.
The fractionation was accomplished using a 10 mm X 23 cm glass
column that was slurry packed (with hexane) with 60/200 mesh
silica gel activated at 210 C for 24 hours. The aliphatic
fraction was eluted with 30 ml of hexane and the aromatic
fraction was eluted with 45 ml of hexane/benzene (1:1). Both the
aliphatic and the aromatic fractions were analyzed using the GC
methods described above.
62
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Subsamples of the final concentrated extract were subjected
to mass spectral analysis using a Hewlett-Packard Gas
Chromatography/Mass Spectrometry (GC/MS) system provided by the
U.S. Coast Guard Mobile Analytical Laboratory. The analytical
procedure is given in their Fucus oil analysis protocols
(Hildebrand, 1989).
Subsamples (5-15 mis) of the final concentrated extract were
also removed, filtered through sodium sulfate, and placed in
tared watch glasses. After passive evaporation of the solvent,
the oil residue weight was determined.
Changes in oil composition were determined using three data
analysis procedures:
The branched hydrocarbons pristane and phytane were used as
internal standards, under the assumption that they were slow
to degrade, and weight ratios of C17:Pristane and
CIS:Phytane were calculated as indicators of biodegradation.
The total weight of all alkanes appearing on the
chromatograph, normalized to the total residue weight of
oil, were compared on a sample by sample basis.
Assuming that hopane and norhopane were not biodegraded,
weight ratios with other identifiable hydrocarbons were
calculated.
Nutrient Analysis
Water samples taken for nutrient analysis were filtered
(Whatman glass fiber filter) and then placed in 150 ml plastic
screw capped bottles. The bottles were immediately frozen with a
dry ice-antifreeze solution. Water samples taken offshore were
collected with a clean bucket and subsamples were taken for
nutrient analysis. Water samples from the beach were collected
behind or in front of an ebbing or flooding tide, using a
commercial root feeder. The root feeder was outfitted with
rubber tubing and a peristaltic pump to allow interstitial pore
water to be sucked into the feeder tube and sampled at the top of
the feeder tube. The feeder was inserted approximately 20 cm
into the mixed sand and gravel. Pore water was flushed through
the feeder for one minute prior to sampling.
Nutrient concentrations were determined using the following
standard methods:
Nitrate-
Nitrate was determined by reduction to nitrite followed by a
colorimetric assay for nitrite (see below). Nitrate was reduced
to nitrite by passage through a column containing copperized
cadmium filings. The resulting solution contained total nitrite
63
-------
that was equivalent to the sum of the initial nitrate and nitrite
in the sample; nitrate was determined by difference. The
procedure for nitrate was derived from the non-automated
technique described in Parsons, Maita, and Lalli (1984).
Detection limits for nitrate and nitrite were expected to be
about 0.05 and 0.01 jiM, respectively. An estimate of the
precision for the nitrate measurements at the 20 iM level in the
samples was calculated as the mean of n determinations ±0.5
(mean/n2) in
Nitrite —
Nitrite was determined by the Geiss reaction in which
sulfanilamide and N- ( 1-Naphthyl ) ethylenediamine dihydrochloride
(NNED) react with nitrite in an aqueous acidic solution to form
an intensely pink diazo dye with an adsorption maximum at 540-543
nm.
Ammonium —
Ammonium was determined by the Berthelot reaction in which
hypochlorite and phenol react with ammonium in an aqueous
alkaline solution to form indophenol blue, an intensely blue
chromophore with an absorption maximum at approximately 637-640
nm. Based on the information in Parsons, Maita, and Lalli (1984)
and Whitledge, Malloy, Patton, and Wirick (1981), the detection
limit for ammonium was expected to be approximately 0.1 /iM. An
estimate of precision at the 1 MM level was calculated as the
mean of n determinations + 0.1 (mean/n2) in
Phosphate —
Phosphate (i.e., orthophosphate) was determined as
phosphomolybdic acid, which has an absorption maximum at 880-885
nm in its reduced form in the presence of antimony (Parsons,
Maita, and Lalli 1984) . The detection limit for phosphate was
expected to be about 0.03 juM. An estimate of the precision at
the 3 MM level was calculated as the mean of n determinations +
0.03 (mean/n2) in units of
Total Kjeldahl Nitrogen (TKN)~
TKN was measured by heating the sample in a sulfuric acid
solution containing KySO4 and HgSO4 and comparing colorimetrically
with standards and blanks using a Technicon AutoAnalyzer (EPA
method 365.4) .
Microbiological Analysis
Numbers of oil-degrading microorganisms were measured by
extinction to dilution procedure using oil as the carbon source.
The samples for microbiological analysis were a subset of the
samples taken for analytical analysis. A 5 g portion of the
analytical sample for the sand and gravel beach was transferred
to a pre-weighed sterile dilution bottle.
64
-------
The defined nutrient medium (DNM) used in these tests
contained (per liter of distilled water): NaCL, 24 g;
MgS04.7H20, 1.0 g; KCl, 0.7 g; KH2P04, 2.0 g; Na2HP04/ 3.0 g; and
NH4N03, 1.0 g. The pH of the medium was adjusted to 7.4 with l.O
N NaOH following autoclaving. The DNM was distributed in 4.5 ml
portions to sterile dilution tubes. The initial dilutions were
prepared by adding 5.0 g wet weight of sand and gravel subsample
to the prepared dilution bottles containing 50 ml autoclaved DNM.
Following vigorous mixing (the sample was rapidly shaken by hand
for 15 seconds), a 0.5 ml sample of the initial dilution was used
to prepare a dilution series from 102 to 1010. Each tube was then
amended with 20 /*! of weathered Prudhoe Bay crude oil collected
from an oil-contaminated beach in the Prince William Sound. The
tubes were scored at 21 days of incubation. Tubes that showed
visible microbial turbidity or changes in the physical form of
the oil (oily droplets converted to stringy and flaky particulate
material) were considered positive. The tubes were scored
independently by two individuals. Numbers of oleic-acid
degrading bacteria were determined using standard plate counting
procedures on minimal-salts agar medium supplemented with 1%
oleic acid.
Measurements of Microbial Activity—
Evolution of 14C02 from phenanthrene-9-14C, hexadecane-l-14C,
and naphthalene-l-14C was used to measure the activity of
indigenous petroleum-degrading microorganisms as influenced by
the addition of Inipol and watei-soluble fertilizers. Duplicate
5.0 g samples of beach material (1-5 mm diam.) obtained from
oiled beaches with and without Inipol or water-soluble fertilizer
treatments were added to 10 ml artificial salt-water medium
(ASWM) in clean, sterile 100 ml Wheaton bottles. Each bottle was
spiked with 0.1 /iiCi of radiolabeled substrate and crimp sealed
with a Teflon-lined septum. Following 0, 12, 24 and 48 hr
incubation in the dark at ambient temperature (ca. 15 °C), vessels
were sacrificed and the amount of radiolabeled C02 released from
acidified medium was determined. Medium was acidified to pHO.O
with HC1, the headspace was flushed for 10 min., and CO2 was
trapped in 5.0 ml of 1 N NaOH. Subsamples (0.5 ml) of NaOH
trapping solution were added to 10.0 ml Ready-safe liquid
scintillation cocktail, and the amount of radioactivity present
was determined by liquid scintillation. Trapping efficiency was
determined by recovery of 14Na2C03 from acidified medium. Quench
was accounted for internally.
Ecological Monitoring
Water samples collected offshore in cubitainers were
transported to the laboratory in Valdez and analyzed for several
parameters that might be affected by bioremediation research
efforts. Analysis included measurements reflecting possible
eutrophication, release of oil from the beaches, toxic effects
65
-------
from the fertilizers themselves, and the presence of mutagenic
oil residues. Procedures for these measurements are as follows:
Eutrophication Measurements—
Seven biological and chemical indicators of eutrophication
were monitored routinely through the fertilizer addition periods:
Chlorophyll—One-liter water samples were filtered through
glass fiber filters, and the filters were extracted with a
solution of 90% acetone and 1 N NaOH. After overnight incubation
in the refrigerator, samples were centrifuged and the optical
density of the supernatant was determined at 750 nm (total
absorbance) and 665 run (chlorophyll a). Phaeophytin was
determined by rereading the optical densities after the addition
of 10% HC1.
Primary Productivity—Photosynthetic productivity by
phytoplankton was estimated by incorporation of 14C-bicarbonate.
Plankton samples collected in the field were transported to the
Valdez laboratory, incubated in BOD bottles in an outside
waterbath, filtered, and frozen. Prior to July 5, 1989, samples
were then sent to the U.S. EPA Environmental Research Laboratory
ERL/Gulf Breeze for analysis using a liquid scintillation
counter. Once the liquid scintillation counter was operational
at the Valdez laboratory, on July 5, primary productivity samples
were counted there.
Bacterial Abundance—Estimates of the numbers of bacteria
per ml of water in the water column were determined using
acridine orange direct counting with fluorescent microscopy
(Hobbie et al., 1977). Water samples were filtered through black
Nucleopore 0.2 u pore size filters and stained with buffered
acridine orange solution (Fisher chemical). A minimum of 200
bacterial cells were counted in 5 to 10 grid fields in the
microscope.
Bacterial Productivity—The thymidine incorporation method
of Fuhrman and Azam (1982) was used to measure bacterial
productivity. Triplicate water samples were spiked with 5 Ml of
H-methyl thymidine (1.1 pCi; 2.86 nM final concentration),
incubated for 20 minutes and then extracted with 5 ml of cold 10%
trichloroacetic acid (TCA). Samples were filtered through 0.22
urn Millipore filters, washed with cold TCA, and the radioactivity
on the filter was measured in a liquid scintillation counter.
Microflacrellate Abundance—Microflagellate abundance was
estimated with epifluorescence direct counts using the method
described by Caron (1983).
Dissolved Organic Carbon. Particulate Carbon, and
Particulate Nitrogen—Ten ml water samples for dissolved organic
carbon (DOC) analysis were filtered through precombusted glass-
66
-------
fiber filters (Whatman GF/F). Filtrates were sealed in glass
ampoules, frozen and stored until they were analyzed at ERL/Gulf
Breeze. To remove inorganic carbon, 1 ml water samples are
acidified with 5 /il concentrated phosphoric acid and bubbled with
N2 gas for 10 minutes. DOC was measured in an Ionics model 555
high combustion temperature TOC analyzer equipped with a platinum
catalyst.
Seawater samples (500 ml to 1000 ml) were filtered through
pre-combusted glass-fiber filters (Whatman GF/F) for subsequent
analysis of particulate carbon and nitrogen. Filters were dried
at 50"C and shipped to ERL/Gulf Breeze for analysis. Particulate
carbon and nitrogen were measured simultaneously with a Carlo
Erba Model NA 1500 CHNS analyzer.
Stable Isotope of Carbon and Nitrogen—
For analysis of stable isotopes of carbon and nitrogen in
seawater, filters were prepared as was described above for
particulate carbon and nitrogen analyses. These filters, benthic
algae, and mussels were collected and shipped to Texas A&M for
analysis. Samples were dried and then combusted in quartz tubes
with cupric oxide at 900°C. Co2 and N2 gases were isolated by
cryogenic distillation. Stable carbon and nitrogen isotopes were
measured by mass spectroscopy.
Caged Mussels
At each station designated for mussel monitoring, four cages
filled with 25 mussels (Mytilus edulis) each were deployed to
measure the uptake of petroleum hydrocarbons that might be
released into the water column following application of
fertilizers on the beaches. The mussels were collected from
Tatitlek Narrows, an area of Prince William Sound that was not
affected by the oil spill. The mussels were sampled weekly from
the cages throughout the summer. At each sampling, 3 mussels
from each cage were sacrificed and the tissues were removed from
the shell and frozen. The frozen tissues were returned to the
laboratory, where the tissues from all 3 mussels from a single
cage were extracted by homogenizing and spiking approximately 20
g of tissue with appropriate surrogates, digested with 6 N KOH at
35°C for 18 h. The sample was then serially extracted with ethyl
ether. The eluate was dried with sodium sulfate, concentrated,
and cleaned using the EPA Method 3611 alumina column cleanup
procedure to remove matrix interferences. The combined saturated
and aromatic fractions collected from the cleanup column were
concentrated and optionally split in aliquots for analysis.
Field Toxicitv Tests
Application of fertilizer poses a potential toxic risk to
marine biota if water concentrations of oleophilic fertilizer or
ammonia approach 50 mg/1, the LC50 for the most sensitive species
67
-------
tested in the laboratory toxicity tests. To characterize the
extent to which toxic concentrations might develop in the course
of, or following, application of fertilizers to oiled shorelines,
toxicity tests were conducted using field water samples and a
testing scheme similar to that used to test acute toxicity of
industrial effluents. The data provided insight into the rate at
which fertilizers entered the marine environment from test
beaches and the amount of dilution required to mitigate toxic
effects.
Water samples were collected at specified intervals before
and after application of Inipol and the Sierra Chemicals slow-
release granules to shorelines in Passage Cove. These samples
were sent to a consulting laboratory for 48 hr toxicity tests
with oyster larvae Crassostrea aicras. Endpoints monitored for
these tests were larval survival to test termination and
percentage of larvae that exhibited abnormal development.
One water sample (field control) was collected at the field
reference site, immediately outside of the test area, just before
the initiation of fertilizer application. At the beach where
fertilizer was applied (a 100 m stretch of shoreline), water was
collected at 0.5 m depth (just above the bottom) immediately off-
shore. Water samples were collected immediately before
fertilizer application (pre-application, which was 2 hours before
low tide), following the completion of application (2 hours after
low tide), and again after 1 hr, 3 hr, 6 hr, 12 hr, and 18 hr
intervals. Sampling stopped at this time in order to return
samples for shipping. All water samples were maintained at 4°C
until toxicity tests began.
Oyster larvae toxicity tests were conducted with a standard
dilution series (used for effluent toxicity tests: 100%, 56%,
32%, 18%, and 10%) prepared for each water sample collected after
application. Because the salinity of site water was 26 ppt,
field samples were adjusted to 28 ppt by addition of 90 ppt brine
solution before test dilutions were prepared. The salinity
adjustment accounted for approximately 3% dilution and was
selected as the minimum change necessary to ensure that salinity
was sufficient to sustain normal development of oyster larvae.
(This dilution was not accounted for in the subsequent reporting
of sample concentrations.) The same brine was diluted to 28 ppt
and tested as a "hypersaline control" to characterize the
adequacy of the brine mixture as a test solution. Laboratory
seawater was diluted from 32 ppt to 28 ppt and tested as a
seawater control.
68
-------
SECTION 7
FIELD TEST RESULTS - SNUG HARBOR
VISUAL OBSERVATIONS
Test beaches at Snug Harbor were moderately contaminated.
Visually, the cobble plots had a thin coating of dry, sticky,
black, oil covering rock surfaces and gravel areas under the
cobble. Oil did not penetrate more than a few centimeters below
the gravel surface. In mixed sand and gravel plots, oil was well
distributed over exposed surface areas and commonly found 20-30
cm below the surface. In many areas of the test plots, small
patches of thick oil and mousse could be found. This material
was very viscous and mixed with extensive amounts of debris.
Approximately 8-10 days following oleophilic fertilizer
application to the cobble beach plot, reductions in the amount of
oil on rock surfaces were visually apparent. It was particularly
evident from the air where the contrast with oiled areas
surrounding the plot was dramatic, etching a clean rectangle on
the beach surface. The contrast was also impressive at ground
level; there was a precise demarkation between fertilizer-treated
and untreated areas.
Close examination of this treated cobble plot showed that
much of the oil on the surface of the rocks was gone. There were
still considerable amounts of the oil under rocks and in the
mixed gravel below these rocks. The remaining oil was not dry
and dull as was the oil in other areas of the beach, but appeared
softened and more liquid. It was also very sticky to the touch,
with no tendency to come off the rocks. At the time of these
observations, no oil slicks or oily materials were observed
leaving the beach during tidal flushing.
The mixed sand and gravel beach treated with oleophilic
fertilizer also appeared to have reduced amounts of oil in
8-10 day period. However, differences between treated and
untreated plots were not as dramatic as on the similarly treated
cobble beach. Loss of subsurface oil in treated areas was also
visually apparent. Reduction of oil contamination was
particularly evident at sampling times, as noticeably less oil
remained on sampling equipment used on this beach plot.
At this time, all other plots appeared as oiled as they did
at the beginning of the field study. There were essentially no
visual indications of oil removal on plots treated with slow-
release fertilizer briquettes.
69
-------
Over the next two to three weeks, the cleaned rectangle on
the cobble beach remained clearly visible. Oil below the rocks
remained but was less and less apparent and untreated reference
plots appeared relatively unchanged. The oleophilic-treated
mixed sand and gravel plot actually showed a greater loss of oil,
appearing increasingly cleaner.
Six to eight weeks after fertilizer application the contrast
between the treated and untreated areas on the cobble beach
narrowed. This was due to reoiling from subsurface material
concurrent with the slow removal of oil on the beach material
surrounding the plot. However, it was evident that the total
amount of oil on the treated plots had decreased substantially
relative to reference plots. The corresponding mixed sand and
gravel plot was also reoiled but to a lesser extent. All other
plots still had observable oil contamination but generally less
than that seen at the beginning of the study.
Toward the end of the summer season the area used for the
bioremediation study became steadily cleaner, including most of
the areas surrounding the test plots. This was attributed to
several storms and more frequent rainfall. A heavily
contaminated area to the south which was never treated, remained
heavily contaminated by all visual criteria.
NUTRIENT CONCENTRATIONS
Table 7.1 shows the ammonia concentrations found in
interstitial water for the treatment and reference plots. The
initial background ammonia concentrations (T=0) were low, and
uniform throughout the plots. One to two days after application
(T=l) of the fertilizers, an increase in the ammonia concentra-
tions was evident only in the plots treated with the oleophilic
fertilizer. However, concentrations within the zones were highly
variable. Based on the literature and laboratory nutrient
release experiments described in Section 6, a pulse of ammonia
was expected following application.
In contrast, ammonia concentrations in the plots treated
with the slow-release briquettes remained at background levels.
This is not unreasonable, because nutrient release studies with
the briquettes showed nitrogen was released entirely as TKN,
probably as urea. The absence of elevated NH4 concentrations
suggests that, on the beaches, hydrolysis of urea by
microorganisms leads to immediate uptake of the resulting ammonia
by bacteria or algae.
Eight to 10 days after application of the fertilizers (T=2),
ammonia concentrations were above background only in the sand and
gravel plot treated with oleophilic fertilizer. Ammonia
concentrations in plots treated with briquettes were comparable
70
-------
Table 7.1. Ammonia Concentrations in Interstitial Water Samples
T=0 (before application)
Tide
Zone
High
Mid
Low
Block
NH. (uM N)
6/08/89 6/10/89 6/10/89
OSW SCW
6/9/89
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2.0
2.1
2.0
1.9
2.0
2.1
2.4
2.3
2.1
2.2
NS
NS
NS
NS
NS
2.1
2.3
2.3
2.1
2.2
2.1
2.0
2.2
2.0
2.0
2.8
2.5
2.7
2.6
2.6
3.0
2.6
2.5
2.7
2.7
2.6
2.7
2.7
2.6
2.6
NS
NS
NS
NS
NS
2.3
2.3
2.2
2.2
2.2
2.0
2.4
2.1
2.6
2.3
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
T=l (1-2 days post application)
Tide
Zone
High
Mid
Low
Block
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
ESR
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
6/09/89
OSO
NS
NS
NS
NS
NS
2.6
92.0
8.5
4.8
27.0
22.0
460.0
9.4
2.4
123.4
6/12/89
OSW
NS
NS
NS
NS
NS
0.4
0.7
0.2
0.2
0.4
1.1
0.9
0.5
1.0
0.9
6/12/89
SCW
1.2
DL
0.5
0.4
0.5
0.2
2.2
DL
0.3
0.7
0.8
0.6
0.5
0.4
0.6
6/10/89
SCO
57.0
300.0
9.9
3.8
92.7
410.0
61.0
2.8
6.5
120.0
190.0
2.9
2.4
3.0
48.8
SCR
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
71
-------
Table 7.1. (Continued)
T=2 (8-10 days post application)
Tide
Zone
High
Mid
Low
T=3 (30
Tide
Zone
High
Mid
Low
Block
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
days post
Block
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
ESR
NS
NS
NS
NS
NS
DL
DL
DL
DL
DL
0.5
1.1
1.1
0.8
0.8
6/18/89
OSO
NS
NS
NS
NS
NS
36.0
30.0
30.0
2.8
24.7
19.0
29.0
3.9
0.9
13.2
6/18/89
OSW
NS
NS
NS
NS
NS
DL
0.3
0.3
DL
0.2
DL
0.3
0.6
DL
0.2
6/18/89
sew
DL
DL
DL
DL
DL
DL
DL
DL
DL
DL
1.3
0.3
0.6
0.5
0.7
application)
7/7/89
ESR
0.6
0.4
0.5
0.7
0.6
0.4
0.4
0.4
0.7
0.5
0.6
0.4
0.4
0.6
0.5
7/7/89
OSO
0.3
0.4
0.3
0.5
0.4
0.3
0.2
0.4
0.4
0.3
0.5
0.4
0.4
0.4
0.4
7/7/89
OSW
0.5
0.6
0.9
0.4
0.6
0.5
0.5
0.4
0.5
0.5
0.5
0.5
0.4
0.6
0.5
7/7/89
sew
1.0
0.4
0.6
0.6
0.6
0.6
0.8
0.8
0.8
0.8
4.2
1.0
0.8
0.8
1.7
6/19/89 6/19/89
SCO SCR
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.4
0.4
0.6
0.5
0.5
1.0
1.0
0.6
0.6
0.8
0.8
0.9
0.6
0.7
1.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7/7/89 7/7/89
1.4
0.2
0.8
0.2
0.6
0.9
1.4
0.5
1.0
1.0
0.5
0.6
1.0
1.2
0.8
72
-------
Table 7.1. (Continued)
T=4 (6 weeks post application)
Tide 7/17/89
Zone Block ESR
High 1 1.0
3 1.2
5 1.3
7 1.0
Mid
Low
ESR
SCR
OSO
SCO
OSW
SCW
NS
DL
ND
Avg
8
10
12
14
Avg
15
17
19
21
Avg
= Control
= Control
1.
1.
1.
0.
1.
1.
1.
1.
1.
1.
1.
Mixed
Cobble
1
3
0
8
0
0
0
0
3
7
2
Sand
7/16/89 7/16/89 7/16/89
OSO OSW SCW
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS
0.
1.
1.
0.
1.
0.
1.
0.
0.
0.
and
9
2
0
9
0
9
1
8
9
9
Gravel
= Oleophilic Fertilizer-Treated
= Oleophilic Fertilizer-Treated
= Water-Soluble
= Water-Soluble
NS
0.
0.
1.
0.
0.
1.
1.
1.
1.
1.
NS
8
7
0
8
8
0
0
1
0
0
l
1
1
1
1
1
1
1
1
1
Mixed Sand
.3
.2
.3
.3
.3
.3
.3
.2
.3
.3
and
7/16/89
SCO
NS
NS
NS
NS
NS
1.
1.
1.
1.
1.
1.
1.
1.
1.
1.
8
0
0
5
3
3
3
3
3
3
Gravel
Cobble
Fertilizer-Treated
Fertilizer-Treated
Mixed
Cobble
Sand
and Gravel
= No Sample Taken
= Detection Limit
= No Data
Available
SCR
1.5
1.4
1.2
1.5
1.4
1.3
1.3
1.0
1.2
1.2
1.1
1.2
1.4
1.3
1.2
73
-------
to the reference plot. At approximately 4 and 6 weeks after the
fertilizer application (T=3 and T=4, respectively), no substan-
tial difference in the ammonia concentrations was apparent
between the treatment and the reference plots.
Table 7.2 shows nitrate/nitrite concentrations found in
interstitial water for the treatment and reference plots. One to
2 days following application, notable concentrations of nitrate
were found in samples taken from the briquette-treated beaches.
Eight to 10 days after application (T=2), sand and gravel beaches
treated with oleophilic fertilizer showed substantially higher
levels of nitrate/nitrite nutrients than did the reference plots.
Plots treated with water-soluble fertilizer showed only slightly
elevated concentrations. One month after fertilizer application
(T=3), nitrate/nitrite levels in the treated plots were only
slightly higher than in the reference plots, particularly for the
cobble beach treated with briquettes. Neither the Inipol or the
briquettes contain nitrate or nitrite. Thus, the presence of
these nutrients have been the result of ammonia conversion to
nitrite by nitrification.
Samples taken in July from streams near Eagle and Otter
Beaches showed measurable levels of inorganic nutrients. The
stream to the south of Eagle Beach had 5.2 MM nitrogen as
nitrate. Stream samples taken adjacent to Otter Beach contained
an average of 4.8 MM nitrogen as nitrate. A sample of snow
collected from a snow pile 300 yards southeast of Eagle Beach (a
result of a winter avalanche) contained 2.8 MM of nitrogen as
ammonia, 0.54 MM of phosphorus as phosphate, and l.l MM of
nitrogen as nitrate. Although these concentrations were
relatively low, they indicate that snow-melt and runoff may serve
as important sources of nutrients for limited sections of the
shoreline, particularly in the spring and early summer. Even
though some of the test plots were located near the streams,
nutrient concentrations in the plots were probably unaffected.
This was an unlikely source of the nitrate found in the treated
beaches, as no elevated nitrate/nitrite was detected in reference
beaches having equal exposure to the freshwater. Also, no
nitrate/nitrite was found at T=0 in any of the plots.
On June 19, the briquette bags were repositioned, and all
the bags were placed in the mid- and low-tide zones of the plots.
This resulted in the fertilizer being submerged a longer time,
enhancing nutrient transport in these zones. In general, this
repositioning did not have a detectable impact on nutrient
distribution on the beaches; i.e., nutrient concentrations in the
zones showed no new trends. It was still apparent that minimal
dispersion of the nutrients was occurring from the briquettes in
areas of the shoreline not subjected to routine tidal washing.
Precipitation during the month of June was probably insufficient
to effectively transport nutrients released from the bags of
briquettes located in the high-tide zone.
74
-------
Table 7.2.
Samples
Nitrate/Nitrite Concentrations in Interstitial Water
Total Concentrations of N03 + NO2 (/iM N)
T=l (1-2 days before application)
Tide
Zone
High
6/08/89 6/10/89 6/10/89
Block ESR OSO OSW SCW
6/9/89
Mid
Low
T=2 (8-10 days post application)
Block
6/18/89 6/18/89
ESR OSO
High
Mid
Low
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
NS
NS
NS
NS
NS
2.8
1.5
0.6
4.1
2.2
1.9
1.4
1.7
5.7
2.7
NS
NS
NS
NS
NS
12.0
17.0
29.0
14.2
18.0
11.0
25.0
6.1
5.3
11.8
6/18/89
OSW
NS
NS
NS
NS
NS
5.7
1.4
0.5
8.3
4.0
2.5
4.3
2.4
2.4
2.9
6/19/89
SCW
DL
1.8
2.2
7.0
2.8
8.6
14.0
24.0
19.0
16.4
14.7
17.7
16.4
19.5
17.1
6/19/89
SCO
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NS
NS
NS
NS
NS
13.6
16.3
18.8
9.2
14.5
6.6
5.1
67.5
18.0
23.3
7.8
1.2
1.3
8.5
4.7
20.8
35.8
36.0
38.7
33.1
48.2
29.4
56.5
42.6
44.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
75
-------
Table 7.2. (continued)
T=3 (30 days post application)
Tide
Zone
High
Mid
Low
Block
1
3
5
7
Avg
8
10
12
14
Avg
15
17
19
21
Avg
7/7/89
ESR
0.6
DL
0.6
DL
0.3
0.1
DL
1.1
1.3
0.6
DL
0.1
1.3
0.3
0.4
7/7/89
OSO
0.7
0.7
0.5
1.0
0.7
2.5
2.8
0.5
0.6
1.6
2.7
4.1
1.7
1.6
2.5
7/7/89
OSW
0.2
DL
0.6
3.6
1.0
0.6
0.4
0.3
0.4
0.4
0.8
1.7
1.5
0.1
1.0
7/7/89 7/7/89 7/7/89
SCR
0.6 3.1 0.2
0.9 2.7 0.7
1.7 7.1 2.2
1.4 1.4 3.7
1.2 3.6 1.7
1.7 4.3 4.0
9.6 4.5 3.4
11.0 2.8 1.9
11.0 3.1 2.2
8.3 3.7 2.9
4.4 4.4 3.6
7.2 7.2 3.2
2.9 2.9 2.7
2.9 2.9 4.1
4.4 4.4 3.4
ESR = Control Mixed Sand and Gravel
SCR = Control Cobble
OSO = Oleophilic Fertilizer-Treated Mixed Sand and Gravel
SCO = Oleophilic Fertilizer-Treated Cobble
OSW = Water-Soluble Fertilizer-Treated Mixed Sand and Gravel
SCW = Water-Soluble Fertilizer-Treated Cobble
NS = No Sample Taken
DL = Detection Limit
ND = No Data Available
76
-------
CHANGES IN OIL RESIDUE WEIGHT AND COMPOSITION
Data analysis for oil residue weight and chemistry in
samples taken from beach plots in Snug Harbor has not yet been
completed. Over 1100 samples have been analyzed and the
resulting information is being incorporated into the data base.
Six different approaches for analyzing trends in the data are
being used. These involve analysis through time of the
following;
Oil residue weights (methylene chloride extractable
material),
Ratios of C17/pristane and C18/phytane,
Gas chromatographic profiles of aliphatic hydrocarbons,
Total concentrations of aliphatic hydrocarbons,
• Average individual aliphatic hydrocarbon concentrations
and,
• Relationship of degradation extent to oil residue
weight.
For oil residue weights and ratios of C17/Pristane and
CIS/Phytane, data is presented as mean values. All data from the
cobble beaches has a top and bottom component. Top refers to the
oil extracted from the surfaces of the cobblestones and bottom
refers to the mixed sand and gravel below the cobble.
As expected, oil distribution in the beach plots was
heterogeneous. Sampling procedures, although carefully
standardized, could not guarantee a constant volume or surface
area of sample each time, particularly in cobble beaches.
Standard deviations around the means are therefore large in most
cases, making determinations of statistical significance
difficult. Several statistical approaches are currently being
evaluated to assist in interpretation of the data. Some general
statements, however, can be made at this time.
Residue Weight
Changes in the mean residue concentrations through time for
all plots are shown in Figures 7.1 to 7.4. Each data point on
the figures is the mean of the number of samples available at
this time (maximum samples equals 21 for any sampling time).
Where a small number of samples has been included, standard
deviations may be quite large.
77
-------
8000 ,
00
D)
^
O)
"3
o
c
o
o
0)
D
2
'55
o>
DC
REFERENCE
— O— WATER SOLUBLE
OLEOPHILIC
5/28 6/08 6/17 6/25 7/08
7/29
Sampling Date
Figure 7.1 Mean Residue Concentration at Snug Harbor Mixed Sand and Gravel Plots, All Zones.
-------
O)
c
o
"J3
CO
C
0)
O
C
o
o
o
D
T3
'(/>
o
DC
REFERENCE
— O— WATER SOLUBLE
OLEOPHILIC
5/28
6/08 6/17 6/25 7/08
7/29
Sampling Date
Figure 7.2 Mean Residue Concentration at Snug Harbor Mixed Sand and Gravel Plots, Mid and Low Tide Zones.
-------
00
o
D)
2
o
o
c
o
o
o
3
73
'
O
DC
4000 -
3000 -
2000 -
1000
REFERENCE
— O— WATER SOLUBLE
OLEOPHILIC
6/08
6/25 7/08
7/29
8/26 9/08
Sampling Date
Figure 7.3 Mean Residue Concentration at Snug Harbor Cobble Plots, Top, All Zones.
-------
2000 n
OO
O)
^
D)
E
c
o
'*3
(0
o
c
o
o
0)
3
73
1500 -
~ 1000 -
-------
In the mixed sand and gravel plots (Figure 7.1), residue
weights showed a decreasing trend over time for the reference
plot and the briquette-treated plot. The data is
variable. As much as a 5-fold decrease in residue weight was
apparent in the briquette-treated plot with some indication that
the overall rate of decrease was more rapid than in the reference
plot. This difference may be attributable to nutrient addition
but it could not be verified by statistical analysis. Although
several factors could control changes in oil residue weights, the
large decreases may indicate extensive oil degradation.
The residue weights in the mixed sand and gravel beach
treated with oleophilic fertilizer were very low at the time of
fertilizer application and did not appear to decrease
substantially thereafter. The initial concentration of oil in
this plot was in the same range as that seen in the other treated
and untreated plots toward the end of July.
When the mid and low tide zones of the mixed sand and gravel
plots are considered (Figure 7.2), the same general trends are
apparent. However, the relative difference in rate of residue
weight loss in the briquette-treated plot compared to the others
is even more pronounced.
Data for the residues of oil on the cobble rock surfaces are
shown in Figure 7.3. Unfortunately, information from several
sampling periods are not yet complete. However, it would appear
that oil residue weights decreased dramatically over the two
weeks following application of the oleophilic fertilizer. This
corresponds with the visual observation. Information on the
reference plot during this time period is not available and
therefore it is not known if decreases in residue waste were as
extensive.
In the cobble plots, oil concentrations in the mixed sand
and gravel under the cobble was initially very low (Figure 7.3).
Essentially no change in oil residue weights was apparent in any
plot or in any zone within a plot.
Ratios of branched and straight chain hydrocarbons
Changes in the C17/pristane and C18/phytane ratios through
time for all plots are shown in Figures 7.5 to 7.10. Each data
point on the figures is the mean of the number of samples
available (maximum samples equals 21 for any sampling time).
Where a small number of samples has been included, standard
deviations may be quite large.
82
-------
oo
U)
1.5 -u_
(0
OC
&
"5
1.0 -
0.5 -
O
0.0
REFERENCE
— O — WATER SOLUBLE
OLEOPHILIC
5/25 6/08 6/17 6/25 7/08
7/29
Sampling Date
Figure 7.5 Mean C17 / Pristane Ratio at Snug Harbor Mixed Sand and Gravel Plots, All Zones.
-------
OO
REFERENCE
— O — WATER SOLUBLE
OLEOPHILIC
6/08 6/25 7/08
7/29
8/26 9/08
Sampling Date
Figure 7.6 Mean C17 / Pristane Ratio at Snug Harbor Cobble Plots, Top, All Zones.
-------
OO
.2 1.0,
03
DC
0)
K 0.5 .
O o.o
REFERENCE
— O — WATER SOLUBLE
OLEOPHILIC
6/08 6/17 6/25 7/08
7/29
Sampling Date
Figure 7.7 Mean C17 / Pristane Ratio at Snug Harbor Cobble Plots, Bottom, All Zones.
-------
oo
(0
CC
-------
1.5 4
CO
OC
o
c
CO
1.0 i
oo
O
0.5 -
0.0
REFERENCE
O— WATER SOLUBLE
OLEOPHILIC
6/08 6/25 7/08
7/29
8/26 9/08
Sampling Date
Figure 7.9 Mean of C18 / Phytane Ratio at Snug Harbor Cobble Plots, Top, All Zones.
-------
oo
CO
(0
cr
-------
In mixed sand and gravel plots (Figures 7.5 and 7.8), the
ratios showed a general decrease through tine for the reference
plot and the briquette-treated plot. Decreases in these ratios
are traditionally considered good predictors of biological
degradation of oils. As much as a 2-fold decrease in the ratio
was apparent in the briquette-treated plot with some indication
that the overall rate of decrease was more rapid than in the
reference plot. Examination of the low and mid tide zones taken
together (data not shown) reflected the same trend. This
difference may be attributable to nutrient addition but the
effect could not be verified by statistical analysis.
At the July 8 sampling, the ratios appeared to have
increased. Reoiling of the beaches or, more likely, degradation
of the internal standards, pristane and phytane, could be
responsible for this increase.
Data for the C17/pristane and C18/phytane ratios on the
cobble rock surfaces are shown in Figure 7.6 and 7.9. As
mentioned above, information for some sampling periods is not
complete at this time. It would appear that in all plots, ratios
decreased through time. Nonetheless it was evident that
biodegradation of the oil (i.e., significant change in the
ratios) was occurring in the oleophilic-treated cobble plot at
about the time visual loss of oil from the beaches was observed
(week 2 to 4). Since a similar decrease in ratios was occurring
in the reference cobble plot, yet no visual loss of the oil was
apparent in the field, it would appear that the oleophilic
fertilizer was having a more extensive effect on the
biodegradation processes that is, as yet, undefined.
In the oleophilic-treated mixed sand and gravel plots
(Figures 7.5 and 7.8), the ratios decreased as well but seemingly
at a much slower rate. Data from the mixed sand and gravel under
the cobble (Figures 7.7 and 7.10) showed little change in the
ratios through time. The ratios were initially very low compared
to ratios measured in oil samples from other plots. This is
surprising; i.e., despite very low concentrations of oil in these
samples (see Figure 7.1), it was not expected that the ratios
would necessarily be low as well. This may reflect a more rapid
biodegradation of the oil due to its low concentration and its
distribution over a large surface area (see section below which
provides data for this relationship). The C18/phytane ratio in
samples from the briquette-treated plot showed a decrease through
time but again this has not yet be statistically verified.
89
-------
Gas Chromatoaraphic Profiles
For the C17/pristane ratios there was a possible faster
change in the oleophilic-treated plot relative to the reference
plot, although this is not the case for the C18/phytane ratio.
Note, however that the data point at the 6:25 sampling represents
information from only one block. Other measures of degradation
and data analysis were examined to further explain striking
visual differences of oil disappearance. Examination of gas
chromatographic profiles provided a qualitative indication of
important changes that could be later verified through more
quantitative measures.
Gas chromatographic profiles were therefore recreated by
computer. Representative examples of these illustrations are
shown in Figure 7.11a and b. All lines on the profiles represent
concentrations of aliphatic hydrocarbons (approximately 12
through 28) normalized to the oil residue weight. As a floating
concentration scale was used in the initial analysis to
accommodate all profiles (Figure 7.11a,b), changes in relative
concentrations for the hydrocarbons can be visualized by
comparing the overall profile of the peaks to a profile typical
of a relatively undegraded but weathered oil. This is shown as
the solid "mountain" line in the figure. However, comparing
absolute peak height is not meaningful. Blank graphs indicate
that data for that block was unavailable.
Data from two sampling times, two and four weeks after
application of the oleophilic fertilizer to the cobble plot, are
shown in Figure 7.11. The gas chromatographic profiles are for
oil extracted from the surface of the rocks. These profiles
attest to the heterogeneity of oil composition within a plot.
Further, low molecular weight aliphatic hydrocarbons have
decreased, indicating significant biodegradation has occurred.
This is important as this degradation corresponds with the
observed loss of oil from the oleophilic treated plots in the
field. Visual impressions of differences between the tidal zones
(top line - high tide zone; middle line - mid tide zone; bottom
line = low tide zone) can also be examined. Samples analyzed
from the low tide zone of the cobble plots receiving the
oleophilic fertilizer (Figure 7.lib) may indicate more loss of
hydrocarbons than samples from the other zones.
The recreated profiles can also be illustrated without the
floating concentration axis (i.e., all the same scale). Examples
comparing the mixed sand and gravel plots treated with oleophilic
and slow release fertilizers are shown in Figures 7.12a though f.
These figures show analysis of samples taken prior to fertilizer
application, and 2 to 4 weeks following fertilizer application.
It is apparent that there is considerably less oil and more
degradation of the alkanes (change in the peak profile) in the
samples taken from the oleophilic-treated plots in the 4 week
90
-------
Snug Harbor - Cobble Surface - Oleophilic Fertilizer - 2 Weeks After Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK =
BLOCK = 6
BLOCK =
0)
3
"O 1 7 IB
0)
OC BLOCK = 8
f-R U
O)
3
M
C
O
VO
u
CO ej
is ai
C
0)
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C
O
O
BLOCK=15
ta
17 IB
BLOCK = 9
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U
IJ
U
0*
04
u
u
K
/
/
/
/
/
/
(-"
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r\
h\
r
i
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BLOCK=16
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U
OJ
a;
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ai
a*
17 18
BLOCK=1O
17 IB
BLOCK=11
17 18
BLOCK=12
BLOCK=13
17 IB
BLOCK=14
\
17 18
BLOCK=17
17 IB
BLOCK=18
BLOCK=19
17 IB
BLOCK=2O
BLOCK = 21
14
U
u
17
U
U
14
U
U
II
u
17 18
17 18
17 IB
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.11 a. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble two weeks following application of oleophilic fertilizer at Snug Harbor. Blanks
indicate data not available. Solid line profile estimates peak heights of alkanes in oil that
has undergone minimal biodegradation. Note floating concentration scale.
-------
Snug Harbor - Cobble Surface - Oleophilic Fertilizer - 4 Weeks After Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK =
BLOCK=6
BLOCK=7
vo
NJ
TD
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BLOCK=8
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BLOCK=15
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BLOCK=11
17 18
BLOCK=12
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BLOCK=13
1718
BLOCK=14
17 18
BLOCK=16
17 18
BLOCK=17
17 18
BLOCK=18
17 18
BLOCK=19
17 18
BLOCK=2O
17 18
BLOCK = 21
17 18
17 IB
17 18
17 18
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.11b. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble four weeks following application of oleophilic fertilizer at Snug Harbor. Blanks
indicate data not available. Solid line profile estimates peak heights of alkanes in oil that
has undergone minimal biodegradation. Note floating concentration scale.
-------
Snug Harbor - Below Cobble - Oleophilic Fertilizer - Before Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK=5
BLOCK = 6
BLOCK=7
vo
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BLOCK=8
17 18
BLOCK=9
17 IS
BLOCK=1O
17 18
BLOCK=11
17 18
BLOCK=12
17 18
BLOCK=13
17 18
BLOCK=14
o.o
3.O
2.S
2.O
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1.0
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17 18
BLOCK=15
17 18
BLOCK = 16
17 IB
BLOCK=17
17 18
BLOCK=18
17 18
BLOCK=19
17 18
BLOCK=2O
17 18
BLOCK=21
3.O
2.3
2.0
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Jill mi,::
17 18
17 18
17 18
17 18
17 18
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.12a. Recreated gas chromatographic profiles from samples of oil extracted from the mixed sand
and gravel under the cobble prior to application of oleophilic fertilizer at Snug Harbor.
Blanks indicate data not available. Note all concentrations are on the same scale.
-------
Snug Harbor - Below Cobble - Oleophilic Fertilizer - 2 Weeks After Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK=5
BLOCK = 6
BLOCK=7
vo
0)
3
5
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BLOCK=8
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BLOCK=9
17 18
BLOCK=10
17 18
BLOCK=11
3.0
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1.5
1.O
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0.0
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3.0
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1.5
1.O
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2.9
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0.9
. O.O
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BLOCK=15
BLOCK=16
17 18
BLOCK=17
BLOCK=18
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3.O
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O.O
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. 0.0
Hill
3.O
2.5
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O.S
. O.O
17 18
BLOCK=12
17 18
BLOCK=13
17 IB
BLOCK=14
3.O
2.3
2.0
1.9
I.O
0.3
0.0
3.O
2.9
2.O
1.3
I.O
0.3
. O.O
3.O
2.3
2.O
1.9
I.O
O.9
O.O
17 18
BLOCK=19
17 18
BLOCK=2O
17 18
BLOCK = 21
3.0
2.9
2.O
1.5
1.0
O.S
O.O
Mil
17 ia
17 18
17 18
17 18
17 18
17 18
17 IB
n-Alkanes (n-C12 to n-C32)
Figure 7.12b. Recreated gas chromatographic profiles from samples of oil extracted from the mixed sand
and gravel under the cobble two weeks following application of oleophilic fertilizer at Snug
Harbor. Blanks indicate data not available. Note all concentrations are on the same scale.
-------
Snug Harbor - Below Cobble - Oleophilic Fertilizer - 4 Weeks After Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK=5
BLOCK=6
BLOCK=7
3.0
2.S
2.0
1.5
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BLOCK=8
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17 18
3.O
2.5
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1.5
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Mill 0.5
Jlllilllllljjlhl I nn
17 18
BLOCK=9
3.O
2.5
2.0
1.5
1.0
iilllllllllllllllll 1 n n
17 18
3.O
2.5
2.0
1.5
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1 ll °'5
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17 18
BLOCKs 1O
3.0
2.5
2.0
1.5
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..iiilliiiiiiiiiiii i !'!
17 18
3.0
2.5
2.0
1.5
Jl I. 1 1.0
. Jiillllllli i ::
17 18
BLOCK=1 t
3.0
2.5
2.O
1.3
1.0
. iiJiiiiiiiiiin . ::
17 18
3.O
2.5
2.0
1.5
1 1°
..iiilliiiiiiiii,i . :::
17 ia
BLOCK= 12
3.0
2.5
2.O
1.5
.,nllililillliii i ::
17 ia
3.O
2.5
2.O
1.5
| 1.0
111 . 0.5
.illlllhljlllhiii . nn
17 18
BLOCK= 13
3.0
2.5
2.O
1.5
I.O
Iliiiiiiiiiiii , !'!
17 18
1
Jllllllllllllml ,
17 18
BLOCK= 14
..lllllllllll.lllll .
17 18
BLOCK=15
BLOCK=16
BLOCK=17
BLOCK=18
BLOCK=19
BLOCK=2O
BLOCK=21
3.0
2.5
2.0
1.5
I.O
O.S
0.0
3.0
2.5
2.0
1.5
I.O
O.S
0.0
17 18
..lllllllllili
17 ia
3.0
2.5
2.O
1.5
I.O
0.5
O.O
lllllllllili
3.O
2.5
2.0
1.5
1.0
O.S
. O.O
...III
3.O
2.5
2.0
1.5
I.O
0.5
. O.O
3.O
2.5
2.O
1.5
I.O
O.S
O.O
3.O
2.5
2.O
1.5
I.O
O.S
0.0
17 18
17 18
17
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.12c. Recreated gas chromatographic profiles from samples of oil extracted from the mixed sand
and gravel under the cobble four weeks following application of oleophilic fertilizer at Snug
Harbor. Blanks indicate data not available. Note all concentrations are on the same scale.
-------
VO
3
TO
'«
0>
DC
O)
O)
c
o
°«—
(0
o
c
o
o
Snug Harbor - Below Cobble - Water Soluble Fertilizer - Before Application
BLOCK=1
BLOCK =
BLOCK=3
3.O
2.S
2.O
1.5
t.O
OS
o.o
1
3.O
2.S
2.0
t.S
1.0
O.S
_ 0.0
1
Illl
|
1
1
Illl
17 18
BLOCK =
17 ia
BLOCK=9
17 18
BLOCK=10
17 18
BLOCK=15
17 18
BLOCK=16
17 18
BLOCK=17
3.0
2.3
2.O
1.5
1.O
O.i
0.0
BLOCK=4
BLOCK=5
BLOCK=6
BLOCK=7
3.O
2.3
2.0
1 .3
1.0
O.3
O.O
1
,ll
3.0
2.3
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1.3
1, '•••
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.1
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ll f'°
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,1
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O.O
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2.5'
2.0
1.5
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UN „„
3.0
2.5
2.0
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1.0
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o n
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111
ill
Illl
III Illl
2.O
1.5
|||| 1.0
| II HI o.S
Illllll no
.ll
17 18 17 18 17 18 17 18
BLOCK=11 BLOCK=12 BLOCK=13 BLOCK=14
3.O
2.3
2.0
1.5
1.O
0.5
0.0
1
3.O
2.3
2.0
1.3
1.O
0.3
0,0
.1
3.0
2.3
2.0
1.3
1.O
O.S
i o.o
1
3.O
2.3
2.0
1.3
1.O
O.S
o.o
17 18
BLOCK=18
17 18
BLOCK=19
17 18
BLOCK=20
BLOCK=21
j
3.O
2.3
2.O
1.5
1.O
O.S
- 0.0
1
..I
3.O
2.S
2.O
1.5
III '°
O.S
, HI 0.0
ll
III
3.0
2.5
2.0
1.5
1.0
O.S
0.0
,,l
I
ll
III
3.0
2.3
2.O
1.3
1.0
O.S
1
1
|
,11
3.0
2.3
2.0
1.3
Illl 10
1 "
i!'' I *> <*•
|
.11
3.O
2.5
2.0
1.5
III '°
ll"
I
17 18
17 18
17 18
17 18
17 18
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.12d. Recreated gas chromatographic profiles normalized to oil residue weight from samples
of oil extracted from the mixed sand and gravel under the cobble prior to application of
water soluble fertilizer briquettes at Snug Harbor. Blanks indicate data not available.
Note all concentrations are on the same scale.
-------
Snug Harbor - Below Cobble - Water Soluble Fertilizer - 2 Weeks After Application
BLOCK=1
BLOCK =
BLOCK=3
BLOCK=4
BLOCK=5
BLOCK=6
BLOCK =
0)
tn
o>
DC
O)
D)
3
3.0
2.5
2.0
1.5
1.O
O.S
O.O
..,1
in tt ••in
3.0
2.5
2.0
1.5
HIM '••
B "
1 II 1 I /i n
Ml 1 O.CM
...1
3.O
2.5
2.O
1.5
bl.O
in.. ! •» °
.,1
3.O
2.5
2.0
1.5
1 1 1-°
II 1 °'S
!! ! •>«•
,1
3.0
2.5
2.O
1 '•*'
ll, '-°
Hi:::
...i
3.O
2.5
2.0
1.5
ll , '"
°*
!! ! on
...i
3.O
2.5
2.O
1.5
i
ll '•"
1 "
II 1 on
,,\
c
0)
O
c
O
O
17 IB
BLOCK=8
17 18
BLOCK=9
17 IB
BLOCK=1O
17 18
BLOCK=11
17 IB
BLOCK=12
17 IB
BLOCK=13
17 IB
BLOCK=14
3.0
2.5
2.O
1.5
t.O
0.5
O.O
,1
..I
3.O
2.S
2.O
1.5
1.0
O.S
O.O
...ll
3.0
2.5
2.0
1.5
1.0
0.5
O.O
3.O
2.5
2.O
1.5
1.O
O.S
O.O
..I
I.
1
3.0
2.5
2.O
1.5
1.O
0.5
O.O
17 IB
BLOCK=15
17 18
BLOCK=16
17 18
BLOCK=17
17 IB
BLOCK=18
17 18
BLOCK=19
17 IB
BLOCK=2O
17 18
BLOCK=21
3.O
2.5
2.0
1.5
1.O
O.5
O O
3.O
2.5
2.O
1.5
I.O
• 0.5
Illlllillllllllllll 1 0^
3.O
2.5
2.0
1.5
I.O
O.S
• iniililllllllllll 1 ^ n
,1 1
3.0
2.5
2.O
1.5
1 1 '•'•
"
1 1 n n
..,1
3.O
2.5
2.O
1.5
III, '"
"
III 1 n n-
.,1
3.O
2.S
2.O
1.5
1 1 ' °
0.5
\.'r. 00
I
..,ll
3.O
2.5
2.O
1.5
ll '°
"
! i »•<>
ll
II
17 18
17 IB
17 18
17 18
17 18
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.12e. Recreated gas chromatographic profiles from samples of oil extracted from the mixed sand
and gravel under the cobble two weeks following application of water soluble fertilizer
briquettes at Snug Harbor. Blanks indicate data not available. Note all concentrations are
on the same scale.
-------
Snug Harbor - Below Cobble - Water Soluble Fertilizer - 4 Weeks After Application
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK =
BLOCK=5
BLOCK = 6
BLOCK = 7
vo
00
Q>
o>
oc
O)
O)
c
O
"*•
CO
«•<
0)
O
c
O
O
17 18
BLOCK=8
17 18
BLOCK=9
17 18
BLOCK=10
17 18
BLOCK=11
17 18
BLOCK=12
17 18
BLOCK=13
17 18
BLOCK=14
3.O
2.3
2.0
1.5
1.O
O.S
0.0
3.0
2.5
2.0
1.5
I.O
O.9
O.O
3.0
2.5
2.O
1.S
I.O
0.5
. O.O
3.0
2.S
2.0
1.5
I.O
O.S
O.O
0.0
17 IB
BLOCK=15
17 18
BLOCK=16
17 18
BLOCK=17
17 18
BLOCK=18
17 18
BLOCK=19
17 18
BLOCK=2O
17 18
BLOCK=21
3.0
2.5
2.0
1.5
I.O
0.5
O.O
3.O
2.5
2.0
1.5
1.0
O.S
O.O
..... J.li.lllm.l
3.O
2.5
2.O
1.5
I.O
O.S
O.O
3.O
2.5
2.O
1.5
I.O
0.5
O.O
. h.
3.O
2.5
2.O
1.5
I.O
0.5
O.O
3.O
2.5
2.0
1.5
I.O
OS
0.0
3.0
2.5
2.O
1.5
I.O
0.5
O.O
.III
17 18
17 18
17 18
17 18
17 18
17 18
17 18
n-Alkanes (n-C12 to n-C32)
Figure 7.12f. Recreated gas chromatographic profiles from samples of oil extracted from the mixed sand
and gravel under the cobble four weeks following application of water soluble fertilizer
briquettes at Snug Harbor. Blanks indicate data not available. Note all concentrations are
on the same scale.
-------
sampling period. There was a similar change in the hydrocarbon
profiles from samples taken in the briquettes-treated beach.
Note also that the pristane and phytane were decreasing as fast
as the C17 and CIS alkanes respectively indicating the use of
these hydrocarbons as conserved internal standards was more
highly suspect. Minimal changes in the ratios may not therefore
be indicative of extensive overall degradation. As the data is
further analyzed, more of these profiles will become available
for examination.
Average Individual Aliphatic Hydrocarbon Concentration
Another approach for data analysis is to examine the mean
individual aliphatic hydrocarbon concentrations for all blocks
within a sampling period. Figures 7.13 through 7.15 present bar
charts of mean n-alkane and pristane and phytane concentrations
normalized to the extractable oil residue weight.
Several interesting trends are apparent when the three sets
of figures are compared. For the reference beach (Figure 7.13 a-
d), the relative concentration of pristane and phytane appeared
to be somewhat unchanged over time, whereas C17 and C18 alkanes
decreased approximately 50% in the last two sampling periods.
The high variability and the limited amount of available data
provide only a very rough estimation.
For the oleophilic fertilizer treated beach plot (Figure
7.14 a-d), both the pristane and phytane and the individual
hydrocarbons decreased over time. The decrease for the
individual hydrocarbons was similar to that observed in the
reference plot. Interestingly, the possible decrease in pristane
and loss of phytane may indicate that oil biodegradation in the
oleophilic fertilizer treated plots was more extensive, including
branched hydrocarbons. Preliminary results from the slow release
fertilizer-treated cobble beaches (Figure 7.15 a,b) mirrored the
degradation pattern of the reference beach.
Removal of the marker compounds (pristane and phytane) makes
sole reliance on C17/pristane and C18/phytane ratio data tenuous.
Therefore, additional analyses of available data and limited
GC/MS analyses of selected extracts for residual aromatics and
other marker components (e.g., norhopane and hopane) must be
completed before final evaluation of all treatment processes.
Total Aliphatic Hydrocarbon Residues
Relative differences in the concentrations of aliphatic
hydrocarbons can be further analyzed by examining the total
(summed) aliphatic hydrocarbon residues. Figure 7.16 shows the
total hydrocarbon residues (median values) through time for the
cobble plots, treated (oleophilic and slow release) and untreated
99
-------
o
o
Figure 13a
-
3
,
Figure 13b
Snug Harbor-Mixed Sand and Gravel-Untreated Beach
Sampling Date: 6/8/89
: c : c : : : : c : :
I I I I i I I I I It
> > I I • I • • I ll
N-alkane
Sampling Date: 6/25/89
linn,
ceccccicicc::
11:1111111111
t l > l < r i • ' • 9 l
c c
I I
Figure 13c
It
«|
u
z a)
II
Sampling Date: 7/3/89
: c c c c c • c
::ccecccc
I 1 I > I I I I I
I I
> i
N-alkane
Figure 13d
(0
0>.X
Sampling Date: 7/29/89
..ii illinium
< i
t 9
N-alkane
N-atkane
Figure 7.13a-d. Mean weight of alkanes normalized to the total oil residue weight extracted
from the beach material; control mixed sand and gravel beaches.
-------
Snug Harbor-Mixed Sand and Gravel-Oleophilic Fertilizer
Figure I4a
II
Is
CO '
Sampling Date: 6/8/89
..lllllllllllllll ..
: c c c c c
114)1'
c c : t i c i c c cc
illliliti II
C'lliltri 01
N-alkane
Figure 14b
Sampling Date: 6/25/89
N-alkane
o
ou
Figure 14c
I '
Sampling Date: 7/8/89
•ill.I.I.I
c c c : c
i i • « i
l.l.i
c c c
I 1
1 I
N-alkane
Figure 14d
Sampling Date: 7/29/89
.Illlllllllllllli I
cciciccc:
i i i i > • > > I
N-alkane
Figure 7.14a-d. Mean weight of alkanes normalized to the total oil residue weight extracted from
the beach material; oleophilic-fertilizer-treated mixed sand and gravel beaches.
-------
Snug Harbor-Cobble-Water Soluble Fertilizer
o
K)
Figure 15a
n •
IS
oO
z o
U
Figure 15b
Z 4,
s ,
Sampling Date: 6/8/89
I
: e c t c c t c t c : c t ; t : c < cc
«< ' I l I I I I l I l II
N-alkane
Sampling Date: 7/29/89
lllllllllllllh ••
111111
N-alkane
Figure 7.15a-b. Mean weight of alkanes normalized to the total oil residue weight extracted from
the beach material; water soluble-fertilizer treated (fertilizer briquettes) cobble
beaches.
-------
O>
D)
C
O
1
0>
u
C
o
o
2
o
CO
180
160 "I
140
120
100
80
60
40 -
20 -
-2
SNUG HARBOR
Cobble Top
i i
4 6
Time in Weeks
I
8
Reference
Oleophilic
Water Soluble
10
12
14
Figure 7.16. Median of Total Concentration of Oil on Treated and Untreated
Cobble Plots at Snug Harbor, All Zones
-------
(reference). The data are for oil extracted from rock surfaces.
These hydrocarbons as a group showed a decrease in concentration
through time but little can be concluded at this time because of
a lack of data points in the earlier sampling times. However,
the data analysis technique does have promise in helping to
evaluate the effect of the fertilizers.
Degradation Extent/Oil Residue Weight Relationships
During sampling of the beaches it was obvious that globs of
viscous, sticky oil were present in some areas. Where these
globs were encountered, there was concern that spike
concentrations of undegrated oil would mask evidence of
degradation. Examination of the data indicated that changes in
the C17/pristane and C18/phytane ratios were most apparent in the
samples containing less total oil. This is reasonable if one
realizes that at low concentrations, the surface area-to-oil
residue weight ratio is large, as it is when oil is dispersed
into the beach material as small droplets or films.
Effectiveness of biodegradation will increase as the oil surface
area increases. With higher concentrations of oil, the same
degradation rate is probably occurring, but the surface area-to-
oil amount is much less. Because the oil is in bigger globs, the
degraded oil on the surface is diluted by the undegraded oil
during sampling and homogenization. If this observation is
valid, it should be possible to normalize the extent of
degradation to the amount of oil present. Figures 7.17 through
7.20 show that when the C17/pristane and C18/phytane ratios are
plotted against their respective residue weights, a direct
relationship exists. This data is from plots prior to fertilizer
treatment. Regression analysis of the data gave r-values around
0.8 (alpha = 0.0001). By comparing slopes of this relationship
from two different sampling periods, the effect of biodegradation
can be seen. The slopes increased by 2 and 3 fold in the space
of 2 weeks. With more degradation the slope will continue to
steepen to a limit where the data points begin to cluster closer
to the origin. This relationship may have application in further
analyzing data from treated and untreated plots. Initial
attempts to normalize the ratios with the oil residue weight to
reduce variability of the data have, to date, been ineffective.
The approach, however, seems promising and further work will
evaluate its usefulness.
104
-------
0)
3
a
o
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
.Fresh Prudhoe
Bay Crude Oil
slope
0.81
0.26
Log Residual Weight (mg/kg)
Figure 7.17. C17/Pristane Ratio versus LoglO Residue Weight
Two Weeks Before Fertilizer Application (5/28/89)
105
-------
2
2
?•
&
00
*•
o
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Fresh Prudhoe
Bay Crude Oil
r = 0.79
Slope = 0.28
345
Log Residual Weight (mg/kg)
Figure 7.18. C18/Phytane Ratio versus Log 10 Residue Weight
Two Weeks Before Fertilizer Application (5/28/89)
106
-------
o
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Fresh Prudhoe
Bay Crude Oil
r
slope
0.80
0.49
3 4
Log Residual Weight (mg/kg)
Figure 7.19. C17/Pristane Ratio versus Log 10 Residue Weight
at Time Zero of Fertilizer Application (6/8/89)
107
-------
0)
a.
•
b
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
.Fresh Prudhoe
Bay Crude Oil
r = 0.78
slope = 0.74
• •
Log Residual Weight (mg/kg)
Figure 7.20. C18/Phytane Ratio versus Log 10 Residue Weight
at Time Zero of Fertilizer Application (6/8/89)
108
-------
MICROBIOLOGY
Determinations of numbers of oil-degrading bacteria present
in beach materials were made at each sampling of Snug Harbor,
using all 21 sediment samples taken from each test plot for
sediment chemistry. Numbers of degraders were assessed by ser-
ially diluting each sample in a minimal salts medium containing
ammonium and phosphate, adding a small quantity of oil to each
dilution, and incubating the dilutions for 21 days. The highest
dilution series showing degradation is then scored, and a cal-
culation is made based on dilution to extinction of the total oil
degraders in the undiluted sample. Although similar in design to
a single tube MPN procedure, the dilution to extinction procedure
should not be mistaken for such.
Results from these studies are shown in Table 7.3. The
values reported are the Iog10 mean and standard deviation of 18
to 21 dilution series for each mixed sand and gravel plot (no
cobble beach material was analyzed). When a control plot was
sampled on 2 separate days, the results represented 8 to 10
dilution series per day. Table 7.3 has been keyed to indicate
the number of determinations within a plot in which every dilu-
tion in the series was positive for oil degradation. The greater
the number of positive dilutions, the greater the underestimation
of the relative oil-degrading population.
Results suggested that an increase in oil-degrading
microorganisms occurred within the oleophilic fertilizer-treated
plots between the 0 time and 9 days after application. The
results from the water-soluble treatment showed the same trend,
but the differences in both cases were not statistically
supportable. For unexplained reasons, oil degraders increased
more than 100- to 200-fold on day 31 in control and water-soluble
fertilizer-treated plots.
It was concluded from the available data that an increase in
oil-degrading microorganisms may have occurred as a result of
fertilizer application but, it could not be statistically
varified. The apparent increase in organism populations in the
fertilized plots at day 9 corresponds to the high level of
nutrients seen immediately following the application of nut-
rients. In these tests, the presence of high numbers of oil-
degrading bacteria in the control beaches made differences in the
numbers of degrading organisms between treatments subtle and
difficult to detect.
109
-------
Table 7.3. Relative Concentrations (Log10 of the Cell Numbers/g of
Beach Material) of Oil-Degrading Microorganisms in Snug Harbor
Mixed Sand and Gravel Test Plots*
Sampling Dateb Fertilizer
Before Application Days Control Water Soluble Oleophilic
6/8/89 0 6.58 5.95
±1.00 +/-1-29
6/11/89 1 6.16 5.80
±0.89 ±0.91
After Application
6/17/89 9 6.24* . 6.62* 6.91**
±1.53 ±1.19 +/-1-21
6/24/89 16 5.96 5.86 5.96
±0.83 ±1.15 +/-1-10
7/8/89 30 6.61 5.86
±1.34 +/-0.67
7/9/89 31 8.47* 9.39
±1.33 ±1.12
"No. of dilution series positive in all dilution tubes (0-25%);
*(25-50); **(50-75).
b Samples on 6/8/89 and 6/11/89 are preapplication of the
fertilizer.
110
-------
ECOLOGICAL MONITORING
The monitoring component of the project was designed to
identify ecological effects of nutrients added to the shore zone
on planktonic microorganisms. Sampling stations were established
in nearshore locations next to both treated and untreated
(reference) beaches in Snug Harbor (see Sections 5 and 6) and at
locations outside of Snug Harbor. Samples were collected on 9
occasions; once prior to the addition of fertilizer, 2 days after
addition, and 1, 2, 3, 4, 5, 6, and 8 weeks after addition.
After week 5, the stations 10 m from shore were no longer sampled
in order to accommodate the workload from an additional study
site. Data analyzed after week 5 indicated no significant loss
in assessment capability resulted from this decision.
Nutrients
Ammonia, nitrite, nitrate, and phosphate analyses have been
completed on water samples taken through week 7. Nutrient
concentrations showed no increases in waters adjacent to treated
shorelines as illustrated by ammonia and phosphorus data in
Tables 7.4 through 7.7. These data provide evidence that
fertilizers applied to the Snug Harbor shoreline either remained
within the beach matrix as applied, were taken up by microbial
biomass, or have been diluted to background concentrations within
1 m of the shoreline. In any event, the potential for
stimulating plankton biomass from nutrient enrichment along the
shoreline was not evident from these data.
Chlorophyll Analyses
Chlorophyll analyses of phytoplankton samples were used to
monitor for changes in the abundance of algae. Increased
chlorophyll concentrations would indicate nutrients had washed
from the beach and had been incorporated into algal biomass, if
nutrient enrichment stimulated algal growth in Snug Harbor. None
of the chlorophyll data indicated that algal populations within
Snug Harbor were stimulated by fertilizer applications beyond the
extent of variability observed in week to week sampling (Figure
7.21). Problems with obtaining sufficient extract volumes to
obtain optical densities appropriate for the spectrometer, and
procedural problems in quantifying extinction values contributed
to a great deal of variability in the first three data sets.
Pending additional analyses and evaluation of QA data, results
stated herein should be considered preliminary. However, results
to date demonstrate that nearshore concentrations were similar to
those offshore, and there were no consistent differences between
samples collected near treated beaches and reference areas.
Although statistically significant differences were observed
between treated and untreated samples on some dates, these
differences were not greater than those observed at control sites
week to week (i.e., the normal ecological variability).
Ill
-------
Table 7.4. Ammonia Nitrogen (MM N/l) from Nearshore Water Over
Gravel Beaches at Snug Harbor. Mean of Four Replicates (standard
deviation). (Method detection limit = 0.13 /iM N/l.)
Control Oleophilic Water Soluble
(Rodney Beach) (Otter Beach) (Otter Beach)
Sample Date 1m 10 m 1m 10 m 1m 10 m
6/10/89
6/14/89
6/21/89
6/28/89
7/5/89
7/12/89
7/23/89
8/9/89
1.5
(0.05)
0.68
(0.10)
0.92
(0.03)
0.21
(0.06)
0.51
(0.11)
0.80
(0.32)
0.13
(0.00)
0.13
(0.00)
1.5
(0.08)
0.65
(0.05)
1.02
(0.06)
0.15
(0.02)
0.52
(0.03)
0.73
(0.19)
«... *
^ mm
1.6
(0.05)
0.52
(0.09)
0.74
(0.03)
0.13
(0.00)
0.56
(0.09)
0.57
(0.11)
0.13
(0.00)
0.13
(0.00)
1.7
(0.06)
0.58
(0.10)
0.83
(0.05)
0.20
(0.10)
0.57
(0.10)
0.50
(0.05)
—
—
1.5
(0.22)
0.61
(0.08)
0.73
(0.03)
0.13
(0.00)
0.74
(0.16)
0.63
(0.08)
0.13
(0.00)
0.13
(0.00)
1.8
(0.17)
0.58
(0.10)
0.74
(0.06)
0.20
(0.14)
0.53
(0.09)
0.96
(0.57)
Sample not collected.
112
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Table 7.5. Ammonia Nitrogen (MM N/l) from Nearshore Water Over
Cobble Beaches at Snug Harbor. Mean of Four Replicates (standard
deviation). (Method detection limit - 0.13 /*M N/l.)
Sample Date
6/10/89
6/14/89
6/21/89
6/28/89
(0
7/5/89
7/12/89
7/23/89
8/9/89
a — Sample r
Control
(Fred Beach)
1m 10 m
2.1 1.8
(0.12) (0.00)
0.73 0.70
(0.03) (0.08)
0.99 0.91
(0.08) (0.06)
0.24 0.35
.06) (0.26)
0.61 0.65
(0.12) (0.19)
0.62 0.70
(0.18) (0.20)
0.13 — *
(0.00)
0.13
(0.00)
lot collected.
Oleophilic
(Seal
1 m
1.5
(0.05)
0.45
(0.06)
0.96
(0.04)
0.22
(0.13) (0
0.52
(0.03)
0.79
(0.16)
0.13
(0.00)
0.13
(0.00)
W
Beach)
10 m
1.5
(0.10)
0.55
(0.12)
0.82
(0.03)
0.13
.00)
0.50
(0.05)
0.75
(0.14)
—
— —
ater Solubl
(Seal
1 m
1.4
(0.27)
0.64
(0.06)
0.87
(0.09)
0.22
(0.11) (0.
0.44
(0.21)
0.86
(0.17)
0.13
(0.00)
0.13
(0.00)
e
Beach)
10 m
1.4
(0.08)
0.48
(0.06)
0.88
(0.10)
0.18
07)
0.47
(0.05)
0.78
(0.08)
—
•—
113
-------
Table 7.6. Phosphate (MM P/l) From Nearshore Water Over Gravel
Beaches at Snug Harbor. Mean of Four Replicates (standard
deviation) . (Method detection limit = 0.20 /iM P/l for sample
date 6/10/89, 0.02 MM P/l thereafter.)
Sample Date
6/10/89
6/14/89
6/21/89
6/28/89
7/5/89
7/12/89
7/23/89
8 — = Sample
Control Oleophilic Water Soluble
(Rodney Beach) (Otter Beach) (Otter Beach)
1m 10 m 1m 10 m 1m 10 m
0.20
(0.00)
0.10
(0.00)
0.44
(0.00)
0.25
(0.00)
0.27
(0.04)
0.23
(0.03)
0.08
(0.00)
0.20
(0.00)
0.13
(0.03)
0.40
(0.03)
0.25
(0.00)
0.27
(0.04)
0.29
(0.03)
__a
0.34
(0.27)
0.18
(0.04)
0.29
(0.04)
0.15
(0.03)
0.36
(0.04)
0.22
(0.04)
0.08
(0.00)
0.20
(0.00)
0.15
(0.00)
0.28
(0.11)
0.17
(0.02)
0.23
(0.03)
0.32
(0.03)
—
—
0.20
(0.00)
0.15
(0.04)
0.34
(0.03)
0.20
(0.00)
0.37
(0.05)
0.25
(0.03)
0.10
(0.03)
0.26
(0.12)
0.12
(0.05)
0.35
(0.04)
0.16
(0.00)
0.28
(0.04)
0.22
(0.00)
—
--
not collected.
114
-------
Table 7.7. Phosphate (MM/PI) from Nearshore Water Over Cobble
Beaches at Snug Harbor. Mean of Four Replicates (standard
deviation). Method detection limit = 0.20 /*M P/l for sample date
6/10/89, 0.02 nH P/l thereafter.)
Control
(Fred
Sample Date 1 m
6/10/89
6/14/89
6/21/89
6/28/89
7/5/89
7/12/89
7/23/89
a — = Samples
0.22
(0.03)
0.16
(0.02)
0.36
(0.02)
0.18
(0.02)
0.29
(0.05)
0.38
(0.00)
0.10
(0.03)
Oleophilic Water Soluble
Beach) (Seal Beach) (Seal Beach)
10 m 1m 10 m 1m 10 m
0.20
(0.00)
0.15
(0.00)
0.31
(0.03)
0.28
(0.03)
0.30
(0.04)
0.34
(0.03)
__»
0.20
(0.00)
0.15
(0.00)
0.35
(0.04)
0.16
(0.04)
0.32
(0.03)
0.25
(0.03)
0.09
(0.01)
0.20
(0.00)
0.12
(0.03)
0.25
(0.03)
0.15
(0.03)
0.25
(0.03)
0.23
(0.05)
—
0.22
(0.03)
0.14
(0.04)
0.26
(0.00)
0.20
(0.04)
0.34
(0.03)
0.25
(0,06)
0.09
(0.01)
0.20
(0.00)
0.14
(0.06)
0.27
(0.04)
0.24
(0.05)
0.30
(0.07)
0.22
(0.00)
—
not collected.
115
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SNUG HARBOR
Ill* 1114
GRAVEL OLEOPHIUC
*/!• «/M (III t/tl lit M« fill IK
OMAVEL WATEM SOLUBLE
w
Figure 7.21 Phytoplankton Chlorophyll Concentrations {Mean + SD) in 4 Replicates
of Snug Harbor Water Samples Collected After June 7 and 8,1989,
Fertilizer Additions to Gravel Shorelines
116
-------
Differences between nearshore (1 m) and offshore (10 m) samples
and fertilized and reference shoreline samples were within the
expected day-to-day variation common for phytoplankton data.
Phvtoplankton Primary Productivity
Phytoplankton productivity was used as a functional measure
of the photosynthetic activity of algal cells. It allowed an
evaluation of whether the algal population sampled was viable and
active, nutrient limited or enriched. Comparisons of
photosynthetic rates obtained on different sampling dates are not
valid as the light conditions during incubation could have been
different enough to significantly affect productivity estimates.
Only treatment-versus-treatment and treatment-versus-reference
comparisons were valid for each sampling date. Overall,
differences between treated and reference samples appear small,
inconsistent, and within the range of expected ecological
variability (Figure 7.22). Samples from 6/21 and 6/28 showed a
consistent increase in productivity for treated shoreline samples
compared to reference samples, however, all samples were within a
factor of two. If elevated primary productivity was caused by
nutrient addition, the absence of a change in nearshore
chlorophyll concentration suggested that biomass was not
increasing faster than dilution and transport by tidal exchange
was depleting it.
Bacterial Abundance
Mean bacterial abundances in water column samples from Snug
Harbor varied from 0.51 to 2.49 x 109 cells per liter, reported
in Figure 7.23. Due to sampling error, data were lost for all
samples collected prior to fertilizer addition. One week after
nutrient additions, bacterial numbers near fertilized shorelines
were higher than the second day after fertilizer application.
Bacterial numbers near reference shorelines did not change.
Bacterial numbers near treated beaches returned to background
levels 1 week after treatment. Because bacterial numbers near
treated beaches were no greater than numbers near reference
beaches, the increase from day 1 to week 1 were not considered
ecologically significant. Changes of this magnitude reflect
natural system variability. Other than a decrease from slightly
elevated bacterial numbers in early June, no trends in bacterial
abundance were associated with shoreline treatments or time over
the monitoring period.
Bacterial Productivity
Bacterial productivity was estimated by the incorporation of
3H-thymidine in water samples transported, prepared, and
incubated at the ecology laboratory at Valdez. Because the
abundance of cells alone may not represent the viability of
117
-------
SNUG HARBOR
i i i i • « i ! i i I I
{ 6 ] 3 | I \ 5 \ I !
8 3 3 ! 8 5 I I i
§ "
I -
i u
3 3 8 3
: I ; ! : ! ; i ; ! ; i
5 5 ! 3 ! B ! = ! 3 5 *
3 5 3 5 8 3 I J I 3 i j
Figure 7.22 Primary Productivity Estimates (as C Uptake) (Mean + SD) for 4
Phytoplankton Replicates from Snug Harbor Collected After June 7
and 8,1989, Fertilizer Applications to Shorelines
118
-------
SNUG HARBOR
li
!••
II
1C
14'
U<
U<
l.»
1J'
M •
\ «:
7-549
t" u
"
u
• i • ! • § ; i ; I
6 i 5 3 ' i j g ? i
5 5 8 5 8 5 \ |
• i
\
5 ? 3
8 3 5 3 8 3 j j j | I ]
S = \ 3
2 I 3 5
S S 6 g 5
5 j j i j
Figure 7.22 (Continued)
119
-------
SNUG HARBOR
tn4 tin till r;t r/«i rut «t
GRAVEL WATER SOLUBLE
i/i* i/<4 tm tut nt rm nit tl
:
i
GRAVEL OLEOPHIUC
-------
planktonic microbes, bacterial productivity was estimated to
allow an evaluation of functional activity of this community and
the effect of nutrient enrichments.
Bacterial productivity data revealed no consistent changes
or trends associated with fertilizer application to the shoreline
(Figures 24 and 25). Although data showed greater productivity
during the first two sampling periods compared to subsequent
sampling, this difference was seen in amples from reference
sites as well as treated sites. This data probably represented a
seasonal trend rather than a treatment effect. None of these
differences appeared to be ecologically significant.
Microflaaellate Abundance
Samples for microflagellate abundance were used as an
estimate of the population of grazers that consume bacteria in
microbiological food chains. Increases in their abundance at the
fertilizer-treated sites would indicate that bacterial biomass
was being directly incorporated into the next step in the food
chain at a rate reflective of nutrient enrichment. None of the
initial samples indicated any measurement effect from the
fertilizer applications; therefore, analyses of additional
microflagellate samples was stopped to minimize costs and
streamline sample processing.
Dissolved Organic Carbon. Particulate Carbon. Particulate
Nitrogen
Water samples for these analyses have been processed through
the EPA Valdez laboratory and shipped to US EPA ERL, Gulf Breeze
for analysis. Only preliminary analyses have been completed as
of this date.
Stable Isotope Ratios of Carbon and Nitrogen
Biological samples for stable isotope analyses were sent to
Texas A&M University for analysis.
Caged Mussels
Analyses of mussel tissues is still proceeding, only 20% of
the samples have been analyzed to date. These samples represent
a cross-section of the stations and times sampled at the Snug
Harbor study site. An inspection of available results to date
indicate that slightly more than half of the samples analyzed had
no detectable PAH residues (<0.05 ug/g) and, when present, total
PAH concentrations were always less than 1 ug/g (PPM). The
predominant PAH present in samples with residues was
benzo(a)pyrene. This compound is not prominent in Prudhoe Bay
crude oil and is more likely an indicator of the presence of
diesel combustion products from the myriad of vessels working in
121
-------
SNUG HARBOR
GRAVEL OLEOPHILIC
IM4 till «'»•
MUMMIES
lit
7/11
I'll «lt
MMMIDATIS
rni
GRAVEL WATER SOLUBLE
•/it ft*
UMPUMTn
Figure 7.24 Abundance of Bacterial Cells (x10 ) (Mean + SD) from 4 Replicates of
Snug Harbor Water Collected on Various Dates After June 7 and 8,
1989, Fertilizer Applications to Shorelines
122
-------
SNUG HARBOR
^
COBBLE OLEOPHILIC
^•M^M^M
• 'imnm
O •>•!«•
UMM1DATU
(ill I/II
[DATES
COBBLE WATER SOLUBLE
8 asa I
Figure 7.25 Bacterial Productivity (Mean + SD) as Measured by Tritiated Thymidine
Uptake from 4 Replicates of Snug Harbor Water Collected on Various
Dates After June 7 and 8,1989, Fertilizer Applications to Shorelines
123
-------
Prince William Sound on oil spill clean-up efforts. None of the
mussel tissue data to date indicates any enhanced residues from
bioremediation activities. A definitive assessment must await
completion of analytical work on a greater number of samples.
DISCUSSION AND CONCLUSIONS
Data from the bioremediation field demonstration in Snug
Harbor have been collected and are being processed and analyzed.
Although all evaluations are not yet complete, the following
general discussion and conclusions can be made.
• Visual inspection of beaches treated with oleophilic
fertilizer showed that in approximately 2 to 3 weeks oil was
removed from the treated shorelines. The effect was most
apparent on cobble beaches, where initially much of the
surface oil was removed. No visible decrease in the oil
occurred on the beaches treated with the slow-release
fertilizer briquettes or the reference beaches. This
removal continued on oleophilic-treated plots, eventually
leading to the disappearance of oil from the surfaces of all
beach material.
• No oil slicks or oily materials were observed in the
seawater following application of the fertilizers. Based on
the analyses to date, no oil or petroleum hydrocarbons have
been detected in mussels contained in cages just offshore
from the fertilizer treated beaches. Thus removal of oil
from the beaches did not appear to be chemically mediated.
Analysis of oil extracted from reference beach plots showed
that loss of oil residue weight and changes in chemical
composition of the oil were substantial and progressed
steadily through time. This suggested that natural
biodegradation of the oil occurred at a surprising rate.
Indeed, nutrient analysis of tidal and fresh water that
washed test beaches showed the presence of significant
quantities of ammonia, nitrate and phosphate. Thus, if
biodegradation rates (and possibly the extent of
biodegradation) are limited by the availability of nitrogen
and phosphorous in the Prince William Sound, natural
processes are doing an effective job of bioremediation by
the continual low level supply of these essential nutrients.
• Analyses of oil extracted from beach samples taken from
plots treated with oleophilic and slow-release fertilizer
briquettes showed that decreases in oil residue weight and
changes in oil composition (as measured by a variety of
approaches) may have been stimulated by the fertilizer
addition. This is reasonable given the detection of the
above ambient concentrations of ammonia and nitrate
following fertilizer application. However, due to the very
124
-------
heterogenous distribution of oil on the beaches, imprecise
methods for sampling unconfined gravel and cobble, and high
amounts of natural oil biodegradation, it has been difficult
to statistically verify that nutrient addition caused
enhanced biodegradation. Since only a portion of the total
data set has been analyzed, many options are under
investigation to explain and interpret field test results.
Preliminary indications suggest that the standard measures
of biodegradation, changes in the ratios of specific
branched and straight-chained alkanes may be inadequate.
This is because pristane and phytane, which were thought to
degrade slowly, were in fact readily degraded in some cases,
thus making them very unpredictable as conserved internal
standards. Alternate measures of biodegradation will have
to be developed, including the use of chemical analyses for
different fractions of the crude oil.
Samples of the oil from fertilizer-treated beaches,
particularly from cobble surfaces, taken at about the time
when the oil was visually disappearing, showed substantial
changes in hydrocarbon composition, indicating extensive
biodegradation. This suggests that biodegradation was
affecting removal of the oil, both through direct
decomposition and possibly through the production of
biochemical products (bioemulsifiers) known to be produced
by bacteria as they consume oil and hydrocarbons as sources
of food.
Extensive monitoring studies indicated that the addition of
fertilizer to oiled shorelines caused no ecologically
significant increases in planktonic algae or bacteria or any
measurable nutrient enrichment in adjacent embayments.
125
-------
SECTION 8
FIELD TEST RESULTS - PASSAGE COVE
VISUAL OBSERVATIONS
Original oil contamination in Passage Cove was heavy.
Following complete physical washing, oil was well distributed
over most of the surface of all cobble and all gravel under the
cobble. The oil in appearance was black, dry, and dull with
considerable stickiness. It was spread as a thin layer over the
beach material. Relatively few patches of pooled oil or mousse
were present but where they were present, the oil was thick and
viscous. Oil was also found at depth in the beach, generally 30
to 40 cm below the surface. It was well distributed within the
beach material.
Within approximately two weeks following application of
oleophilic fertilizer and slow release granular fertilizer, it
became apparent that the treated beach was considerably cleaner
relative to the reference plots. In contrast to the observations
at Snug Harbor, not only did the rock surfaces look cleaner but
the oil under the rocks and on the gravel below was also
disappearing. In another two weeks, oil could be found only in
isolated patches and at 10 cm and below in the subsurface. At no
time were oil slicks or oily material seen leaving the beach
area. During this time no loss of oil from the rock surfaces was
apparent in the reference plot.
The beach treated with fertilizer solution from the
sprinkler system behaved in a very similar manner to the
oleophilic/granule-treated plot; that is, it became clean. The
only difference was that it lagged behind the oleophilic/
granular-treated beach by about a 10-14 days. By the end of
August, both beaches—the oleophilic and fertilizer solution
treated—looked equally clean. In contrast, the reference plot
appeared very much as it did in the beginning of the field study.
Oil in the subsurface still remained in all plots. However,
visually in the fertilizer-treated plots oil was apparent only
below 20-30 cm of depth.
NUTRIENT CONCENTRATIONS
At the time this interim report was completed, nutrient data
from Passage Cove was still being processed and therefore, could
not be included.
CHANGES IN OIL RESIDUE WEIGHT AND COMPOSITION
Data analysis for oil residue weight and chemistry in
samples taken from beach plots in Passage Cove has not yet been
127
-------
completed. Over 600 samples have been analyzed and the resulting
information is being incorporated into the data base. Approaches
for analyzing trends in the data are the same as those used for
Snug Harbor (see Section 7).
Data for oil residue weights and ratios of C17/Pristane and
C18/phytane, have been analyzed only for oil extracted from the
surface of the cobbles (referred to as top), not for oil from
gravel under the cobble. Some general statements, however, can
be made at this time.
Residue Weight
Changes in the mean residue concentration with 75th and 25th
quartiles through time for all plots are shown in Figure 8.1.
Each data point is the mean of the number of samples available at
this time (maximum samples equals 21 for any sampling time). The
reference plot showed a slow decrease in oil residue weights over
the first three weeks followed by a somewhat more rapid decrease.
The plot treated with fertilizer solution applied by a sprinkler
system started at approximately the same oil concentration as the
reference plot but over time dropped rapidly and then leveled
off. Oil in the oleophilic granule-treated plot showed a slow
steady decrease through time, perhaps with a noticeable drop
between week 1 and 3.
Although none of these trends have yet been satisfactorily
verified, results do appear to show a notable effect of the
fertilizer solution. It corresponds with the visual observation
and with similar observations in microcosms (see Section 9) where
controlled nutrient addition was also maintained. The lack of a
large decrease in oil residue weight in the oleophilic
fertilized-treated beach contrasts with the visual observations.
Ratio of Branched and Straight Chain Hydrocarbons
Information on changes in the C17/pristane and C18/phytane
ratios is available only for the oil extracted from the cobble
surface. The data is presented in Figures 8.2 and 8.3. The
results generally mirror trends observed with oil residue
weights. Ratios typically decreased through time. The plot
treated with the fertilizer solution applied by sprinkler system
showed the most dramatic change in ratios with a 2-3 fold
decrease in the first week. Ratios from the reference plot
decreased steadily over the two week period, the change in
C18/phytane ratio being more pronounced than the C17/pristane
ratio (note initial values for the C17/pristane ratio were very
low). The increase was probably not due to reoiling, as oil
residue weights did not increase during the same time period. It
is possible that degradation of the pristane and/or phytane
occurred, rendering this measure of degradation inadequate.
128
-------
K>
VO
D)
REFERENCE
— O— WATER SOLUBLE
WATER SOLUBLE
AND OLEOPHILIC
7/22
8/06 8/13
9/04
Sampling Date
Figure 8.1 Mean Residue Concentration at Passage Cove Cobble Plots, Top, All Zones.
-------
0.4 n
~ 0.3 -
CO
DC
0>
c
0.2-
—o —
REFERENCE
WATER SOLUBLE
WATER SOLUBLE
AND OLEOPHILIC
O 0.1 -
7/22
8/06 8/13
9/04
Sampling Date
Figure 8.2 Mean C17 / Pristane Ratio at Passage Cove Cobble Plots, Top, All Zones.
-------
0.7-u
REFERENCE
WATER SOLUBLE
WATER SOLUBLE
AND OLEOPHILIC
7/22
8/06 8/13
9/04
Sampling Date
Figure 8.3 Mean C18 / Phytane Ratio at Passage Cove Cobble Plots, Top, All Zones.
-------
As with much of the Passage Cove data, this information is
still being analyzed. It will require more time and probably
special statistical analyses, to verify the indicated trends.
Gas Chromatoqraphic Profiles
Unfortunately only a few computer recreated gas
chromatographic profiles are available. Examples using floating
concentrations on the plots are shown in Figures 8.4 and 8.5.
Comparing profiles in the fertilizer solution-treated beach
(sprinkler system) prior to, 2 and 3 weeks following application,
it is apparent that both the amount of oil analyzed and the
relative concentrations of the alkanes in the profile changed
quickly and dramatically through time. Thus, under conditions
where microbial communities experience repeated and controlled
exposure to nutrients, degradation of oil on the rock surfaces
occurred within 1 week. The extensive degradation apparent at 3
weeks, the point in time when oil was visually disappearing from
the rocks, suggests that degradation of other fractions
(aromatics, waxes, asphaltenes, polars) of the oil may be
occurring. Additional chemical and mass spectral analysis of the
oil will provide insight into this supposition.
Profiles from two sampling times from the reference plot are
compared (Figure 8.5). Degradation does not appear as extensive,
yet it occurred to a significant extent.
Total Alkane Concentrations
Examination of changes in the total (summed) aliphatic
hydrocarbons (normalized to oil residue weight) as determined
from the gas chromatographic profiles (Figure 8.6) show how
rapidly degradation proceeded in Passage Cove. Again, the
absence of complete statistical analyses makes definitive
statements difficult. The visual impression is that the
reference beach may have had the slowest degradation and the
oleophilic/slow release granule fertilizer combination (in terms
of total aliphatic hydrocarbons degraded) may have been the most
rapid. The initial rise in total hydrocarbons in this latter
plot remains unexplained. However, if the worst case is
considered and the assumption is made that the sampling on the
week following application (week 1) is a fluke, the overall
decrease in total hydrocarbons is still as fast as that seen in
the fertilizer solution-treated plot. Thus, as more data are
analyzed, evidence for nutrient enhanced bioremediation may
become stronger.
132
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Passage Cove - Cobble Surface - Water Soluble Fertilizer - Before Treatment
BLOCK=1
BLOCK=2
BLOCK=3
BLOCK=4
BLOCK=5
BLOCK=6
BLOCK=7
0>
0)
DC
I.O
O.9
O.B
O.7
O.6
O.S
O.4
0.9
0.2
0.1
0.0
I.O
O.9
O.B
0.7
O.B
O.S
O.4
0.3
O.2
O.I
0.0
I.O
O.9
0.8
O.7
0.6
0.5
O.4
0.3
O.2
O.I
O.O
BLOCK=8
BLOCK=9
BLOCK=1O
o.e
o.s
0.4
0)
u
c
o
o
0.0
17 is
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Figure 8.4a. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble before application of water soluble fertilizer (sprinkler system) on Kittiwake Beach in
Passage Cove. Blanks indicate data not available. Solid line profile estimates peak heights
of alkanes in oil that has undergone minimal biodegradation. Note floating concentration scale.
-------
Passage Cove - Cobble Surface - Water Soluble Fertilizer - 2 Weeks After Application
BLOCK=1
BLOCK=2
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BLOCKS?
-------
Passage Cove - Cobble Surface - Water Soluble Fertilizer - 3 Weeks After Application
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Figure 8.4c. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble three weeks after application of water soluble fertilizer (sprinkler system) on Kittiwake
Beach in Passage Cove. Blanks indicate data not available. Solid line profile estimates
peak heights of alkanes in oil that has undergone minimal biodegradation. Note floating
concentration scale.
-------
Passage Cove - Cobble Surface - Untreated - Before
Fertilizer Application to Nearby (Treated) Beaches
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Figure 8.5a. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble at the control beach (Raven Beach) before application of water soluble fertilizer at
Passage Cove. Blanks indicate data not available. Solid line profile is an estimated
line connecting peak heights of alkanes in oil that has undergone minimal biodegradation.
Note floating concentration scale.
-------
Passage Cove - Cobble Surface - Untreated - 2 Weeks
After Fertilizer Application to Nearby (Treated) Beaches
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n-Alkanes (n-C12 to n-C32)
Figure 8.5b. Recreated gas chromatographic profiles from samples of oil extracted from the surface of
cobble at the control beach (Raven Beach) two weeks after application of water soluble
fertilizer (sprinkler system) to nearby beaches at Passage Cove. Blanks indicate data not
available. Solid line profile estimates peak heights of alkanes in oil that has undergone
minimal biodegradation. Note floating concentration scale.
-------
CO
OO
REFERENCE
WATER SOLUBLE
WATER SOLUBLE
AND OLEOPHILIC
7/22
8/06 8/13
9/04
Sampling Date
Figure 8.6 Median of Total Aliphatic Hydrocarbon Concentrations (Normalized to Oil Weight)
on Treated and Untreated Passage Cove Cobble Plots, Top, All Zones.
-------
MICROBIOLOGY
The number of oil-degrading bacteria present on beach
materials has also been determined for Passage Cove. Samples of
beach material were taken from grids 1, 3, 5, 7, 8, 10, 12, 14,
15, 17, 19, and 21. Numbers of degraders were assessed by a
modification of the dilution to extinction method used for Snug
Five replicate dilution series were prepared from the *p244XHa
initial 1:10 dilution. The relative numbers of bacteria in each
sample was an average of the five replicate dilution series.
Results from these studies are shown in Table 8.1. The
values reported are the Iog10 normal mean and standard deviation
of 11-12 dilution series for each mixed sand and gravel sample.
Results suggested that no consistent increase in oil-degrading
microorganisms occurred as a result of fertilizer application.
This means that even in the plot treated with nutrient solutions
from a sprinkler system, where nutrient exposure to the bacteria
should be optimized, no increase in oil-degrading microorganisms
occurred. This could be the result of a relatively constant
sloughing of microbial biomass from the surfaces of the beach
material, perhaps as caused by tidal flushing action. Grazing by
protozoans could also keep the microbial numbers at a specific
density. The presence of high numbers of oil-degrading bacteria
in the reference beaches made differences in the numbers of
degrading organisms between treatments subtle and difficult to
detect.
In early August, several beaches that had not been impacted
by the oil spill were sampled to determine realtive levels of
oil-degrading microorganisms. Samples were collected from the
high, mid, and low tide areas at each beach. The bacterial
densities are shown in Table 8.2. The range in concentration of
oil degrading organisms was much greater than that observed for
oil impacted beaches. It is clear that the number of oil
degraders in uncontaminated areas was 1000-100,000 times lower
than in contaminated areas. Thus the presence of oil causes a
significant enrichment of oil degrading microorganisms.
The rate of mineralization of C14labeled substrates is
being used to determine the physiological competence of microbial
populations to degrade specific crude oil components. The
ability to utilize these components will be compared to the
treatments at test sites.
Three C14labeled constituents of crude oil were used:
naphthalene, phenanthrene, and hexadecane. The rate of conver-
sion of each of these compounds to 14CO2 will establish the
activity of the microorganisms as opposed to the number of micro-
organisms. Preliminary mineralization data from the Passage Cove
sampling site indicated that the initial sampling times for 14C02
were early; no 14C02 was produced. Due to the number of samples
139
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Table 8.1. Relative Concentration (Log10 of the cell number/g of
beach material) of Oil-Degrading Microorganisms in Passage Cove.
Sampling Date Plots
Fertilizer-Treated
Water Oleophilic &
Before Application Reference Soluble Water soluble
07/22/89 6.44 6.31 6.44
±1.44 ±1.36 ±1.33
After Application
08/06/89 5.32 5.78 5.71
±1.12 ±1.45 ±0.67
08/19/89 6.60 5.47 5.66
±1.83 ±1.34 ±0.35
140
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Table 8.2. Relative concentratrion (Log10 of the cell numbers/g
of beach material and standard deviation) of Oil Degrading
microorganisms in Samples from Beaches that were Not Impacted by
Oil.
Site High Tide Mid Tide Low Tide
Tatitlek 2.41 4.31 6.11
±.58 ±1.14 ±2.05
Fish Bag <1.51 <1.31 <2.71
Snug Corner Cove 2.31 2.51 <1.11
±.54 ±.55
Hell's Hole <2.11 2.51 <.91
±.89
Commander Cove 4.51 <1.31 3.11
±1.14 ±.45
141
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being processed and the current capability to analyze them, only
four sampling times were possible. The first studies indicated
that after 48 hours of incubation, significant amounts of 14CO2
were produced. Therefore, further studies will use an extended
incubation period of 3 to 5 days.
ECOLOGICAL MONITORING
The same environmental parameters were monitored at the
Passage Cove study site as were monitored at the Snug Harbor-
study site, using a somewhat modified strategy for sample site
location. Sample stations were located along the central axis of
the embayment and along 3 nearshore areas where fertilizers were
applied (see Sections 5 and 6). Reference sites for the Passage
Cove study were established outside of the embayment along the
eastern shore of northern Knight Island. Water from the central
sites of Passage Cove was sampled at 0.5 m and 5 m depths,
whereas the nearshore stations (1 m offshore of low tide) were
sampled at 0.5 m depths. Fertilizers were applied on July 25 and
26, 1989, to selected plots along the shoreline. Samples were
collected prior to application of fertilizer along the shoreline,
3 days after application, and then at weekly intervals for 6
weeks after application.
Nutrients
Only limited data are available from analyses of water samples
from Passage Cove for ammonia, nitrite, nitrate, and phosphorus.
Assessment of eutrophication resulting from fertilizer additions
must await additional sample analyses.
Chlorophyll Analysis
Phytoplankton chlorophyll data showed little change over the
course of the study period (Figure 8.7). No trends consistent
with nutrient effects were observed. An increase observed on
8/27 was seen in the 0.5 m sample from all mid-channel stations
and the reference site.
Phytoplankton Primary Productivity
Results from the pre-treatment sample (7/21), Day 3 (7/28), and
Weeks 1 (7/31) and 2 (8/2) are shown in Figure 8.8. These data
showed no trends toward greater primary productivity for Passage
Cove stations as a result of nutrient additions, except on 7/31.
Primary productivity estimates on this date showed greater values
for stations 5, 6, and 7, the nearshore stations along the
treated shoreline. This increase was not observed 1 week later,
and it was not borne out in the chlorophyll data. If primary
productivity was enhanced along the shoreline due to nutrient
input, the effect on plankton growth was not sufficient to
overcome dilution and transport due to tidal exchange, i.e.,
142
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PASSAGE COVE
7.11 r-it r >i 41 411 4-11 4-ir 4.4
Figure 8.7 Mean Chlorophyll Measurements (+ SD) From 4 Replicate Plankton
Samples Taken at Passage Cove Study Sites Before and After July 25,
1989, Fertilizer Applications to Shorelines
143
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PASSAGE COVE
STATION 5
I
r-ii r.i« /.it «.> 4-14 4-11 i.ir 1.4
•AMPUOATt
Figure 8.7 (Continued)
144
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PASSAGE COVE
s s s
I -
«.«
„
7-28-89
5 ! s i s i • i
« s i i i ; i j
- 2 I ! !
Figure 8.8 Mean Primary Productivity Measurements (+ SD), as C-Uptake From 4
Replicate Plankton Samples Taken at Passage Cove Study Sites Before
and After July 25,1989, Fertilizer Applications to Shorelines
145
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there was no persistent increase in plankton chlorophyll. These
conclusions will be reevaluated once all data are available.
Bacterial Abundance
The mean number of bacterial cells per liter of water at
Passage Cove sample sites ranged from approximately 0.4 to 1.2 x
109 over the 7-week sample period (Figure 8.9). All stations
followed the same general pattern of greater numbers on the first
three sample dates with lesser abundances thereafter. No trends
were observed for nearshore and offshore comparisons, treated
versus control comparisons, or 0.5 m to 5.0 m sample comparisons.
Fertilizer additions had no stimulatory effect on bacterial
numbers.
Bacterial Productivity
Bacterial productivity, measured by bacterial uptake of
tritiated thymidine, demonstrated considerable variability
between sample dates with no consistent trends through time or
with fertilizer treatments (Figure 8.10). The samples with
prominently increased productivity usually occurred on the same
dates for all samples, with similar trends at upper and lower
depths. There were no trends consistent with effects of nutrient
addition.
Caaed Mussels
Analyses of mussel tissues is still proceeding, only 20% of
the samples have been analyzed to date. These samples represent
a cross-section of the stations and times sampled at the Passage
Cove study site. An inspection of available results to date
indicate that slightly more than half of the samples analyzed had
no detectable polycyclic aromatic hydrocarbons (PAH) residues
(<0.05 /ig/g) and, when present, total PAH concentrations were
always less than 1 Mg/9 (ppm)• The predominant PAH present in
samples with residues was benzo(a)pyrene. This compound is not
prominent in Prudhoe Bay crude oil or its degradation products,
but is more likely an indicator of the presence of diesel
combustion products from the myriad of vessels working in Prince
William Sound on oil spill clean-up efforts. None of the mussel
tissue data to date indicates any enhanced residues from oil
degradation resulting from bioremediation activities. A
definitive assessment must await completion of analytical work on
a greater number of samples.
Field Toxicity Tests of Oleophilic Fertilizer at Passage Cove
Water samples were collected at specified intervals before
and after the July 25, 1989, application of Inipol and slow
release granules to the treated shoreline. These samples were
sent to a consulting laboratory for 48-hr toxicity tests with
146
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PASSAGE COVE
7-ti r-t* r.ji (.1 i.i« 4.11
r ti »•!• i.11 4-1 4-14 4 >i 4->r
Figure 8.9 Abundance of Bacterial Cells (x10) (Means + SD) From Water Samples
Taken at Passage Cove Study Sites Before and After July 25,1989,
Fertilizer Applications to Shorelines
147
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PASSAGE COVE
7-II f.ll 7-JI 1-1 1-14 t-tl • •!»
Figure 8.9 (Continued)
148
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PASSAGE COVE
r 11 »•»• r-li (-1 ••<« «-ii • •»» ••«
r-ii r-it T.JI 1.1 (.it t.ii t.if i.«
!•!! '•»• r-11 I.a «-H *•!! ••>! • 4
Figure 8.10 Bacterial Productivity Measurements (Means + SD), From Tritiated
Thymidine Uptake by Water Samples Taken at Passage Cove Study
Sites Before and After July 25,1989, Fertilizer Applications to
Shorelines
149
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PASSAGE COVE
J-J1 T-lt I-II «•! «.|4 I.II LIT 1.4
Figure 8.10 (Continued)
150
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oyster larvae, Crassostrea gigas. Endpoints monitored for these
tests were larvae survival to test termination and percentage of
larvae that exhibited abnormal development. The data are given
in Table 8.3.
Test acceptability criteria dictated that for each series
tested, control survival must be greater than 70% with
abnormality less than 10%. All laboratory control, field
control, and pre-application samples met these criteria. The
greater survival of larvae in laboratory seawater controls
relative to hypersaline controls, field controls, and pre-
application samples may be related to minor toxic components in
field samples or in the brine solution. These differences were
not statistically significant when compared by Dunnett's
procedure. The percentage of abnormal larvae varied little among
the four control samples.
Tests with water samples collected at the field site after
Inipol application indicated survival values of less than 70% and
rates of abnormal development greater than 10%, suggesting the
presence of toxic components. Because the survival of larvae was
greater than 50% for all water samples except the 18 hr sample,
an LC50 could be computed for the 18 hr sample only. Toxicity
associated with the other samples was assessed through the use of
Dunnett's procedure to determine if observed effects were
significantly greater than mortality or abnormal development
rates for the field control and pre-application samples, which
are the proper samples for comparisons with test site treatments.
None of the values for post-application samples, except the 18 hr
test samples, were significantly different from the field control
survival and pre-application survival of 70% and 74%,
respectively. In addition, none of the percentages of abnormally
developed larvae for these samples was significantly different
from those of the field control and pre-application samples, 8.4%
and 10.4%, respectively. Comparison with laboratory seawater
controls showed that significant effects occurred for several
samples, but these comparisons combine Inipol toxicity with
residual toxicity in site water at Passage Cove prior to
fertilizer additions.
The water sample taken 18 hours post treatment killed 61% of
the oyster larvae during the toxicity test. A 48 hr LC50 of 58%
of full-strength water was calculated using the dilution series.
Thus, when full-strength site water was diluted to 58% of its
original concentration, it would kill 50% of the oyster larvae
during a 48 hr test. The 95% confidence interval for the LC50 is
46% to 75%. The full-strength site water collected at 18 hours
had significantly greater numbers of abnormal larvae compared to
site controls and laboratory controls. Dilution to 56% of the
full strength concentration would produce abnormal larvae at a
rate not significantly different than the field control and pre-
application samples.
151
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Table 8.3. Larval Survival and Development After 48 hours in
Salinity-Adjusted Prince William Sound Water.
Sample Designation
Lab Seawater Control
Hypersaline Control (28 ppt)
Field Control
Pre-Application 74% 10.4%
Survival AbnormalTidal Stage
92% 9.5%
75% 7.8%
70% 8.4%2-hr pre-low
2-hr pre-low
Inipol Application 10 AM - 2 PM
1-hr Post Application 62% 14.2%3-hr post-low
3-hr Post Application 87% 16.1%near high tide
6-hr Post Application77%10.5%mid-tide, outgoing
12-hr Post 58%10.1%mid-tide, incoming
18-hr Post Application 39%31.4%mid-tide, outgoing
152
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Discussion and Conclusions
Toxic effects from misapplication of Inipol or immediate
release from the shoreline during initial tidal flooding were not
seen. Test results indicated that application of Inipol to oiled
shorelines at the Passage Cove test site resulted in water
concentrations that caused abnormal development and mortality of
oyster larvae only during the sampling that occurred 18 hours
after application. The 48 hr LC50 for this sample was 58% of
full-strength site water. The increase in abnormal development
associated with this sample was mitigated by dilution to 56% of
full-strength.
Apparently, more toxicity was associated with the second
flooding of the Inipol-treated shoreline than the initial
flooding. This was unexpected. No unusual weather or oil
movements that could have caused this effect were observed
following the Inipol application. In the absence of Inipol
additions, test site water produced survival and abnormality
rates that were marginally above acceptance criteria. This may
demonstrate residual toxicity problems that exist along oiled
shorelines unless definitive clean-up actions are taken.
If we attribute all the observed toxicity to release of
Inipol from the treated shoreline upon re-flooding by incoming
tides, then the release rate can be estimated.
a) Using the application rate of 293 g Inipol/m2,
concentrations of 4,500 mg/1 would be expected if 100% of
the applied Inipol was immediately released into water over
the treated beach, with minimal dilution.
b) The LC50 for the most toxic sample, the 18 hour post
application sample, was 58% of full-strength, ie, an
exposure resulting in 50% mortality from the field sample.
c) Using 50 mg/1 as the LC50 for oyster larvae and Inipol, any
field sample that gives 50% mortality should have 50 mg
Inipol/1. Thus, a 58% dilution of 18 hr water would get
concentrations down to 50 mg/1.
e) Thus, the initial concentration in the 18 hr sample may have
been 90 mg/1 Inipol (dilution to 58% yielded 50 mg/1). This
concentration was 2% (90 mg/1 divided by 4,500 mg/1) of the
"no-dilution and 100% release" assumption.
f) This crude estimate of the release rate (2%) is within the
range of expectations for initial releases of Inipol
following application.
153
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DISCUSSION AND CONCLUSIONS
Much of the data from the Passage Cove study is still being
processed. However, several points can be discussed at this
tine.
The biological cleaning effect of oleophilic fertilizer
observed in Snug Harbor also occurred in Passage Cove. However,
the effect was perhaps more dramatic in that oil from all areas
of the treated plots disappeared. It is possible that the
homogeneous distribution of oil over a large extensive surface
area by physical washing promoted the biological degradation of
the oil in the presence of the fertilizer.
Application of nutrients from the sprinkler system proved to
be the most efficient system for exposing oil-degrading bacteria
to nutrients in a controlled and reproducible manner. As a
result, oil degradation was extensive enough to cause removal of
the oil from the surfaces of the beach materials. Since there
were no chemicals involved in this treatment except inorganic
nutrients, it would appear that biodegradation activities were
responsible for the oil removal and these activities were
enhanced by the nutrient addition.
The action of oleophilic fertilizer probably involved a
stimulation of microbial degradation activities through sustained
and controlled nutrient addition. The shorter time for this
stimulation may have resulted from the softening of the oil
caused by the mild surfactants in the fertilizer increasing
bioavailability.
No occurrence of eutrophication was revealed by extensive
monitoring studies, nor was oil released from nutrient addition.
154
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SECTION 9
SUPPORTING STUDIES
MICROCOSMS
Background
The purpose of these studies was to provide supplemental
information to the field demonstration project. In the event of
a major storm event, or some other unforseen complication,
significant amounts of data from the field demonstration project
could be lost. Microcosm studies that were designed to simulate
the field demonstration project could, therefore, provide a basis
from which scale-up decisions could be based.
In addition, microcosm studies allow the testing of
bioremediation concepts under idealized conditions to provide
complementary data and information to the field demonstration
projects. For example, if biodegradation of oil occurs in
microcosms operated at constant and slightly elevated
temperatures, the study could act as a prelude to what would
happen in the field where conditions are less constant. It is
also desirable to demonstrate that changes in the composition of
the oil caused by biodegradation correspond with significant
decreases in the weight of the oil. In the field, high spatial
variability of oil concentrations may prevent this observation.
However, in microcosms, oil concentrations can be standardized
and thus indications of weight loss can be readily obtained.
Finally, the field verification of microcosm results lend
weight to results from other microcosm tests which cannot be
coupled with a field demonstration component. Such related, but
non-field verified, microcosm tests can be used with confidence
in making decisions about other approaches to the bioremediation
of oil-contaminated beaches.
Methods
Microcosms were constructed on board the F.V. AUGUSTINE to
simulate treatment and control plots in the field demonstration.
Six tanks (representing the six plots) were used to hold 9 two-
gallon polyethylene containers per tank. A schematic of the
microcosm system is shown in Figure 9.1. Twenty-seven of the
containers were filled with homogenized sand and gravel obtained
from the same area as the sand and gravel used for in situ
containers, and mixed in the same manner. The remaining 27
containers were first filled about one-fourth full with
homogenized sand and gravel, and then filled with oiled cobble.
The microcosm containers had four one-inch holes in the bottom to
155
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Mixed Sand Gravel
Cobblestone
Pump C J
Control
Soluble
Soluble
Nutrients
Oleophilic
Seawater
Seawater
PUMPS
Beach
Material
ain
««*— c
1
-z-jp-l
1
:-i-:-=-z-
^^
-----j^---
h~T.J
1
Figure 9.1. Schematic Diagram of the Microcosms
-------
allow percolation of the water through the beach material as the
tanks filled. Seawater from the harbor was pumped into the
tanks, held for 6 hours (high tide) and withdrawn to simulate
tidal cycles. The tanks, therefore, remained dry for 6 hours.
This cycle was then repeated over the next 12 hours, simulating 2
tidal cycles. Within each tank with nine containers, three
replicate containers were sacrificed at three intervals. These
were analyzed to characterize the remaining oil. Intermittent
samples were taken for nutrient analyses.
Fertilizer was added to the microcosms on June 16th. The
oleophilic fertilizer was applied by portable backpack sprayers.
Enough fertilizer was applied to coat the exposed surface of the
beach material in the microcosms. For the water-soluble
fertilizer, 80 IBDU briquettes were placed in a container such
that water entering the microcosm flushed over the briquettes. '
However, since ammonia concentrations in the microcosms were
never above background during the first week of operation, the
briquettes were replaced with small bags filled with commercial
granular fertilizer (N:P:K, 16:5:5 not slow-release), to ensure
adequate levels of nutrients were maintained. This approach
continuously produced ammonia concentrations around 400-700 mg/1
at each filling of the microcosms.
Results
The first set of mixed sand and gravel microcosms was
sampled on July 7 (22 days post application) and a set of cobble
microcosms was sampled on July 11 (26 days post application) and
July 26 (41 days post application). Visual observations at the
time of sampling indicated that the oleophilic fertilizer-treated
cobble microcosms appeared to have the least oil on the surface,
but the difference from other treated and control microcosms was
not dramatic. The cobble microcosms treated with the oleophilic
fertilizer had a mottled appearance, suggesting that the oil on
the surface had been partially removed or degraded. Oil was
apparent under the rocks, but it was very black and viscid. This
consistency appeared to be due to the oleophilic fertilizer
dissolving into the oil.
Amounts of surface oil in the control and water-soluble
fertilizer-treated microcosms appeared the same. Cobble systems
showed some rocks with clean surfaces, but generally fewer than
in the oleophilic fertilizer-treated systems. Oil on the rock
surfaces appeared gray and dried. Oil under the rocks was drier
and less fluid than oil observed in the oleophilic fertilizer
treated microcosms.
After sampling the microcosms, it was noted that the inside
walls of the water-soluble fertilizer microcosms and the
reference microcosms were spotted with oil smudges. This was not
the case in the oleophilic fertilizer-treated set, where the
157
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walls generally appeared free of oil. Small particles of white
waxy material were also observed throughout the sand and gravel
in the oleophilic fertilizer-treated set of microcosms, even with
the daily influx of fresh seawater. This material may have been
residual oleophilic fertilizer.
Discussion and Conclusions >
Results from chemical analyses sampling dates are available.
In a sand and gravel microcosm sampled on July 7 (22 days post
fertilizer application), the C17/pristane and C18/phytane ratios
in the oleophilic fertilizer-treated microcosms were the same as
those in the untreated reference microcosms (Table 9.1). Ratios
in the oil from microcosms treated with the water soluble
fertilizers were almost half of those for the other microcosms.
There was also approximately 20% less oil residue by weight.
These data suggest that the more rapid degradation of oil was
occurring in the water-soluble fertilizer treatments, assuming
oil concentration and composition were approximately the same in
all microcosms at the start of the experiment (data not yet
available). Because of the large amount of readily degradable
carbon added with the oleophilic fertilizer, enhanced degradation
of the oil may not occur until after much of the carbon is
degraded. In the cobble microcosms sampled on July 11, similar
results were observed (Tables 9.2 and 9.3). The most active
degradation, in terms of loss of weight and change in
composition, appeared to be in the water-soluble fertilizer-
treated systems. Degradation of oil in the oleophilic
fertilizer-treated systems was about the same as in the untreated
systems. Oil residue weights in the former gravel systems were,
on the average, 6 times those in the reference microcosms. This
indicates that a component of the oleophilic fertilizer may
contribute to the residue weight.
Analysis of the data from the July 26 sampling of a cobble
microcosms is only partially complete. For those samples
available, mass spectral analysis was performed. A summary of
the results is given in (Tables 9.4 and 9.5). In contrast to the
July 11 data, C17/pristane and C18/phytane ratios for July 26
samplings indicated that the control microcosms were degrading
oil faster than the water-soluble fertilizer-treated microcosms
(Table 9.4). This is in contrast to the initial sampling in
which the results showed greater degradation in the water-soluble
fertilizer-treated microcosms. However, the hydrocarbon ratios
may yield false indications of limited degradation for samples in
which marked degradation of the oil has occurred if pristane is
degraded along with straight chain hydrocarbons. Additionally,
more degradation may be occurring in the oleophilic fertilizer-
treated microcosms than the pristane or phytane ratios suggest.
158
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Table 9.1. Chemical Analysis of Mixed Sand and Gravel Microcosms
Sampled 17 Days After Initiation of Fertilizer Application.
Residue Weight
Treatment (ma/ka) C17/Pristane C18/Phvtane
Control 1 1570 0.5 0.8
Control 2 913 0.4 0.6
Control 3 790 0.4 0.6
Average 1091 0.4 0.7
Oleophilic 1 1490 0.4 0.7
Oleophilic 2 1360 0.4 0.8
Oleophilic 3 795 0.4 0.7
Average 1215 0.4 0.7
Soluble 1 913 0.3 0.4
Soluble 2 916 0.1 0.3
Soluble 3 845 0.3 0.3
Average 891 0.2 0.3
159
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Table 9.2. Residue Weight of Oil in Cobble Microcosms Analyzed
26 Days After Fertilizer Application
Treatment
Control 1
Control 2
Control 3
Average
Residue Weights (ma/kg)
Top Bottom
Cobble Cobble
1120
1090
722
1116 977
Gravel
889
1090
1030
1993
Oleophilic 1
Oleophilic 2
Oleophilic 3
Average
1770
1260
2340
1790
1910
2460
3550
1640
6350
5580
6960
6297
Soluble 1
Soluble 2
Soluble 3
Average
161
1240
383
595
1310
725
664
900
1020
714
814
849
160
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Table 9.3. Ratios of Hydrocarbons in Oil From Cobble Microcosms
Analyzed 26 days After Fertilizer Application
Treatment
Control 1
Control 2
Control 3
Average
Top
Cobble
0.8
0.9
C17/Pristane
Bottom
Cobble
0.7
0.6
0.8
0.7
C18/Pristane
Top Bottom
Gravel Cobble Cobble Grave
0.3 1.3 1.1 0.5
0.4 1.3 1.0 0.5
0.4 1.3 1.1 (Oil
0.4 1.3 1.0 0.5
Oleophilic 1 0.9
Oleophilic 2 1.0
Oleophilic 3 .1.0
Average 1.0
0.8
0.9
0.9
1.0
0.9
0.8
0.9
1.3
1.2
1.2
1.4
1.5
1.4
1.5
1.4
1.4
Soluble 1
Soluble 2
Soluble 3
Average
0.2
0.6
0.6
0.5
0.3
0.1
0.2
0.5
0.6
0.5
0.4
1.1
0.8
0.4
0.3
0.3
0.5
0.7
0*5
0.5
161
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Table 9.4. Comparison of C17/Pristane Ratios and C17/Norhopane
Ratios as Measures of Oil Degradation in Samples Taken From
Cobble Microcosm 42 Days After Initiation Of Fertilizer
Application.
C17/ C17/ Pristane/ Norhopane/
Microcosm Pristane Norhopane Norhopane Hopane
Control .19 1.03 5.44 .78
Water Soluble .49 .22 .44 .75
Standard 1.7 17.50 10.68 .78
162
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Table 9.5. Use of Dibenzothiophene Peaks/Norhopane Ratios as
Relative Measures of the Degradation of Aromatic Components in
Oil Sampled From Cobble Microcosms 42 Days After Initiation of
Fertilizer Application.
Dibenzothiphene Peaks'/Norhopane Ratios
Microcosms1* Peak 1 Peak 2 Peak 3
Control 1
Control 2
Control 3
Water Soluble 1
Water Soluble 2
Water Soluble 3
Oleophilic 1
Oleophilic 2
Oleophilic 3
Standard
.40
.49
.46
.08
.10
.11
.82
.81
.85
1.06
.54
.66
.70
.13
.12
.13
1.21
1.15
1.17
1.84
.60
.71
.71
.13
.19
.17
1.06
1.01
.99
1.54
* In the mass spectral analysis of oil, C-2
dibenzothiophenes and their homologs show a series of peaks at
mass ion 212. Three prominent peaks (labled here 1, 2, and 3)
were selected for comparison.
b Average of three replicates
163
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Further gas chromatography/mass spectrometry data provided
sufficient data to evaluate this possibility. By extracting and
analyzing all microcosm samples in the same manner, compounds
that did not change in concentration in any of the treatments
were identified. Two compounds, norhopane and hopane were
identified. Their concentrations did not change, and the ratio
of norhopane to hopane remained constant at 0.76 (Table 9.4.).
Constructing C17/norhopane and pristane/norhopane ratios
indicated that C17 was degraded 5 times more effectively in the
water-soluble fertilizer-treated than in the control microcosms
(Table 9.4). Surprisingly, pristane was also degraded in both
the control and water-soluble, microcosms, thereby supporting the
suggestion that C17/pristane ratios were inadequate indicators of
biodegradation. It was concluded that norhopane may be a better
choice of a very slowly degraded oil component to be used as an
internal marker for the undegraded oil components.
The ratios of the three major dibenzothiophene peaks to the
very poorly degraded hydrocarbon, norhopane, were also examined
using mass spectral analysis (Table 9.5). Further differences
between the treatments were observed. Water-soluble fertilizer-
treated microcosm samples showed the greatest degree of
degradation of the dibenzothiophene isomers. Interestingly, the
ratios for the oleophilic treatment indicated little change in
the dibenzothiophene isomers, compared with the ratios observed
in a Prudhoe Bay crude oil standard. These observations are
consistent with the C17/pristane data from previous samplings,
which also indicated that oil degradation in the oleophilic
treatment was less active than the degradation in both the water-
soluble and control treatments. The dibenzothicphene to hopane
ratio may be useful to estimate degradation of the sulfur-
heterocyclics in oil. Ratios with other aromatic compounds
(phenanthrene, fluorene, etc.) may also provide a similar tool to
evaluate the homocyclic aromatic fraction.
From these initial microcosm results, it can be concluded
that if sufficient nutrients are supplied to the microorganisms,
then enhanced biodegradation of the oil will occur. Because the
microcosms represent the test systems that best reflect field
conditions, a similar response could be expected in the field, if
nutrient concentrations can be maintained at adequately high
levels. The microcosm studies also showed that pristane and
phytane are readily biodegraded and as such they are not good
markers for assessing changes in oil composition. Mass spectral
analysis may provide other markers to use in this regard.
164
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LABORATORY BIODBGRADATION SCREENING EVALUATIONS'
Background
Studies have shown that oil biodegradation can be enhanced
by the addition of inorganic nutrients under controlled
laboratory conditions. While it could be assumed that similar
enhancement would occur in the field for oil spilled in Prince
William Sound if degradation was limited by nutrient
availability, laboratory studies using samples of weathered oil
and beach material from the Prince William Sound were needed to
verify this assumption. Laboratory flask studies were designed
to investigate the validity of this assumption, using various
nutrient sources, inocula, and temperatures. The results of
these studies will be used to help interpret the results of field
observations.
Methods
Flask studies used samples of Prince William Sound water
and/or oiled beach material. All flasks were incubated with slow
shaking at constant temperature. At each sampling, flask
contents were sacrificed and extracted with methylene chloride.
Extracts were dried and then analyzed by flame ionization
detection gas chromatography (GC/FID). Experiments were
conducted as follows:
• Effects of different inocula: Samples of artificially
weathered Prudhoe Bay crude oil (30% weight loss induced by
distillation) (1% by weight) were placed in sterile
Bushnell-Haas medium, a defined nutrient medium containing
0.03% nitrogen and 0.04% phosphorous. This mixture was
added at a rate sufficient to provide nitrogen and
phosphorus equal to 3.5% and 4.1% by weight of oil,
respectively. This mixture was used uninoculated (control)
or inoculated with either a 10% inoculum of water from the
Alyeska ballast treatment facility or seawater from Prince
William Sound. All flasks were incubated at 15"c for 16
days before the oil composition was analyzed.
• Effect of incubation temperature: Artificially weathered
Prudhoe Bay crude oil was added to sterile Bushnell-Haas
medium and inoculated with 10% Prince William Sound water.
Flasks were incubated for 38 days at 15° and 5°C before the
oil composition was analyzed.
* Experiments conducted by Exxon Researchers at Research
Laboratories in New Jersey and Texas.
165
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Relative effectiveness of Inipol: Two sets of flasks
containing artificial seawater and 1% by weight of
artificially weathered Prudhoe Bay crude oil were made; one
set was then poisoned with 50 mg/1 HgCl2. Inipol, at 10% of
the oil concentration, was added to a poisoned and a
nonsterile flask. Water-soluble fertilizer (Woodace; N:P:K
= 14:3:3) was added to a nonsterile flask at a rate
sufficient to produce a mixture of fertilizer and oil that
had 0.4% added N and 0.09% added P, Inipol (10%) and
fertilizer were added to a second nonsterile flask. All
flasks were inoculated with 10% Prince William Sound water,
and incubated for 16 days before the oil composition was
analyzed.
Optimal Inipol concentration: Flasks for this study
contained artificial seawater and 1% of artificially
weathered crude oil. Inipol, at concentrations of 3, 10, 20
and 50% of the oil concentration was added to the flasks.
Flasks were inoculated with 10% and water from the Alyeska
ballast water treatment facility, and incubated for 16 days
at 15°C before the oil composition was analyzed.
• Effect of temperature on Inipol enhancement: Flasks for
this study contained artificial seawater and artificially
weathered crude oil. One flask received 10% Inipol and
another received fertilizer. Both flasks were incubated at
5", 15°, and 20°C for 38 days before the oil composition was
analyzed.
• Inipol enhanced oil degradation on rock surfaces: Oiled
beach material from Prince William Sound was placed in
flasks and covered with Inipol at concentrations
approximating 10% of the oil concentration. Untreated oiled
beach material was used as a control. A poisoned control of
oiled rocks and Inipol was established using 50 mg/1 HgCl2.
All beach materials were covered with artificial seawater.
Flasks were incubated at 15°C for 16 days before the oil
composition was analyzed.
Results
Indigenous organisms have an ability to degrade weathered
crude oil if provided with adequate nutrients. Initial
experiments showed that organisms in both the Prince William
Sound seawater and in the water from the Alyeska ballast water
treatment facility were able to substantially degrade
artificially weathered crude oil in the presence of high levels
of nitrogen and phosphorus (3.5 and 4.1% with respect to oil,
0.03% N and 0.04% P by weight of water) (Figure 9.2). There was
a substantial decrease in the amount of dichloromethane
extractable material, and substantial degradation of both the
166
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Bushnell-Hass Broth
(3.5% N, 4.1% P)
15°C,16days
Alyeska
Inoculum
15.0 M.O 45.0 «0.0 75.0 M.O 10S.O 120.0 13S.O 1SO.O
MINUTES
hNo
loculum
15.0 M.O 4S.O M.O 7S.O W.O 105.0 120.0 1».0 1M.O
MINUTES
Seawater
Inoculum
i8.o M.O «.o «e.o n.o «o.o ios.0 120.0 iss.o iso.o
MINUTES
Figure 9.2 Gas Chromatographic Profiles Showing the Effect of Different Inocula
on Degradation of Artificially Weathered Prudhoe Bay Crude Oil.
167
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resolvable fractions and the unresolvable fractions on GC
analysis. Very little organic carbon remained in the aqueous
phase after dichloromethane extraction once the precipitated
organisms were allowed to settle out.
Biodegradation proceeded much more effectively at warmer
temperatures, but there was significant biodegradation at 5°C in
the presence of water soluble fertilizers (Figure 9.3).
Inipol EAP 22 stimulated the biodegradation of crude oil.
Flask experiments revealed that the extent of biodegradation
increased with the concentration of Inipol (Figure 9.4).
Water soluble fertilizers and Inipol had at least an
additive, and perhaps a synergistic effect on biodegradation
(Figure 9.5).
Inipol EAP 22 shows a sharper temperature dependency than
water soluble fertilizers, and at 2*C there was very little
biodegradation when Inipol EAP 22 was used alone (Figure 9.6).
Inipol EAP 22 stimulated the biodegradation of oil on Prince
William Sound beach material (Figure 9.7). Rocks treated with
Inipol and then incubated at 15°C became significantly cleaner
after 14 days; all the resolvable peaks had disappeared in the GC
analysis, and 50% of the total dichloromethane-extractable
material had disappeared; furthermore, the rocks were clean to
the touch. This biodegradation was not accompanied by a
detectable lowering of interfacial tension between oil and brine,
indicating that the microorganisms were not producing significant
amounts of surfactants under the conditions tested.
Discussion and Conclusions
These studies showed that weathered oil can be degraded by
organisms indigenous to the Prince William Sound, and that
addition of either oleophilic or water-soluble fertilizer
accelerated the degradation of weathered oil. Oil degradation
from the flasks was also temperature dependent, and was a
function of the concentration of added nutrient, as long as an
adequate inoculum was present. These data indicate that either
oleophilic or water-soluble fertilizers can be used to enhance
biodegradation of weathered oil; they also suggest that a further
enhancement may be possible by using the two types of fertilizer
together.
168
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Bushnell-Hass Broth
(3.5% N, 4.1% P)
38 days
15.0 30.0
60.0 75.0 90.0 105.0 120.0 135.0 150.0
MINUTES
15.O 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0 150.0
Figure 9.3. Gas Chromatographic Profiles Showing the Effect of Temperature on
the Degradation of Artificially Weathered Prudhoe Bay Crude Oil.
169
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Artificial Seawater, 15°C, 16 Days
3% Inlpol
10% Inlpol
0.00 f.OO 10.00 11.00 20.00 21.00 JO.00 11.00 40.00
MINUTES
0.00 f.OO 10.00 11.00 20.00 25.00 90.00 IS.00 40.00
MINUTES
20% Inlpol
50% Inlpol
0.00 500 10.00 14.00 20.00 2f.OO 30.00 JI.OO 40.00
MINUTES
0.00 1.00 10.00 11.00 20.00 2500 M.OO li.OO 40.00
MINUTES
Figure 9.4. Gas Chromatographic Profiles Showing the Effect of Different
Concentrations of Inipol (% of Oil Concentration) on the
Degradation of Artificially Weathered Prudhoe Bay Crude Oil.
170
-------
Artificial Seawater, Poisoned, 15°C, 16 days
15.0 30.0 45.0
60.0 75.0 90.0
MINUTES
105.0 120.0 135.0 150.0
15.0 30.0 45.0 60.0 75.0 00.0 105.0 120.0 135.0 150.0
Oil & 10% Inipol
Figure 9.5. Gas Chromatographic Profile Showing the Effect of Different Fertilzers,
Under Poisoned and Unpoisoned Conditions, on the Degradation of
Artificially Weathered Prudhoe Bay Crude Oil.
171
-------
Artificial Seawater, Active, 15°C, 16 days
Oil + Soluble Fertilizer
(0.4% N, 0.08% P)
45.0 60.0 75.0 90.0
MINUTES
105.0 120.0 135.0 150.0
Oil +10% Inipol
(0.7% N, 0.06% P)
15.0 30.0 45.0 60.0
75.0 00.0 105.0 120.0 135.0 150.0
MINUTES
Oil * Inipol and Soluble Fertilizer
»-r-f-T'»~»— »"»"r
-r-»-»-r-»-r •» -»-r- r-r-T" »-T-r-|
15.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0 150.0
MINUTES
Figure 9.5. (Cont.)
172
-------
Artificial Seawater, 10% Inipol, 38 days
15.0 30.0 45.0 60.0 75.0 80.0 105.0 120.0 135.0150.0
MINUTES
15.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0150.0
MINUTES
15.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0 135.0150.0
MINUTES
Figure 9.6. Gas Chromatographic Profiles Showing the Effect of Temperature on the
Degradation of Artificially Weathered Prudhoe Bay Crude Oil Treated
with Inipol.
173
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Oiled Beach Material, Artificial Seawater, 15°C, 16 Days
No Nutrients
(O.OSg Oil)
15.0 30.0 46.0 60.0
75.0 90.0
MINUTES
10S.O 120.0 136.0 160.0
Inlpol, Poisoned
(O.OSg Oil)
16.0 30.0 46.0 60.0
76.0 90.0
MINUTES
106.0 120.0 136.0 160.0
Inipol
(0.025Q Oil)
16.0 30.0 46.0 60.0 76.0 90.0 106.0 120.0 138.0 160.0
MINUTES
Figure 9.7. Gas Chromatographic Profiles Showing the Effect of Inipol, Under
Poisoned and Unpoisoned Conditions, on the Degradation of Oil on
Beach Material Taken from Prince William Sound.
174
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RESPIROMBTRIC ANALYSIS OP BIODEGRADATION
Background
To obtain additional information on the effect of Inipol for
enhancing, under very controlled conditions, the degradation of
different concentrations of artificially weathered oil,
laboratory flask studies were conducted. These studies were
designed to evaluate the inherent ability of the indigenous
Prince William Sound microflora to degrade weathered Prudhoe Bay
crude oil. Water from the Alyeska ballast water treatment plant
was also evaluated as a source of oil-degrading microorganisms to
enhance the natural microbiota for biodegrading the oil.
Analytical respirometry was used as the primary tool for studying
rates of biodegradation of the oil. To corroborate the oxygen
uptake measurements collected in the respirometric reactors,
GC/FID chromatography scans of aliphatic and aromatic
hydrocarbons were performed at various times on samples taken
from batch flasks. This was done to determine which oil
constituents were being biodegraded in the closed systems.
Methods
Nutrient Media—
Two nutrient formulations, Inipol and a defined minimal-
salts medium (OECD), were compared for their ability to support
the growth of hydrocarbon degraders on weathered Prudhoe Bay
crude oil.
Microbial Inocula—
The microbial inocula consisted of seawater from Snug
Harbor, beach material collected from an uncontaminated beach in
Valdez, weathered crude oil from the spill, and indigenous biota
from the Alyeska ballast water treatment plant.
Chemical Analyses—
The oil was fractionated into the aliphatic, aromatic, and
polar fractions using standard silica gel column chromatography.
Composition of the aliphatic fraction was measured by gas
chromatography using flame ionization detection (GC/FID).
Composition of the aromatic fraction was characterized by gas
chromatography/mass spectrometry (GC/MS). Samples were collected
at 0 weeks, 6 weeks, and 26 weeks.
Analytical Respirometry—
Respirometry experiments were carried out in a Voith
Sapromat B-12 respirometer. This instrument consisted of a
temperature controlled water bath containing 12 measuring units,
a recorder for direct plotting of the decomposition velocity
curves, and a cooling unit for conditioning and continuous
recirculation of water bath volume. Each measuring unit
comprised a reaction vessel with a C02 adsorber, an oxygen
175
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generator, and a pressure indicator. Microbial activity created
a vacuum in the reaction vessel, which was recorded by the
pressure indicator. Pressure was balanced by electrolytic oxygen
generation from the dissociation of copper sulfate and sulfuric
acid. The recorder/plotter constructed an oxygen uptake graph
automatically.
Design of the respirometry experiments is summarized in
(Table 9.6). All vessels contained 2 grams of uncontaminated
beach sand from Valdez and 1000 ml of seawater collected offshore
at Snug Harbor. The vessels containing beach material, oil, and
Inipol were charged by first adding the beach material, pouring a
measured amount of oil onto the sand, adding the Inipol to the
oiled rocks, and finally filling the vessel with the Snug Harbor
seawater. All reaction vessels were mixed with stirring turbines
and incubated at 15"c in the dark.
Flask Studies-
Flask microcosm experiments were conducted to provide further
support for the respirometric studies. Each flask contained
20 gm of uncontaminated beach material and 1000 ml of Snug Harbor
seawater. The flasks were charged with the various additives in
the same fashion and order as above. The experimental design for
these experiments is summarized in (Table 9.7). Flasks were
incubated on a shaker at 15°C.
Results
Analytical Respirometry—
Results of the analytical respirometry experiments are
summarized in Figure 9.8. The figure displays cumulative oxygen
uptake as a function of time in the respirometric vessel
containing 1000 mg/1 oil and Inipol (5% by weight of oil) and in
the respirometric vessel containing only Inipol. Oxygen uptake
began in both vessels after only 1.5 days lag period. Maximum
uptake of Inipol occurred by the 9th day, then leveled off at
approximately 150 mg/1. The oxygen uptake rate on weathered oil
with Inipol added was multi-phasic: the first 10 days exhibited
the highest uptake rate, followed by a slower rate for the next
.16 days, a somewhat faster rate for the next 4 days and a much
slower rate after the 30th day. Endogenous oxygen uptake (vessel
with no Inipol or oil) was always close to background (data not
shown).
The vessel containing oil, Inipol, and the Alyeska ballast
water biomass exhibited an oxygen uptake curve that was almost
superimposable on the curve for oil plus Inipol (data not shown)
Thus, in the closed environment of the respirometric vessel, no
176
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Table 9.6. Experimental Design for Respirometric Studies
Reaction Oil Inipol Alyeska
Vesselb Concentration Concentration Ballast W<
(mg/1) _ (ma/11 (ml)
V1,V1R 1000 50
V2,V2R 300 15
V3,V3R 100 5
V4,V4R 1000 50 10
C5 - 50 -
C6 -
C7 1000 50
C8 - 50 -
b V = Vessel
R = Replicate
C = Control
177
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Table 9.7. Experimental Design of Flask Studies.
Flask0
Oil Inipol
Concentration Concentration
(mg/1) (mg/1)
OECD0
Alyeska
Ballast
(ml)
F1,F1R
F2,F2R
F3,F3R
F4,F4R
Cl
C2
10,000 500
10,000 500
10,000
10,000
10,000
10,000
— —
10
+
+ 10
- -
10
e F
R
Flask
Replicate
C = Control
d OECD, a defined minimal-salts medium was composed of the
following constituents added to provide the specified final
concentration (mg/1) in the test solution: KH2P04 (170),
(435), Na2HP04 (668), NH4C1 (50), MgS04.6H?0 (45), CaCl2 (55),
FeCls.6H20 (2.5). It included the following trace elements added
to provide final concentrations (Mg/1) in the test solution:
MnS04 (60.4), H3B03 (114.4), ZnSO4.7HjO (85.6), (NH4)6MO7O34 (69.4),
and FeCls EOT A (200) . To prevent trace nutrient limitation,
either 1 ml/1 of a stock yeast extract solution (15 mg/100 ml),
or the following vitamins, biotin (0.4), nicotinic acid (4.0),
thiamine (4.0), p-aminobenzoic acid (2.0), pantothenic acid
(2.0), pyridoxamine (10.0), cyanocobalamine (4.0), and folic acid
(10.0) .
178
-------
1600
1400
1200
1000
•S 800
o.
0)
O) 600
X
O
400
200
10
20
Oil + Inipol -f Seawater
Inipol
Oil + Inipol Without Seawater
JL
30 40
Time (Days)
50
60
70
Figure 9.8. Cumulative Oxygen Uptake on Weathered Prudhoe Bay Crude Oil
-------
enhancement of oil degradation by an external source enriched
with oil-degrading organisms was detected.
Flask Studies—
Figure 9.9 summarizes the results of GC/FID scans of the
alkane hydrocarbons from three sets of flasks: control
(containing 10,000 mg/1 weathered crude oil and no nutrients)
Inipol-treated (containing (10,000, mg/1) oil and (500 mg/1)
Inipol, and defined minimal salts-treated (containing 10,000 mg/1
oil and OECD). In the control, some minor changes in the alkane
fractions are evident after 6 weeks incubation. Some of these
changes may have been due to biodegradation resulting from
background levels of N and P present in the seawater, oil, or
beach material; adsorption to the flask walls; sampling error; or
a combination of the above. Whatever was the cause, the
magnitude of the changes was relatively insignificant.
Flasks containing oil plus Inipol exhibited complete removal
of all aliphatic components within six weeks. Even the pristane
and phytane fractions were reduced to undetectable levels. The
flask containing the minimal-salts solution also exhibited
complete removal of the straight chain aliphatics. However,
there were still measurable amounts of pristane and phytane
remaining at six weeks, although the levels were significantly
reduced from the controls. These results suggest that Inipol may
have enriched a different type of microbial population than that
enriched by the minimal-salts solution. The Inipol-enriched
organisms were able to break down not only straight chain
components at a very rapid rate but branched-chain components as
well. The organisms enriched by the minimal-salt solution were
also able to degrade the branched chain aliphatics, but at a
reduced rate or after a longer lag period.
The GC/MS traces of the aromatic fractions are presented in
Figure 9.10. In the control, several of the components were
reduced to undetectable levels after six weeks (note fractions H,
Q, S, and T, corresponding respectively to dibenzothiophene, C3-
fluorenes, naphthylene, and Cl-naphthylene). The traces from the
Inipol and minimal-salt solution flasks exhibited virtually
complete removal of all aromatic fractions after six weeks
incubation.
Discussion and Conclusions
Results indicate rapid and virtually complete biodegradation
of all aliphatic and aromatic components of the weathered oil
contaminating Alaskan beaches occurred in nutrient enriched
respirometer vessels and flasks. Oxygen uptake started after
only a 1.5-day lag period and disappearance of aliphatic and
aromatic components occurred within 6 weeks. Different microbial
populations appear to have been enriched by the two types of
nutrient solutions (Inipol and a minimal-salt solution). This
180
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0 weeks
6 weeks
16UUU
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C9 10 11 12 13 14 15 16 17 PR 18 PH 19 20 21 22 23 24 25 26 28 30 32 34 36 38
Numbers of Carbons
Figure 9.9. Gas Chromatographic Profiles of Alkanes at 0 and 6 Weeks
After Initiation of Flask Studies.
181
-------
0 weeks
6 weeks
1OUU
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A. Acenaphthene
B. Acenaphthylene
C. Benzo(a)pyrene
D. Benzo(b)fluoranthene
E. Benzo(g,h,l)perylene
F. Chrysene/Benzo(a)anthracene
G. C1-Chrysenes
H. Dlbenzothlophene
I. C1-Dlbenzothlophenes
J. C2-Dlbenzothlophenes
K. C3-Dibenzothlophenes
L Fluoranthene
M. C1-Fluoroanthenes/Pyrenes
N. Fluorene
0. C1-Fluorenes
P. C2-Fluorenes
Q. C3-Fluorenes
R. lndeno(1,2,3-cd)pyrene
S. Naphthalene
T. C1-Naphthalenes
U. C2-Naphthalenes
V. 03-Naphthalenes
W. C4-Naphthalenes
X. Phenanthrene/Anthracene
Y. C1-Phenanthrenes/Anthracenes
Z. C2-Phenanthrenes/Anthracenes
1. C3-Phenanthrenes/Anthracenes
2. C4-Phenanthrenes/Anthracenes
3. Pyrene
ABCDEFGHIJKLMNOPORSTUVWXYZ123
Aromatics
Figure 9.10.
Gas Chromatographic Profiles of Aromatics at 0 and 6 Weeks
After Initiation of Flask Studies.
182
-------
suggests that perhaps a combination of Inipol and a water-soluble
source of nutrients may ultimately be the appropriate manner of
stimulating rapid bioremediation of crude oil contaminating
Alaskan beaches. Results from ballast water biomass enrichments
suggest that external sources of microbial populations would not
enhance biodegradation, and massive inoculations may not be
warranted, at least in the Alaskan bioremediation effort. The
respirometric data will eventually be quantitatively analyzed to
calculate the kinetics of oil biodegradation.
MECHANISM OF ACTION OF INIPOL-ENHANCED OIL DEGRADATION
Background
A laboratory study was conducted to investigate the
mechanism by which the Inipol fertilizer enhanced oil
degradation. Numbers of oil-degrading microorganisms and oleic
acid-degrading microorganisms were specifically examined along
with changes in oil composition. The study was performed in a
manner which would, to some extent, simulate environmental
conditions; i.e., no shaking and daily water change to simulate
tidal flushing. Results are currently available for oil-
degrading and oleic acid-degrading microbial populations.
Methods
The experimental design is shown in Table 9.8. Studies were
conducted in chemically clean (I-Chem) jars, each containing
approximately 200 g of oiled rocks and either seawater, defined
nutrient medium, or sodium chloride solution (20%). The defined
nutrient medium (DNM) used in these tests contained (per liter of
distilled water): NaCL (24g) MgS04.7H20 (1.0 g) KCL (0.7 g) KH2P04
(2.0 g). Na2HP04 (3.0 g), and NH4NOS (1.0 g). The pH of the
medium was adjusted to 7.4 with 1.0 N NaOH following autoclaving.
For sterile systems, the oil-contaminated rocks were autoclaved
in I-Chem jars. This removed the water from the oil, but did not
remove the oil from the rocks. Inipol application consisted of
dripping 3 ml of Inipol (sterile) over the rock surface and
allowing the treated rocks to incubate for 3 hours before filling
the jars with the appropriate aqueous phase (about 100 ml).
Except for the jar containing unautoclaved seawater, sterile
medium (seawater, defined nutrient medium, or NaCl solution) was
used in each microcosm. Subsamples of 1.0 ml for bacterial
enumeration were collected from all jars at 24-hour intervals.
Oleic acid-degrading bacteria were enumerated on oleic acid-
containing agar plates supplemented with nitrogen and
phosphorous. Oil-degrading bacteria were enumerated by the
dilution to extinction technique described in Section 7. After
collecting bacterial enumeration samples, the aqueous phase from
one set of jars was decanted into a sterile I-Chem jar and
183
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Table 9.8. Experimental Design for Laboratory Microcosm Study
Flask
Seawater
Artificial SeawaterSterile
NaCl
Inipol added
Seawater
Artificial SeawaterNonsterile
NaCl
Seawater
Artificial SeawaterSterile
NaCl
No Inipol added
Seawater
Artificial SeawaterNonsterile
NaCl
184
-------
replaced with fresh sterile medium (fresh seawater was added to
the nonsterile seawater jar). The decanted solution was frozen
for analysis of residual oil components.
Results
The results from these studies indicated that the addition
of Inipol led to a substantial increase in the number of
organisms capable of growth on oleic acid-agar plates (Figure
9.11). High background concentrations of oleic acid-degrading
bacteria were observed in the water even before Inipol treatment.
Since the aqueous phase at each water change was sterilized,
the number of oleic acid degraders possibly reflected those that
sloughed off the oiled rocks during a 24 hour period. However,
no obvious differences were observed for the different aqueous
phases. Similar results were observed in systems that did not
have daily water changes.
Results from the enumeration of oil-degrading organisms
(Figure 9.12.) indicated that in all cases the populations
increased to a high value by day 3 and then decreased to an
intermediate but variable level for the following 6 days.
Similar results were seen in those jars that did not have a daily
water change. Although all the samples showed a peak after 3
days of incubation, jars containing only seawater appeared to
have the fewest microorganisms in the 6 days following the 3-day
peak. Chemical analysis of the water samples is being preformed.
Information on how effectively the enriched oleic acid degraders
can degrade the oil also is forthcoming.
Discussion and Conclusions
Inipol increased the number of oleic acid-degrading bacteria
in flask studies designed to approximate field conditions. This
situation would theoretically result in competition for available
nutrients between oleic acid-degrading and oil-degrading
bacteria. This competition could explain the decrease in oil-
degrading bacteria following their initial rise after initiation
of the experiment. Tests of oleic acid-degrading bacteria are
currently being conducted to determine the percentage which are
also hydrocarbon degraders. Supplying dissolved nutrients in
addition to those nutrients in Inipol did not seem to affect the
oleic acid- and oil-degrading bacterial populations.
185
-------
G Control
S Inipol-Treated
•s.
1 -
Defined 1 7
Nutrient « -
Medium % j
o ;
CO
CD
i
(U
CD
— t
LoglO Bacteria Colony Count
a — N u K u> a -i •
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l l 1 1 1 1 1 l 1 1 1 1 1 i 1
8-
s ;
7-
1 r
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Solution „ I-
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—
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Days
Figure 9.11. Effect of Inipol on the relative numbers of oleic acid-degrading bacteria in jars containing
oiled rocks and seawater, defined nutrient medium, or saline solution. Incubated with
daily change of water.
186
-------
10 —
Seawater | s"
0 .
__ 1—
—
i
I
1
n Control
^ ^ Inipol-Treatec
—
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Days
Figure 9.12. Effect of Inipol on the relative numbers of oil-degrading microorganisms in jars containing
oiled rocks and seawater, defined nutrient medium, or saline solution. Incubated with daily
change of water.
187
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CHEMICAL EFFECT OF OLEOPHILIC FERTILIZER6
Background
The mechanism by which oil is removed from substrates is
important for interpreting the results of biodegradation studies.
Since several substances in the Inipol fertilizer formulation are
known to act as surfactants or to otherwise change the
consistency of oil on rock surfaces, the question arose as to
whether or not Inipol acted to alter the physical characteristics
of the oil such that removal of oil from rock surfaces could
occur in the absence of biodegradation. This study was designed
to evaluate the rock-washing characteristics of Inipol under
conditions that precluded biological activity.
Methods
The test system was designed to address the efficacy of
various chemicals as potential "rock-washers". Each chemical was
applied to fully oiled gravel, and the gravel was then
refrigerated at 5*C for 1 hour. Artificial sea water at 5°C was
then added to cover the gravel, and the gravel refrigerated at
5*C for 6 hours. The gravel was then drained, and the amount of
oil in the water was estimated. A typical test used four washing
cycles.
Results
Inipol EAP 22 removed only 0.6% of the oil in a first test
at a normal application rate of 5% by weight of oil, and 0.84% of
the oil in a test where the Inipol was added to ensure complete
surface coverage. Both tests indicated an insignificant amount
of oil was removed (more than 30% of the oil was removed by some
preparations sold specifically for this purpose). Approximately
45% of the Inipol, by weight, remained with the oiled rock in the
regime used in these, which were designed to remove as much of
the rock-washing material as possible. Similarly, about 50% of
the available nitrogen, (in the Inipol) was released in the first
2 wash cycles, with the remainder being released more slowly.
Discussion and Conclusion
Inipol EAP 22 does not wash oil off rocks at typical Prince
William Sound water temperatures. Based on the results of this
study, it is reasonable to expect that oil removal associated
with Inipol EAP 22 applications is the result of something other
than a physical process. In addition, associated tests by Exxon
* These tests were conducted by Exxon Researchers at
laboratories in Housten and New Jersey.
188
-------
demonstrated that Inipol enhanced biodegradation was not
accompanied by a detectable lowering of interfacial tension
between oil and brine. This suggests that the microorganisms did
not produce significant amounts of surfactants under the
conditions tested.
TOXICITY OF OLEOPHILIC FERTILIZER
Background
Little information is available in the literature regarding
the toxic effects of Inipol on sensitive marine biota. Studies
were designed in response to requests for information regarding
the possible toxic effects of oleophilic fertilizer on indigenous
biota of the Prince William Sound. However, several marine
species commonly used in toxicity testing are known to be more
sensitive than species indigenous to the Prince William Sound.
Therefore, the toxicity testing conducted included both species
that are commonly used in toxicity testing and species from the
Prince William Sound.
Methods
Toxicity of Inipol EAP 22 and weathered oil were tested in 3
ways:
1) To account for worst-case conditions, the Inipol EAP 22 was
tested in a mixture with seawater.
2) Because Inipol EAP 22 is very likely to become bound to oil
after application to an oil-contaminated shoreline, and
because data generated by the manufacturer show that
toxicity of the fertilizer is appreciably decreased in the
presence of oil, a second treatment involved spraying
fertilizer on a layer of oil on seawater.
3) Finally, the oil was tested alone to provide data for
comparisons.
Toxicity tests were conducted by Battelle and E.V.S.
Consultants under contract to the US EPA for the development of
definitive, acute LC50 values for fishes, invertebrates, and
algae (Table 9.9). Organisms tested by Battelle included silver
salmon smolts, herring fry, and mussel larvae. The oleophilic
fertilizer was tested both alone and in seawater plus weathered
Prudhoe Bay crude oil. E.V.S. Consultants conducted similar
tests with an alga, oyster larvae, mysids, grass shrimp, and
sticklebacks, and a sperm cell fertilization test with sand
dollars. Final test results are not yet available at this time
for all test species.
189
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Table 9.9. Results of Laboratory Toxicity Tests with Oleophilic
Fertilizer, Inipol EAP 22, and Various Marine Species. (Values
are 96-hour LC50 estimates unless otherwise noted.)
Organism
Fish
Salmon smolts
Herring
Sticklebacks
Invertebrates
Mussel larvae
Oyster larvae
Mysids
Pandalid shrimp
Algae
Skeletonema
Inipol
2,500 ppm*
200 ppm
100 ppm*
35 ppm (48hr)
>10ppm <100ppm (48hr)
range-finder <100ppm
400 ppm*
Inipol Plus Oil
6,700 ppm
800 ppm*
range-finder underway
70 ppm (48hr)
range-finder underway
range-finder underway
range-finder underway
range-finder underway range-finder underway
* Best estimate from non definitive test.
190
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Results
General trends show that larvae of mussels, oysters, and
juvenile mysids are two orders of magnitude more sensitive than
salmon and approximately one order of magnitude more sensitive
than herring and sticklebacks. When mixed with oil, the toxicity
of Inipol is reduced two- to four-fold.
Discussion and Conclusions
These data were provided to the Shoreline Committee and to
advisory groups in Valdez, Seward, and Homer to assist in the
evaluation of potential toxic effects associated with large-scale
application of Inipol as a clean-up technique. In addition, a
risk assessment procedure was suggested as a means to establish a
benchmark that identifies the concentration where no acute
effects are observed. Such a benchmark would be useful for
comparison with possible environmental concentrations following
shoreline treatment. This method was modified by the Shoreline
Committee in Valdez and Homer and used to assist in approval of
shoreline segments for fertilizer application.
Toxicity of Inipol to marine biota was thought to be
possible as a result of unintentional over-spraying of marine
waters during application, or release of Inipol from the
shoreline into the bay immediately after application. Analysis
of a worst-case example considered the effects of elevated levels
of Inipol in protected embayments with minimal tidal exchange and
maximum shoreline-to-water volume ratios (long, narrow bays with
constricted openings). The standard application rate for Inipol
applied to oiled shorelines was 293 g/m (0.06 Ib/sg ft) in the
bioremediation program. Assuming this rate was applied to 100 m
of shoreline on a 10 m swath marked from the low-tide line to the
upper storm berm, a total of 293,000 g would be used. If all of
the Inipol was washed in a pulse into completely mixed nearshore
water that was 100 m long, 10 m wide, and had an average depth of
1 m (1000 ms), the "worst-case" expected environmental
concentration would be 293 ppm. This value is considerably less
than the 96 hr LC50 value for salmon and is comparable to the
LC50 for herring. Toxicity to marine invertebrates residing in
the area next to shore is possible at these concentrations,
should unrealistic application conditions exist. Any marine
invertebrate exposure that resulted from shoreline applications
would be mitigated by tidal mixing, dilution, and transport out
of the system into the Prince William Sound. Initial
concentrations should decrease by orders of magnitude within one
to two days, to levels considerably less than acutely toxic
concentration measured in laboratory tests. Thus, it is
predicted that the prospect of sustained lethal concentrations
for any biota is very unlikely.
191
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There are no proven analytical methods to quantify Inipol in
seawater, so environmental concentrations of Inipol could not be
measured to compare with worst-case predictions. However, daily
input into nearshore waters was estimated to be in the range of
1% to 10% of the applied material, based on 1) visual
observations of a colored film present after spraying,
2) sustained nutrient enriched pore water observed in the
intertidal zone following nutrient additions, and 3) the lack of
measured nutrient increases in the nearshore zone. If this
estimated input (10%) was diluted with a nearshore (10 m) volume
of water averaging 2 m (a depth consistent with the steep slope
of most shorelines in Prince William Sound), the estimated
environmental concentration would be between 3 and 30 ppm. These
values indicate that peak environmental concentrations would be
less than the laboratory LC50 values of Inipol and oil mixtures
for invertebrates. Peak values would develop immediately after
application, and would be subjected to subsequent tidal mixing,
dilution, and transport. When considered in this light, the
potential for toxic effects of Inipol applied to oiled beaches at
recommended rates appears to be minimal.
Inipol and oil mixtures that may leave the treated shoreline
should have minimal ecological impact based on their propensity
to degrade and the dilution potential of surrounding waters.
Enhanced microbial biomass and available nutrients associated
with mixtures of Inipol and oil should result in their rapid
degradation. In their mixed form, oil and Inipol have less
toxicity than does Inipol by itself for marine biota, as
demonstrated by the reduced toxicity of Inipol in laboratory
tests where it was mixed with oil.
BEACH HYDRAULICS
Background
Numerous hydrological factors could influence the
redistribution of oil on contaminated beaches or affect the
release and distribution of nutrients applied as fertilizers.
Prior to the initiation of these studies, no knowledge of the
flow of water in the highly porous beaches of the Prince William
Sound was available. Beach hydrological studies were conducted
to identify the primary factors that influence the distribution
of fresh and salt water and the dynamics of aqueous flow in these
beaches.
Methods
Hydrological evaluation of Kittiwake Beach at Passage Cove,
Knight Island was implemented through installation of sample
wells, instrument packages, a tide gauge and a weather station.
Concurrently, Kittiwake Beach was used to test the efficacy of
nutrient application via a sprayer using water-soluble
192
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fertilizer. The orientation of wells installed on the beach and
a diagram of the instrument packages installed in the wells are
shown in Figures 9.13 and 9.14, respectively.
Nutrient samples were collected every two weeks between
August 6 and September 12, 1989. Samples were collected using
peristaltic pumps to withdraw water from each of three small
tubes placed alongside the major well casing. At each sample
location these sample tubes extended to specific depths: two
feet below the beach surface, one foot above the bottom of the
well, and the bottom of the well, respectively. Clean 250 ml
polyethylene bottles were filled with water and frozen as soon as
possible after collection. For each sampling period, samples
were collected every three hours over two tidal cycles. It was
not feasible to collect samples over 24 hour cycles due to
weather conditions and the hours of darkness. Salinity and
temperature data were collected in the field concurrently with
nutrient sample collection. Samples were analyzed for ammonium,
nitrite, nitrate, and phosphorous.
Due to the vertical changes in sea level over a tidal cycle,
often a complete series of nutrient samples could not be
obtained. Survey samples were collected in groups. Groups 1
through 7 were collected September 10 and 11, 1989. Sample sets
were collected at about three hour intervals, unless otherwise
indicated.
Results
Tables 9.10 through 9.34 show the salinity, temperature,
ammonium, nitrate, and phosphorous data from sample groups
1 through 25 respectively. The data indicate rapid changes in
salinity and nutrient content in each series of samples over
tidal cycles. The presence of nitrate in many samples indicates
that anaerobiosis was not particularly evident during the sample
period.
Samples taken August 6 and 7 (Tables 9.10 through 9.16) were
collected two days after fertilization had begun. The ammonium
data indicated that fertilizer had penetrated from the surface of
the beach to the bottom of the wells. The ammonium concentration
in interstitial water reached a maximum of 179 -M in the center
of the plot. Salinities were also at a minimum in the same
samples series. The salinity data indicated that the subsurface
water flow was very complex and not easily described. No nitrate
data were available for this series at the time of writing.
Samples taken August 20 and 21 (Tables 9.17 through 9.26)
were collected after a two day hiatus in fertilizer application.
The data showed that ammonium and nitrate persisted in the body
of the beach even after cessation of fertilizer application. The
ammonium data usually showed less than 100 -M concentration.
193
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mean low tide
Figure 9.13. Location of Wells for Beach Hydraulics Experiment at Passage Cove
194
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Strap for removal
Aandura sensor for <
conductivity and temperature"
Tygon tubing for
sample collection
3 PVC tubes attached to inside of
casing extending to precise locations
Beach Surface
Levels given
Band of screen
excluding solids
Sensor package rests firmly
on the bottom screen.
Aandura sensor for pressure,
conductivity, and temperature
6" diameter
Casing capped with
a screw-on cap
8" diameter
Holes or slots in
band encircling pipe
Screen
Figure 9.14. Casing Configuration
195
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Table 9.10. Passage Cove Beach Hydraulics:
4:30 a.m.; High Tide
August 6, 1989;
Pond
Station Number
Salinity (MMho)
Temperature ("C)
NH4 (MM)
N03 (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Tempe r a tur e ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (*C)
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
7
T M
- 15
- 14
- 108
— —
- 0.9
6
T M
0 0
14 14
111 114
- -
3.1 3.7
B
15
14
110
—
1.7
B
0
14
90
-
4.0
T
-
-
-
-
*
T
11
15
81
—
2.2
T
_4_
400
13
1.1
0.2
3
M
11
14
45
-
0.6
2
M
11
15
71
—
1.3
1
M
B
12
13
41
-
0.9
B
13
14
179
—
6.1
B
10
T M
- 16
- 13
- 54
— —
- 0.5
9
T M
0 0
14 14
70 64
— —
0.9 0.7
B
16
13
97
—
0.9
B
0
15
53
—
2.3
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature (*C)
NH4
N03
P04
* T
M
B
(MM)
(MM)
(MM)
= Top - 2
= Middle -
= Bottom -
feet below beach surface
1 foot above bottom of well
bottom of well
196
-------
Table 9.11. Passage Cove Beach Hydraulics: August 6, 1989;
7:30 a.m.; Falling Tide
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( e C )
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ("C)
NH4 (MM)
N03 (MM)
P04 (MM)
7
T M B
9
- 15
- 43
— — —
- 1.1
6
T M B
17 16 16
15 15 15
157 130 151
- - -
4.3 3.7 5.1
340
14
0.8
0.4
3
T M
- -
- -
- -
— —
^ —
2
T M
- 10
- 15
- 57
- -
- 2.3
1
T M
16 16
15 15
33 47
- -
0.9 0.8
B
10
15
29
—
0.3
B
17
15
33
—
1.8
B
14
15
44
-
1.1
10
T M B
_ _ _
- - -
- - —
— — —
^ •» ^
9
T M B
17 17 16
14 14 15
89 93 109
— — —
3.7 2.5 4.1
Offshore
Salinity (MMho)
Temperature (° C)
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
197
-------
Table 9.12. Passage Cove Beach Hydraulics: August 6, 1989;
10:00 a.m.; Low Tide
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
PO4 (MM) 0.2
High Tide Wells
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
7 3 10
TMB TMB TMB
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
629
TMB TMB TMB
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) - 15 14
Temperature (°C) 16 16
NH4 (MM) - 46 46
N03 (MM) - - -
P04 (MM) - 0.9 0.6
Offshore
Salinity (MMho) 13
Temperature (°C) 17
NH4 (MM) 1.2
N03 (MM)
P04 (MM) 0.3
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
198
-------
Table 9.13. Passage Cove Beach Hydraulics:
1:00 p.m.; Rising Tide
August 6, 1989;
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( • C )
NH4 (MM)
NOS (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
7
T M
- 16
- 15
- 48
- -
- 1.2
6
T M
19 15
16 16
83 73
- -
8.5 2.9
* T = Top - 2 feet below beach
M = Middle - 1 foot
B = Bottom - bottom
350
16
35
0.3
3
B T M
15 - 12
15 - 16
52 - 22
— — —
1.6 - 0.7
2
B T M
15 18 14
18 17 17
21 5.3 34
- — -
4.0 1.8 1.3
1
T M
- 15
- 16
- 36
- -
- 1.1
surface
B
12
16
27
—
0.9
B
16
17
38
—
1.3
B
15
17
32
-
0.9
10
T M B
- 15 15
- 16 16
- 7.6 7.1
_ _ _
- 0.9 1.0
9
T M B
12 12 12
18 17 18
52 34 38
— — —
2.2 1.2 1.6
above bottom of well
of well
199
-------
Table 9.14. Passage Cove Beach Hydraulics:
5:10-7:30 p.m.; High-Falling Tide
August 6, 1989;
Pond
Station Number
Salinity (MMho)
Temperature ( " C )
NH4 (MM)
N03 (/iM)
P04 (MM)
Hicrh Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( 8 C )
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
7
T M
- 17
- 15
- 40
— —
- 1.4
6
T M
16 17
15 15
45 40
— —
4.1 3.9
B
17
15
38
—
1.7
B
17
15
51
—
3.0
T
-
-
-
-
^
T
17
14
35
-
2.6
T
4
200
15
13
3.5
3
M
12
16
19
-
0.7
2
M
16
14
32
-
2.1
1
M
B
13
15
21
-
0.8
B
16
15
39
-
3.4
B
10
T M
- 17
- 15
- 17
— —
- 1.0
9
T M
16 16
14 14
31 29
- -
2.1 1.9
B
17
15
-
—
1.0
B
17
14
30
-
2.0
Salinity (MMho)
Temperature (•C)
(MM)
(MM)
NH4
N03
P04 (MM)
Offshore
Time
Salinity (MMho)
Temperature ( • C )
NH4 (MM)
N03 (MM)
P04 (MM)
5:10
20
15
0.06
0.6
7:30
14
13
3.5
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
200
-------
Table 9.15. Passage Cove Beach Hydraulics: August 6, 1989;
9:00 p.m.; Low Tide
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
7 3 10
TMB TMB TMB
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
6
T M
- 18
- 15
- 64
— —
" "
B
17
15
66
—
"
2
T M
- 17
- 15
- 35
— —
" "
B
18
14
32
—
"
9
T M
- 18
- 14
- 43
— —
"
B
18
14
43
—
"
Low Tide Wells
Station Number l
Sample Position* TMB
Salinity (MMho) 18 17 17
Temperature (°C) -
NH4 (MM) 15 23 23
N03 (MM) - - -
P04 (MM) -
Offshore
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
201
-------
Table 9.16. Passage Cove Beach Hydraulics: August 7, 1989;
6:00 a.m.; High Tide
Station Number _ 4_
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number _ 7 _ 3 _ _ ifi _
Sample Position* TMB TMB TMB
Salinity (MMho) - 15 15 - 12 13 - 15 16
Temperature ('C) - 13 13 - 13 13 - 13 13
NH4 (MM) - 28 51 - 13 10 - 3.8 6.9
N03 (MM) ..... - ---
P04 (MM) ... -_- _..
Mid Tide Wells
Station Number _ 6 _ 2 _ 9
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature ("C)
NH4 (MM)
NOS (MM)
P04 (MM)
Low Tide Wells
Station Number l
Sample Position* TMB
Salinity (MMho)
Temperature (° C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature (°C)
NH4 (MM) 8.5
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
202
-------
Table 9.17. Passage Cove Beach Hydraulics:
7:20 a.m.; Falling Tide
August 20, 1989;
Pond
Station Number
Salinity (MMho)
Temperature ( e C )
NH4 (MM)
N03 (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ('C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature ( • C )
NH4 (MM)
N03 (MM)
P04 (MM)
4
320
13
<0.13
<0.04
7 3
T M B T M B
- 11 6
- 14 - 14
- - 24 - 19
- 214 - - 210
6 2
T M B T M B
12 12 12 - 10 12
14 14 14 - 14 14
4 43 46 - 38 33
216 219 224 - 200 230
1
T M B
13 13 13
14 14 14
104 42 40
299 260 221
17
13
1.4
10.8
10
T M B
2
- - 14
- 0.4
- 16
9
T M B
989
14 14 14
24 24 25
115 105 73
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom
of well
203
-------
Table 9.18. Passage Cove Beach Hydraulics: August 20, 1989;
10:00 a.m.; Low Tide
Station Number
Salinity (MMho)
Temperature (* C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (•C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho) - 11
Temperature (°C) 15
NH4 (MM) - - 243
N03 (MM) - - 227
P04 (MM) -
Low Tide Wells
Station Number l
Sample Position* TMB
Salinity (MMho) - 12
Temperature ("C) 15
NH4 (MM) - 64
N03 (MM) - - 282
P04 (MM) - - -
Offshore
Salinity (MMho) 15
Temperature ("C) 14
NH4 (MM) <0.1
NO3 (MM) 0.4
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
204
-------
Table 9.19. Passage Cove Beach Hydraulics:
12:50 p.m.; Rising Tide
August 20, 1989;
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
<0.04
High Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (* C)
NH4
NO,
(MM)
(MM)
P04 (MM)
M
B
M
10
B
M
B
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
6
TMB
- 11 13
- 16 17
- 40 50
- 212 291
^ ^ ^
2
TMB
8
- 16
- 37
- 226
*"* ^m ^
9
TMB
7 9
- 16 18
- 396 48
- 159 145
^ •» ^"
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (e C)
NH4 (MM)
N03 (MM)
P04 (MM)
TMB
16 12 12
15 16 17
24 41 43
82 240 256
Offshore
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
21
18
<0.1
<0.04
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
205
-------
Table 9.20. Passage Cove Beach Hydraulics: August 20, 1989;
4:15 p.m.; High Tide
Pond
Station Number
Salinity (MMho)
Temperature (° C)
NH4 (MM)
N03 (MM) <0.04
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho) 21 19 20 12 8 9 19 19 19
Temperature (°C) 17 18 20 15 15 15 16 16 17
NH4 (MM) - 2 3 25 28 19 <0.1<0.1<0.1
N03 (MM) 8 9 7 74 166 54 25 13 9
P04 (MM) --- ... __-
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
629
TMB TMB TMB
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho)
Temperature (° C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho) 21
Temperature (*C) 15
NH4 (MM) 4.8
N03 (MM) 4.9
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
206
-------
Table 9.21. Passage Cove Beach Hydraulics: August 20, 1989;
9:15 p.m.; Falling Tide
Pond
Station Number
Salinity (MMho)
Temperature ( e C )
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (*C)
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C)
NH4 (MM)
NOS (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature (*C)
NH4 (MM)
N03 (MM)
P04 (MM)
4
5
14
<0.04
7 3
T M B T M B
6 8 -55
- 14 14 - 14 14
- 119 156 - 253 286
6 2
T M B T M B
16 16 16 13 14 18
15 15 15 15 14 14
176 211 216 172 169 151
1
T M B
21
15
20
10
T M B
- - 4
- 14 14
- 27 32
9
T M B
13 14 14
14 14 14
144 161 161
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
207
-------
Table 9.22. Passage Cove Beach Hydraulics: August 21, 1989;
6:30 a.m.; Falling Tide
Pond
Station Number
Salinity (MMho)
Temperature ( * C )
NH4 (/iM)
NO, (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C)
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
7
T M
8
- 13
- -
- 65
•• ^m
6
T M
17 17
14 14
- -
176 189
^ ^
B
10
13
-
84
*"*
B
18
14
-
172
™*
140
13
0.05
3
T M
5
- 13
- -
- 160
^ ^"
2
T M
14 15
13 13
- -
115 97
~ —
1
T M
B
5
13
-
112
^m
B
17
14
-
131
"^
B
10
T M
5
- 13
- -
- 21
^ *B
9
T M
15 15
14 14
- -
119 115
~ ^
B
8
13
-
5
^
B
15
14
-
70
™
Salinity (MMho)
Temperature (e C)
NH4 (MM)
NOS (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
21
13
12
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
208
-------
Table 9.23. Passage Cove Beach Hydraulics: August 21, 1989;
10:00 a.m.; Low Tide
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
NO, (MM) <0.04
P04 (MM)
Hioh Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho) - 15
Temperature (*C) 13
NH4 (MM) - - -
N03 (MM) - - 172
P04 (MM) - - -
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) - 16 15
Temperature ("C) 15 14
NH4 (MM) -
N03 (MM) - 237 245
P04 (MM) -
Offshore
Salinity (MMho) 22
Temperature ("C) 14
NH4 (MM)
N03 (MM) 1.6
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
209
-------
Table 9.24. Passage Cove Beach Hydraulics: August 21, 1989;
12:00 noon; Rising Tide
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM) <0.06
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (* C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho) - 15 14 - 12 9 11
Temperature ("C) - 17 17 - 16 - 15 15
NH4 (MM) --
N03 (MM) - 220 99 - - 144 148
P04 (MM) -__ _._ ...
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) 15 15 15
Temperature ('C) 16 16 16
NH4 (MM) ...
NO3 (MM) 140 92 226
P04 (MM) - - -
Offshore
Salinity (MMho) 20
Temperature (°C) 17
NH4 (MM)
N03 (MM)
PO4 (MM) <0.06
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
210
-------
Table 9.25. Passage Cove Beach Hydraulics: August 21, 1989;
3:45 p.m.; Rising Tide
Pond
Station Number
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ("C)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( • C )
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
4
500
17
0.2
7 3
T M B T M B
19 19 21 19 11 11
18 18 17 16 17 17
0.7 3.2 3.1 48 151 173
6 2
T M B T M B
1
T M B
21
17
0.5
10
T M B
19 19 20
17 17 17
1.7 0.4 5.2
9
T M B
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
211
-------
Table 9.26. Passage Cove Beach Hydraulics: August 21, 1989;
5:00 p.m.; Falling Tide
Station Number
Salinity (MMho)
Temperature (*C)
NH4 (MM)
N03 (MM) <0.04
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( • C )
NH4 (MM)
N03 (MM)
P04 (MM)
7
T M B
- 13 17
- 15 16
_ - _
- 167
v • •
3
T M
- 14
- 15
- -
- 176
"
B
14
15
-
77
^
10
T M
- 13
- 14
- -
- 40
^ ^*
B
16
15
-
26
"
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (° C)
NH4 (MM)
NOS (MM)
P04 (MM)
Low Tide Wells
Station Number l
Sample Position* TMB
Salinity (MMho) -
Temperature (°C) -
NH4 (MM) -
NO, (MM) - 75
P04 (MM) -
Offshore
Salinity (MMho) 21
Temperature (*C) 15
NH4 (MM)
N03 (MM) 6.9
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
212
-------
Table 9.27. Passage Cove Beach Hydraulics: September 10, 1989;
12:20 noon; Falling Tide
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho) 3 -1000 1 800
Temperature (eC) - 14 - 14 14 - 13
NH4 (MM) --- _-_ .__
N03 (MM) . _ - ___ -__
P04 (MM) --- _ - _ ___
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
629
TMB TMB TMB
7 14 14 4 7 12 7 9 15
14 14 13 13 14 13 14 13 14
--- ___ ___
— — — — — — ___
™ * ^ ** ^ ^" ^ ^" ^" ^
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho)
Temperature (* C)
NH4 (MM)
NO, (MM)
P04 (MM)
Offshore
Salinity (MMho) 17
Temperature (°C) 15
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
213
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Table 9.28. Passage Cove Beach Hydraulics: September 10, 1989;
3:10 p.m.; Low Tide
Pond
Station Number
Salinity (MMho)
Temperature (° C)
NH4 (MM)
N03 (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
N03 (MM)
P04 (MM)
7 3 10
TMB TMB TMB
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho) 569 -6 13 334
Temperature ('C) 14 14 14 - 15 14 15 15 14
NH4 (MM) --
N03 (MM) -_- ___ __-
P04 (MM) -__ ___ .__
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) 754
Temperature (°C) 14 14 18
NH4 (MM) - - -
N03 (MM) -
P04 (MM) -
Offshore
Salinity (MMho) 15
Temperature (°C) 14
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
214
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Table 9.29. Passage Cove Beach Hydraulics: September 10, 1989;
6:15 p.m.; Rising Tide
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho) - - 2 --3 --1
Temperature (°C) - 13 - 14 - 13
NH4 (MM) .__ __- __.
N03 (MM) - - -
P04 (MM) - - - __. ___
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( ° C )
NH4 (MM)
NOS (MM)
P04 (MM)
T
9
15
-
—
"
6
M
7
12
-
—
"
B
9
13
-
—
"
T
6
14
-
—
"
2
M
6
14
-
—
"
B
10
14
-
—
"
T
16
14
-
—
"
9
M
9
13
-
—
"
B
10
14
-
—
"
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho) 19
Temperature (°C) 14
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
215
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Table 9.30. Passage Cove Beach Hydraulics: September 10, 1989;
8:45 p.m.; Rising Tide
Pond
Station Number
Salinity (MMho)
Temperature (* C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho) -45 -22 -55
Temperature (*C) - 14 13 - 12 13 - 13 13
NH4 (MM) --- .__ -__
N03 (MM) - - - - - - - - -
P04 (MM) - - -
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (*C)
NH4 (MM)
N03 (MM)
P04 (MM)
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho)
Temperature (e C)
NH4 (MM)
N03 (MM)
P04 (MM)
Offshore
Salinity (MMho) 16
Temperature CC) 13
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - l foot above bottom of well
B = Bottom - bottom of well
216
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Table 9.31. Passage Cove Beach Hydraulics: September 11, 1989;
8:10 a.m.; Rising Tide
Pond
Station Number
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (e c)
NH4 (MM)
N03 (MM)
P04 (MM)
Mid Tide Wells
Station Number 6 2 9
Sample Position* TMB TMB TMB
Salinity (MMho) - - - __8 __6
Temperature ("C) - 15 15
NH4 (MM) -__ ___ __-
N03 (MM) - --
P04 (MM) --
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) 777
Temperature (°C) -
NH4 (MM) - - -
N03 (MM) - - -
P04 (MM) - - -
Offshore
Salinity (MMho) 15
Temperature (*C) 14
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M - Middle - 1 foot above bottom of well
B = Bottom - bottom of well
217
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Table 9.32. Passage Cove Beach Hydraulics: September 11, 1989;
11:45 a.m.; High Tide
Pond
Station Number 4
Salinity (MMho) 350
Temperature (°C) 13
NH4 (MM)
NO, (MM)
P04 (MM)
Hiah Tide Wells
Station Number 7 3
Sample Position* T M B T M B
Salinity (MMho) - 4 5 2 2
Temperature ('C) - 14 13 - 13 13
NH4 (MM) - - - - - -
NO, (MM)
P04 (MM) - - - - - -
Mid Tide Wells
Station Number 6 2
Sample Position* T M B T M B
Salinity (MMho) - 14 14 14
Temperature (°C) 14 13 13
NH4 (MM) - - - - - -
N03 (MM) - - - - - -
P04 (MM) - -
Low Tide Wells
Station Number 1
Sample Position* T M B
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
10
T M B
3 3
- 14 13
9
T M B
Offshore
Salinity (MMho) 14
Temperature (°C) 13
NH4 (MM)
NO, (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B - Bottom - bottom of well
218
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Table 9.33. Passage Cove Beach Hydraulics: September 11, 1989;
3:00 p.m.; Falling Tide
Pond
Station Number 4
Salinity (MMho) 350
Temperature (°C) 14
NH4 (MM)
N03 (MM)
P04 (MM)
Hiah Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature ( * C )
NH4 (MM)
N03 (MM)
P04 (MM)
7 3 10
TMB TMB TMB
Mid Tide Wells
Station Number
Sample Position*
Salinity (MMho)
Temperature (°C)
NH4 (MM)
N03 (MM)
P04 (MM)
T
9
14
-
-
^"
6
M
9
14
-
-
**
B
9
14
-
-
^
T
-
-
-
-
^"
2
M
8
14
-
-
**
B
10
9
-
-
^
T
7
14
-
—
"
9
M
8
14
-
—
"
B
7
13
-
—
"
Low Tide Wells
Station Number l
Sample Position* TMB
Salinity (MMho) 11 9 10
Temperature (°C) 14 14 13
NH4 (MM) -
N03 (MM) -
P04 (MM) - - -
Offshore
Salinity (MMho) 13
Temperature (°C) 14
NH4 (MM)
N03 (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
219
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Table 9.34. Passage Cove Beach Hydraulics: September 11, 1989;
6:30 p.m.; Low Tide
Pond
Station Number 4
Salinity (MMho) 300
Temperature (*C) 12
NH4 (MM)
NOS (MM)
P04 (MM)
High Tide Wells
Station Number 7 3 10
Sample Position* TMB TMB TMB
Salinity (MMho)
Temperature (•C)
NH4 (MM)
NO, (MM)
P04 (MM)
Mid Tide Wells
Station Number 6 2 9.
Sample Position* TMB TMB TMB
Salinity (MMho) - 11 13 8 9 - 8 10
Temperature (*C) - 12 12 - 14 14 - 14 12
NH4 (MM) -__ ... _-.
NO, (MM) _._ ... ___
P04 (MM) -_- ___ -_-
Low Tide Wells
Station Number 1
Sample Position* TMB
Salinity (MMho) 11 11 12
Temperature (*C) 14 14 15
NH4 (MM) - - -
NO, (MM) - - -
P04 (MM) - - -
Offshore
Salinity (MMho) 16
Temperature (*C) 14
NH4 (MM)
NO, (MM)
P04 (MM)
* T = Top - 2 feet below beach surface
M = Middle - 1 foot above bottom of well
B = Bottom - bottom of well
220
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Samples from groups 9 and 10 (Tables 9.18 and 9.19) had NH4
concentrations greater than 100 ~M. The nitrate data showed
definite high concentrations of N03 at virtually every depth
sample. The lowest concentrations were observed in the well
samples on the right side of the plot. This indicates that
wither fertilizer application was uneven or that the freshwater
flow was greater on the right side of the plot. The salinity
tended to support the conclusion that freshwater flow wasp244Xdata
greater on the right side of the plot. The salinity data tended
to support the conclusion that freshwater was more prevalent
under the right side of the plot. Similarly, the lowest nutrient
concentrations were observed when salinities were closest to
seawater. Surface samples collected offshore in water about 0.4
meters deep had very low nutrient concentrations in each case.
Nitrate never exceeded 20 -M immediately outside the plot area,
indicating very little loss of nutrients to the sea, or that
nutrient loss was thoroughly diluted by the time it reached the
sample location. The highest nutrient concentrations were
associated with salinities intermediate between seawater and
freshwater, indicating that the nutrients were partially confined
to the zone where mixing of fresh and salt water occurred. One
possible explanation is that the incoming tide pushes water into
the face of the beach, rather than flowing under and pushing
water up in the body of the beach.
Nutrient data for samples collected September 10 and 11
(Tables 9.27 through 9.34) were not available at the time of
writing. Final conclusions regarding the beach hydraulics
experiments will have to be made after reception and
consideration of the data. Tentative conclusions based on
evaluation of data from tables 9.20 through 9.26 include: 1) the
use of water soluble fertilizers seems to be effective in
distributing nutrients on beaches to support biodegradation of
crude oil, 2) nutrients persist in the body of the beach at least
two days after the last fertilizer application, and 3) fresh and
salt water flow dynamics in beaches are sufficiently complex to
require additional study. In addition, hydrologic studies of
other types of beaches may be required to describe the hydrology
of oil coated beaches in the Prince William Sound in order to
improve the results of bioremediation efforts. However
additional evidence argues that nutrient loss from the plots was
minimal.
Discussion and Conclusions
The data provided by nutrient analysis, salinity
measurements, well instruments (salinity, temperature, water
depth), the tide gauge, and the weather station will be
incorporated into a mathematical model of hydrology of a beach in
Prince William Sound. The model is likely to be very complex due
to the number of variables affecting water movement in this area.
221
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Data analysis, model development, and incorporation of data
into the model are still in progress. Detailed nutrient analysis
and the hydraulic model will be presented in the final report of
this project.
MUTAGENICITY TESTS
Background
The types of health hazards for which monitoring is most
difficult are those that have chronic, delayed effects such as
carcinogenicity, neurotoxicity, and mutagenicity. Fortunately,
for mutagenicity there are short-term in vitro tests that
demonstrate whether or not a pollutant interacts in a detrimental
manner with DNA. Due to the mechanistic research with oncogenes,
available evidence shows that oncogene activity can be initiated
by mutation. Mutation assays, although not definitive, can be
used as screening tests for the presence of potential
carcinogens, When performed in a quantitative, dose-responsive
fashion, one can use these bioassays to detect alterations in the
quantity of mutagens present within complex mixture samples. One
of the methods used to assess potential health effects associated
with this and similar spills, therefore, is the examination of
mutagenicity associated with the oil spill, the weathered oil,
and the products associated with bioremediation. The most
commonly used mutation assay is the Salmonella typhimurium /
mammalian microsome assay developed by Ames.
Experiments were initiated to determine the potential
mutagenic activity associated with biodegradation of oil. An
early pilot study had demonstrated that extracts of spilled oil
are mutagenic in the Salmonella typhimurium bioassay for
mutagenicity. This meant that the removal of genotoxic
components from the oil by biodegradation could be monitored with
this assay.
Methods
The bioassay chosen for the monitoring of these samples is
the spiral Salmonella assay as described by Houk, et. al. This
bacterial assay is a modification of the standard Salmonella
plate incorporation assay. It requires less total material,
accommodates more samples per unit time, and samples do not have
to be solvent-exchanged into dimethylsulfoxide.
Preparation for samples for this assay was accomplished by
extracting the samples using a sonication procedure and
dichloromethane (DCM). The samples are filtered through
silanized glass wool are concentrated to <100 ml using roto-
evaporation. After drying with anhydrous NaS04, all samples are
then concentrated or diluted to a concentration of 10 mg/ml (a
222
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reference point derived from preliminary testing) and stored in a
freezer at -30°C until taken for bioassay.
Results
Due to the characteristics of some complex mixture samples
(e.g., insolubility), the standard assay can sometimes be
impractical. The Alaskan oil samples are examples of mixtures
that are difficult to test in the standard assay. Due to the
physical properties of the samples, therefore, tests were done
using the spiral Salmonella assay. Both the Prudhoe Bay crude
oil and the weathered oils tested were weakly mutagenic using
TA100. The commercial nutrient formulations were negative.
Organic samples collected from the beaches showed varying results
depending upon the type and timing of the treatments. Although
the data is in final analysis, we do know that the mutagenicity
of the organic extracts from both treated and untreated beaches
decreases over time when based upon the amount of extracted
organic material applied to the test. This result means that the
mutagenicity is being lost at a rate greater than the rate at
which the organic material is depleted. Calculations showing the
mutagenic response per area or volume of beach treated have not
yet been done; therefore, we cannot yet compare treated with
untreated beaches.
Discussion and Conclusion
In final analysis, these mutagenicity studies show that
mutagenic toxins associated with spills of Prudhoe Bay crude oil
are lost over time. In conjunction with chemical analysis, these
studies will also help to demonstrate whether or not these
decreases in toxicity are due to bioremediation efforts, or other
natural processes, or to some combination of effects. These
studies will assist in the selecting of appropriate
bioremediation procedures for environmental oil spills.
223
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and Phototrophic Nanoplankton, using Epifluorescence Microscopy
and Comparison with other Products. Applied and Environmental
Microbiology 46:491-498.
Fuhrman, J.A. and F. Azam. 1982. Thymidine Incorporation as a
Measure of Heterotrophic Bacterioplankton Production in Marine
Surface Waters: Evaluation and Field Results. Marine Biology
66:109.
Hildebrand, Robert, 1989. Draft Fucus Protocols. United States
Coast Guard Research and Development, Mobile Laboratory.
Hobble, J.E., R.J. Daley, and S. Jasper. 1977. Use of Nuclepore
Filters for Counting Bacteria by Fluorescence Microscopy.
Applied Environmental Microbiology 33:1225-1228.
Houk, V.S., S. Schalkowsky, and L.D. Claxton, 1989. Development
and Validation of the spiral salmonella assay; an automated
approach to bacterial mutagenicity testing. Mutation Res.
223:49-64.
Parsons, T.R., Y. Maita, and C.M. Lalli. 1984. A Manual of
Chemical and Biological methods for Seawater Analysis. Pergamon
Press, Inc., Maxwell House, Elmsford, N.Y.
Sveum, P., 1987. Accidentally Spilled Gas-Oil in a Shoreline
Sediment on Spitzbergen: Natural Fate and Enhancement of
Biodegradation. Sintef, Applied Chemistry Division, N-7034
Trondheim, Norway. 16 pp.).
Whitledge, Malloy, Patton, and Wirick (1981).
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