INTERIM  REPORT
OIL SPILL BIOREMEDIATION PROJECT
                 Prepared by

        U.S. Environmental Protection Agency
         Office of Research and Development
               February 28,1990

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                       INTERIM REPORT

              OIL SPILL BIOREMEDIATION  PROJECT
P.H. Pritchard, Ph.D.; R. Araujo, Ph.D.; J.R. Clark, Ph.D.;
    L.D.  Claxton,  Ph.D.;  R.B.  Coffin,  Ph.D.;  C.F.  Costa;
    J.A.  Glaser,  Ph.D.; J.R. Haines, Ph.D.; D.T. Heggero;
     F.V. Kremer, Ph.D.;  S.C.  McCutcheon, Ph.D.,P.E.;
          J.E. Rogers, Ph.D.; A.D. Venosa, Ph.D.
           U.S. Environmental Protection Agency
            Office of Research and Development

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       i UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
       •                WASHINGTON. D.C. 20460


                           MAR  t 5  =C20

                                                     OFFICE OF
                                               RESEARCH AND DEVELOPMENT


Rear Admiral David Ciancaglini
Federal On Scene Coordinator
United States Coast Guard
Key Bank Building
601 West 5th Avenue, Suite 500
Anchorage, Alaska  99501

Dear Admiral Ciancaglini:

     At the Alaska bioremediation planning meeting in February,
Captain Dave Zawadzki of your organization requested that the
Environmental Protection Agency  (EPA) define its position on
bioremediation.  Enclosed please find, Interim Report; Oil Spill
Bioremediation Project, which summarizes our conclusions to date.

     Based upon visual observations of our demonstration plots
and analyses of the field and laboratory data from both EPA and
Exxon efforts, we conclude that the application of nitrogen and
phosphorus fertilizers enhances biodegradation of oil from the
contaminated beaches.  The absence of adverse ecological effects
observed from fertilizer application further supports
bioremediation as a feasible clean-up procedure.

     In summation, our findings indicate that fertilizer addition
to enhance biodegradation of oil is effective and environmentally
safe.  Should you have any questions or comments regarding
bioremediation, please address them to Dr. John H. Skinner,
Acting Deputy Assistant Administrator for Research and
Development, at (202) 382-7676.

                        Sincerely yours,
                        Erich W. Bretthauer
                      Assistant Administrator
                    for Research and Development

Enclosure

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                              CONTENTS


                                                             Page

Executive Summary 	    xv

Acknowledgment	    xix

     1.  Introduction 	     1

     2.  Background	     5

     3.  Overview of Activities	    11
              Organization  	    11
              Chronology of Events  	    15

     4.  Site Selection and Characteristics	    19
              Snug Harbor	 .    19
              Passage Cove	    21

     5.  Fertilizer Selection and Characteristics ....    27
              Background	    27
              Selected Fertilizer Formulations  	    27
              Nutrient Release Characteristics -
                Methods	    31
                   Static Tests 	    31
                   Intermittent Submersion Tests  ....    31
                   Field Tests	    32
     Nutrient Release Characteristics - Test
                Results	..-I	    32
                   Woodace IBDU Briquettes  	    32
                   IBDU Granules	    35
                   Osmocote Briquettes  	    39
                   MAGAMP Briquettes  	    39
                   Sierra Chemical Granules 	    43
                   Inipol EAP 22	    43
              Discussion and Conclusions  	    48

     6.  Field Test Design and Methods	    51
              Test Plot Sampling Design	    51
                   Sampling Procedure 	    51
                   Sampling Method  	    53
              Fertilizer Application  	    55
                   Slow-Release Water-Soluble
                     Fertilizers	    55
                   Oleophilic Fertilizer  	    60
                   Sprinkler System 	    61
              Analytical Procedures 	    61
                   Oil Chemistry	    61
                   Nutrient Analysis  	    63
                   Microbiological Analysis 	    64

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              Ecological Monitoring  	     65
              Caged Mussels	     67
              Field Toxicity Tests	     67

7.  Field Test Results - Snug Harbor	     69
         Visual Observations 	     69
         Nutrient Concentrations 	     70
         Changes in Oil Residue Weight and
           Composition	     77
         Microbiology  	    109
         Ecological Monitoring 	    Ill
              Nutrients	    Ill
              Chlorophyll Analyses 	    Ill
              Phytoplankton Primary Production ...    117
              Bacterial Abundance  	    117
              Bacterial Productivity 	    117
              Microflagellate Abundance  	    121
              Dissolved Organic Carbon, Particulate
                Carbon, Particulate Nitrogen ....    121
              Stable Isotope Ratios of Carbon and
                Nitrogen	    121
              Caged Mussels	    121
         Discussion and Conclusions  	    124

8.  Field Test Results - Passage Cove	    127
         Visual Observations 	    127
         Nutrient Concentrations 	    127
         Changes in Oil Residue Weight and
           Composition	    127
         Microbiology  	    139
         Ecological Monitoring 	    142
              Nutrients	    142
              Chlorophyll Analyses 	    142
              Phytoplankton Primary Production ...    142
              Bacterial Abundance  	    146
              Bacterial Productivity 	    146
              Caged Mussels	    146
              Field Toxicity Tests of Oleophilic
                Fertilizer at Passage Cove	    146
         Discussion and Conclusions  	    154

9.  Supporting Studies	    155
         Microcosms	    155
              Background	    155
              Methods	    155
              Results	    157
              Discussion and Conclusions 	    158
         Laboratory Biodegradation Screening
           Evaluation	    165
              Background	    165
              Methods	    165
              Results	    166

                           ii

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                   Discussion and Conclusions  	     168
              Respirometric Analysis of Biodegradation   .     175
                   Background	     175
                   Methods	     175
                   Results	     1/76
                   Discussion and Conclusions  	     180
              Mechanism of Action of Inipol-Enhanced Oil
                Degradation	     183
                   Background	     183
                   Methods	     183
                   Results	     185
                   Discussion and Conclusions  	     185
              Chemical Effect of Oleophilic Fertilizer   .     188
                   Background	     188
                   Methods	     188
                   Results	     188
                   Discussion and Conclusions  	     188
              Toxicity of Oleophilic Fertilizer  	     189
                   Background	     189
                   Methods	     189
                   Results	     191
                   Discussion and Conclusions  	     191
              Beach Hydraulics	     192
                   Background	     192
                   Methods	     192
                   Results	     193
                   Discussion and Conclusions  	     221
              Mutagenicity Tests  	     222
                   Background	     222
                   Methods	     222
                   Results	     223
                   Discussion and Conclusions  	     223
Reference	     224
                               iii

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                              Tables
                                                             Page
Table 2.1.   Calculated Ratios of C17/Pristane and CIS/
             Phytane	    10

Table 3.1.   EPA Bioremediation Project Staff 	    13

Table 4.1.   Description of Demonstration Plots at Snug
             Harbor	    22

Table 4.2    Analysis  of oil extracted from mixed sand and
             gravel samples taken from Otter Beach on May 28,
             1989, two weeks prior to fertilizer
             application	    23

Table 4.3.   Description of Fertilizer Treatment Demonstration
             Plots at  Passage Cove	    25

Table 5.1.   Inipol EAP 22 Chemical  Composition	    30

Table 5.2.   Total Kjeldahl Nitrogen (TKN) Released From
             IBDU Granular Fertilizer in Static Water
             Conditions	    40

Table 5.3.   Release of Ammonia, Total Kjeldahl Nitrogen  (TKN)
             and Total Phosphorus (TP)  from Inipol EAP 22 During
             Intermittent Submersion Experiment 	    49

Table 7.1.   Ammonia Concentrations  in Interstitial Water
             Samples	    71

Table 7.2.   Nitrate/Nitrite Concentrations in Interstitial Water
             Samples	    75

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	    110

Table 7.4.   Ammonia Nitrogen (/iM N/l)  from Nearshore Water Over
             Gravel Beaches at Snug  Harbor.  Mean of four
             replicates (standard deviation).  (Method detection
             limit = 0.13 MM N/l.)   	    112

Table 7.5.   Ammonia Nitrogen (/iM N/l)  from Nearshore Water Over
             Cobble Beaches at Snug  Harbor.  Mean of four
             replicates (standard deviation).  (Method detection
             limit = 0.13 /iM N/l.)   	    113
                                IV

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Table 7.6.
Table 7.7.
Table 8.1.
Table 8.2.
Table 8.3.


Table 9.1.



Table 9.2.


Table 9.3.



Table 9.4.
Table 9.5.
Table 9.6.


Table 9.7.
             Phosphate (/iM 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 /iM P/l
             thereafter.) 	   114

             Phosphate (/xM/Pl) from Nearshore Water Over Cobble
             Beaches at Snug Harbor.  Mean of four replicates
             (standard deviation).  Method detection limit = 0.20
             MM P/l for sample date 6/10/89, 0.02 /iM P/l
             thereafter.)	   115

             Relative Concentration (Log10 of the cell number/g
             of beach material) of oil-degrading microorganisms
             in Passage Cove	   140

             Relative Concentration (Log10 of the cell number/g
             of beach material) of oil-degrading microorganisms
             in samples from Beaches that were not impacted by
             oil	   141

             Larval Survival and Development After 48 hours in
             Salinity-Adjusted Prince William Sound Water .   152

             Chemical Analysis of Mixed Sand and Gravel
             Microcosms Sampled 17 Days After Initiation of
             Fertilizer Application 	   159

             Residue Weight of Oil in Cobble Microcosms Analyzed
             26 Days After Fertilizer Application 	   160
             Ratios of Hydrocarbons in Oil From Cobble
             Microcosms Analyzed 26 days After Fertilizer
             Application  	
                                                              161
             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  	   162

             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	   163
             Experimental Design for Respirometric
             Studies  	 ,
             Experimental Design of Flask Studies
                                                              177
                                                              178

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Table 9.8.   Experimental Design for Laboratory
             Microcosm Study   	   184

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.)  	   190

Table 9.10.  Passage Cove Beach Hydraulics:  August 6, 1989;
             4:30 a.m.; High Tide	   196

Table 9.11.  Passage Cove Beach Hydraulics:  August 6, 1989;
             7:30 a.m.; Falling Tide	   197

Table 9.12.  Passage Cove Beach  Hydraulics:  August 6, 1989;
             10:00 a.m.; Low Tide	   198

Table 9.13.  Passage Cove Beach  Hydraulics:  August 6, 1989;
             1:00 p.m.; Rising Tide	   199

Table 9.14.  Passage Cove Beach  Hydraulics:  August 6, 1989;
             5:10-7:30 p.m.; High-Falling Tide  	   200

Table 9.15.  Passage Cove Beach  Hydraulics:  August 6, 1989;
             9:00 p.m.; Low Tide	   201

Table 9.16.  Passage Cove Beach  Hydraulics:  August 7, 1989;
             6:00 a.m.; High Tide	   202

Table 9.17.  Passage Cove Beach  Hydraulics:  August 20, 1989;
             7:20 a.m.; Falling Tide	   203

Table 9.18.  Passage Cove Beach  Hydraulics:  August 20, 1989;
             10:00 a.m.; Low Tide	   204

Table 9.19.  Passage Cove Beach  Hydraulics:  August 20, 1989;
             12:50 p.m.; Rising Tide	   205

Table 9.20.  Passage Cove Beach  Hydraulics:  August 20, 1989;
             4:15 p.m.; High Tide	   206

Table 9.21.  Passage Cove Beach  Hydraulics:  August 20, 1989;
             9:15 p.m.; Falling Tide	   207

Table 9.22.  Passage Cove Beach  Hydraulics:  August 21, 1989;
             6:30 a.m.; Falling Tide	   208

Table 9.23.  Passage Cove Beach  Hydraulics:  August 21, 1989;
             10:00 a.m.; Low Tide	   209

Table 9.24.  Passage Cove Beach  Hydraulics:  August 21, 1989;
             12:00 noon; Rising Tide	   210

                               vi

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Table 9.25.


Table 9.26.


Table 9.27.


Table 9.28.


Table 9.29.


Table 9.30.


Table 9.31.


Table 9.32.


Table 9.33.


Table 9.34.
Passage Cove Beach  Hydraulics:   August 21,  1989;
3:45 p.m.; Rising Tide	   211

Passage Cove Beach  Hydraulics:   August 21,  1989;
5:00 p.m.; Falling Tide	   212

Passage Cove Beach  Hydraulics:   September 10, 1989r
12:20 noon; Falling Tide	   213

Passage Cove Beach  Hydraulics:   September 10, 1989?
3:10 p.m.; Low Tide	   214

Passage Cove Beach  Hydraulics:   September 10, 1989;
6:15 p.m.; Rising Tide	   215

Passage Cove Beach  Hydraulics:   September 10, 1989;
8:45 p.m.; Rising Tide	   216

Passage Cove Beach  Hydraulics:   September 11, 1989;
8:10 a.m.; Rising Tide	   217

Passage Cove Beach  Hydraulics:   September 11, 1989;
11:45 a.m.; High Tide	   218

Passage Cove Beach  Hydraulics:   September 11, 1989;
3:00 p.m.; Falling Tide	   219

Passage Cove Beach  Hydraulics:   September 11, 1989?
6:30 p.m.; Low Tide	   22O
                               vii

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                             Figures

                                                             Page

Figure 2.1.  Unfractionated Prudhoe Bay Crude Oil 	    7

Figure 2.2.  Prudhoe Bay Crude Oil, Aliphatic Fraction  .  .    8

Figure 2.3.  Prudhoe Bay Crude Oil, Aromatic Fraction ...    9

Figure 3.1.  Project Organization Chart 	   12

Figure 4.1.  Snug Harbor, Knight Island 	   20

Figure 4.2.  Passage Cove, Knight Island  	   24

Figure 5.1.  Cumulative Release Rate From IBDU Briquettes  .   33

Figure 5.2.  Daily Nutrient Release Rate From IBDU Briquettes
             (mg/l/day)	   34

Figure 5.3.  Ammonia Release From IBDU Briquettes at 9 and 21
             Degrees Centrigrade in 3 Different Water
             Sources	   36

Figure 5.4.  Cumulative Release of Ammonia and Total Kjeldahl
             Nitrogen (TKN) From IBDU Fertilizer Granules
             Contained in Bags	   37

Figure 5.5.  Cumulative Release of Ammonia and Total Kjeldahl
             Nitrogen (TKN) From IBDU Granules in Static Flask
             Experiments	   38

Figure 5.6.  Cumulative Release of Ammonia and Total Kjeldahl
             Nitrogen (TKN), and Total Phosphorus from Osmocote
             Briquettes in Static Flask Experiments ....   41

Figure 5.7.  Cumulative Release of Ammonia,  Total Kjeldahl
             Nitrogen (TKN), and Total Phosphorus from MAGAMP
             Briquettes in Static Flask Experiments ....   42

Figure 5.8.  Sampling Point Location for Magnesium Ammonia
             Phosphate Fertilizer Field Test  	   44

Figure 5.9.  Magnesium Ammonium Phosphate Fertilizer Test  .   45

Figure 5.10. Cumulative Release of Ammonia and Nitrate from
             Sierra Chemical Granules in Static Flask
             Experiments	   46
                              Vlll

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Figure 5.11. Cumulative Release of Ammonia and Total Kjeldahl
             Nitrogen (TKN)  from Inipol EAP 22 in Static Flask

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure



Experiments 	 47
6.1. Placement of the Bags of Fertilizer Briquettes
on Otter and Seal Beaches 	 56
6.2. Repositioning of the Bags of Fertilizer
Briquettes on Otter and Seal Beaches 	 57
6.3. Tidal Fluctuations for High Tides, Snug Harbor,
June 6-30, 1989 	 58
6.4. Tidal Fluctuations for Low Tides, Snug Harbor,
June 6-30, 1989 	 59
7.1. Mean Residue Concentration at Snug Harbor Mixed Sand
and Gravel Plots, All Zones 	 78
7.2. Mean Residue Concentration at Snug Harbor Mixed Sand
and Gravel Plots, Mid and Low Tide Zones ... 79
7.3. Mean Residue Concentration at Snug Harbor Cobble
Plots, All Zones 	 80
7.4. Median Residue Concentration at Snug Harbor Cobble
Plots 	 81
7.5. Mean C17/Pristane Ratio at Snug Harbor Mixed Sand
and Gravel Plots, All Zones 	 83
7.6. Mean C17/Pristane Ratio at Snug Harbor Cobble Plots,
Top, All Zones 	 84
7.7. Mean C17/Pristane Ratio at Snug Harbor Cobble Plots,
Bottom, All Zones 	 85
7.8. Mean C18/Phythane Ratio at Snug Harbor Mixed Sand
and Gravel Plots, All Zones 	 86
7.9. Mean C18/Phytane Ratio at Snug Harbor Cobble Plots,
Top, All Zones 	 87
7.10. Mean C18/Phytane Ratio at Snug Harbor Cobble Plots,
Bottom, All Zones 	 88
7.11a Recreated Gas Chromat ©graphic Profiles from Samples
of Oil Extracted from the Surface of Cobble Two
Weeks Following Application of Oleophilic
Fertilizers 	 91

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Figure 7.lib Recreated Gas Chromatographic Profiles from Samples
             of Oil Extracted from the Surface of Cobble Four
             Weeks Following Application of Oleophilic
             Fertilizers	   92

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	   93

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  	   94

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  	   95

Figure 7.12d Recreated Gas Chromatographic Profiles from Samples
             of Oil Extracted from the Mixed Sand and Gravel
             Under the Cobble prior to Application of Water
             Soluable Fertilizer  	   96

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 	   97

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 	   98

Figure 7.13. Mean Weight of Alkanes (mg) Normalized to the
             Total Oil Residue Weight (mg)  Extracted from the
             Beach Material; Control Mixed Sand and Gravel
             Beaches	   100

Figure 7.14. Mean Weight of Alkanes (mg) Normalized to the Total
             Oil Residue Weight (mg) Extracted from the Beach
             Material; Oleophilic Fertilizer Treated Mixed Sand
             and Gravel	   101

Figure 7.15. Mean Weight of Alkanes (mg) Normalized to the Total
             Oil Residue Weight (mg) Extracted from the Beach
             Material; Water Soluble Fertilizer Treated Cobble
             Beaches	   102

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Figure 7.16. Median of Total Concentration of Oil on
             Treated and Untreated Cobble Plots at Snug Harbor,
             All Zones	   103
             C17/Pristane Ratio versus LoglO Residue Weight Two
             Weeks Before Fertilizer Application  	   105

             C18/Phytane Ratio versus LoglO Residue Weight Two
             Weeks Before Fertilizer Application  	   106

             C17/Pristane Ratio versus LoglO Residue Weight at
             Time Zero of Fertilizer Application	   107

             C18/Phytane Ratio versus LoglO Residue Weight at
             Time Zero of Fertilizer Application	   108
Figure 7.17.


Figure 7.18.


Figure 7.19.


Figure 7.20.


Figure 7.21. Phytoplankton Chlorophyll data from Water
             Samples Collected at Snug Harbor Following June 7
             and 8, 1989, Fertilizer Additions to Gravel
             Shorelines	   116

Figure 7.22. Primary Productivity Estimates (as 14C uptake) for
             Phytoplankton Samples from Snug Harbor at Various
             Sample Dates Following the June 7 and 8, 1989,
             Fertilizer Additions 	   118

Figure 7.23. Bacterial Productivity, as Measured by Tritiated
             Thymidine Uptake, for Bacterial Samples Collected on
             Various Sample Dates Adjacent to Gravel Shorelines
             at Snug Harbor	   120

Figure 7.24. Abundance of Bacterial Cells (xlO9)  from
             Water Samples Taken along Gravel Shorelines
             on Various Sample Dates Following the June 7 and
             8, 1989, Fertilizer Additions to Snug Harbor
             Shorelines	   122

Figure 7.25. Bacterial Productivity, as Measured by Tritiated
             Thymidine Uptake, for Bacterial Samples Collected on
             Various Sample Dates Adjacent to Cobble Shorelines
             at Snug Harbor	   123

Figure 8.1.  Median Residue Concentration of Oil on Passage Cove
             Cobble Beaches 	   129

Figure 8.2.  Median of C17/Pristane Ratio Passage Cove Cobble,
             Top, All Zones	   130

Figure 8.3.  Median of C18/Phytane Ratio Passage Cove Cobble,
             Top, All Zones	   131
                               xi

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Figure 8.4a. Gas Chromatographic Profiles of Oil Residue Weights
             Before Plots Were Sprinkler Irrigated with water-
             Soluble Fertilizer 	   133

Figure 8.4b. Gas Chromatographic Profiles of Oil Residue Weights
             Two Weeks After Sprinkler Application of Water-
             Soluble Fertilizer Began 	   134

Figure 8.4c. Gas Chromatographic Profiles of Oil Residue Weights
             Three Weeks After Sprinkler Application of Water-
             Soluble Fertilizer Began 	   135

Figure 8.5a. Gas Chromatographic Profiles of Oil Residue Weights
             on Untreated Plots - Raven Beach 	   136

Figure 8.5b. Gas Chromatographic Profiles of Oil Residue Weights
             on Untreated Plots - Raven Beach - Two Weeks After
             Previous Profiling 	   137

Figure 8.6.  Median Total Aliphatic Hydrocarbon Concentrations on
             Passage Cove Plots	   138

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

Figure 8.8.  Mean Primary Productivity Activity 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
Figure 8.9.
Figure 8.10.
Abundance of Bacterial Cells (xlO9)  From Water
Samples Collected at Passage Cove Study Sites Before
and After Fertilizer Application on July 25, 1989.
Values Are Means (+ SD) of 4 Replicates  ...   147

Bacterial Productivity Measurements From Tritiated
Thymidine Uptake by Water Samples Collected at
Passage Cove Before and After Nutrient Application
to Shorelines on July 25, 1989.  Values are means
(+ SD) of 4 replicates	   149
Figure 9.1.

Figure 9.2.
Schematic Diagram of the Microcosms
156
Gas Chromatographic Profiles Showing the Effect of
Different Inocula on the Degradation of Artificially
Weathered Prudhoe Bay Crude Oil  	   167
                               XII

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Figure 9.3.  Gas Chromatographic Profiles Showing the Effect of
             Temperature on the Degradation of Artificially
             Weathered Prudhoe Crude Oil  	   169

Figure 9.4.  Gas Chromatographic Profiles Showing the Effect of
             Different Concentrations of Inipol (% of Oil
             Concentration) on the Degradation of Artificially
             Weathered Prudhoe Crude Oil  	   170

Figure 9.5.  Gas Chromatographic Profiles Showing the Effect of
             Different Fertilizers, Under Poisoned and Unpoisoned
             Conditions, on the Degradation of Artificially
             Weathered Prudhoe Crude Oil  	   171

Figure 9.6.  Gas Chromatographic Profiles Showing the Effect
             of Temperature on the Degradation of
             Artificially Weathered Prudhoe Crude Oil Treated
             with Inipoll	   173

Figure 9.7.  Gas Chromatographic Profiles Showing the Effect of
             Inipol (Poisoned and Unpoisoned Conditions)  on the
             Degradation of Oil on Beach Material Taken from
             Prince William Sound 	   174

Figure 9.8.  Cumulative Oxygen Uptake on Weathered Prudhoe Bay
             Crude Oill	   179

Figure 9.9.  Gas Chromatographic Profiles of Alkanes at 0 and 6
             Weeks After Initiation of Flask Studies  .  . .   181

Figure 9.10. Chromatographic Scan of Aromatic at 0 and 6 Weeks
             After Initiation of Flask Studies  	   182

Figure 9.11. Effect of Inipol on the Relative Number of Oleic
             Acid-degrading Bacteria in Jars Containing Oiled
             Rocks and Seawater, Defined Nutrient Medium, or
             Saline Solution  	   186

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  	   187

Figure 9.13. Location of Wells for Beach Hydraulics Experiment at
             Passage Cove	   194

Figure 9.14. Casing Configuration 	   195
                              XI11

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                        EXECUTIVE SUMMARY
     The U.S. Environmental Protection Agency's Alaska
Bioremediation Project was initiated in the aftermath of the
March 24, 1989, EXXON VALDEZ oil spill.  The objective of the
project was to demonstrate an alternative cleanup method for oil-
contaminated shorelines based on enhancing natural biodegradation
of the oil through the addition of nitrogen and phosphorus
nutrients.  This enhancement process is a well-recognized and
scientifically sound approach to bioremediation but had never
been tested on a large scale in marine environments.  The project
was managed by EPA's Office of Research and Development with
financial, scientific, and logistical support from the Exxon
Company USA under the authority of the Federal Technology
Transfer Act.

     After planning, mobilizing staff and facilities, and
selecting test sites in Prince William Sound, Alaska, nutrient
application began on June 8, 1989.  Nitrogen- and phosphorus-rich
nutrients were added to the oil-contaminated shoreline sites as
three types of fertilizers: (1) a slow-release formulation in
which water-soluble nutrients leaching from point sources were
distributed over the contaminated beaches by tidal actions;
(2) an oleophilic formulation designed to "dissolve" nutrients
into the oil on the surface of the beach substrata; and (3) a
fertilizer solution in which inorganic nitrogen and phosphorus
were added to seawater and the solution applied to the beaches at
low tide using a sprinkler system.  The liquid oleophilic
fertilizer was sprayed onto the test plots from a hand-pumped
backpack sprayer.  The slow release formulations were in the form
of either briquettes or granules.  Application of these
formulations was accomplished by placing netbags of briquettes on
the shoreline surface in a designated pattern or by broadcasting
the granules on the beaches using a commercial fertilizer
spreader.

     Test plots were established on Knight Island in Prince
William Sound, one in Snug Harbor that was tested early in the
summer, and the other in Passage Cove that was tested late in the
summer.  Snug Harbor, a moderately oiled beach, was selected to
simulate conditions considered typical of a beach following
physical washing of the beach material (the primary cleanup
procedure used by Exxon).  Passage Cove,  a heavily oiled beach
that had been physically washed, served as the definitive test
beach to show effectiveness of the large-scale application of
fertilizers ultimately performed by Exxon.  The shoreline
surfaces were both mixed sand and gravel, and cobblestone.   Beach
materials were sampled before and after application of the
fertilizers and the results compared to untreated reference
beaches.  Samples were processed to determine changes in the
quantity and composition of oil residues following fertilizer

                                xv

-------
application.  Visual changes in the amount of oil remaining on
rock surfaces were also noted.  In addition, monitoring for any
potential adverse environmental effects was performed.  This
included measurements of algal growth (eutrophication) due to any
nutrient accumulation in seawater adjacent to the treated beaches
and any toxicity of the fertilizer to marine species.  Laboratory
and microcosm experiments were conducted to examine nutrient-
enhanced oil degradation under more controlled conditions.

     Following careful discussion of visual observations,
analysis of the field and laboratory data, and two formal
workshops to discuss interpretations and significant findings, we
conclude that the application of nitrogen and phosphorus
fertilizers did enhance biodegradation of the oil and the clean-
up of oil from the contaminated beaches.  Since there were no
adverse ecological effects observed from the fertilizer
application, bioremediation is recommended as a feasible cleanup
procedure.

     Specific conclusions and interpretations of the
demonstration project are as follows:

*    Visual inspection of beaches treated with the inorganic
     nutrients (using the sprinkler system) showed that within
     three weeks following fertilizer application, considerably
     less oil was observed on the rock surfaces than in the
     untreated control beaches.  This condition became more
     pronounced with time and remained visually apparent through
     the end of the summer season (five weeks).

*    No oil slicks were observed in the near-shore seawater
     following application of the inorganic fertilizer,
     indicating oil was not released.

*    Samples of the oil taken from the beach surfaces when the
     oil was visually beginning to disappear showed changes in
     composition indicating extensive biodegradation.

*    Visual disappearance of the oil as a result of inorganic
     nutrient application (sprinkler system) can only be
     attributed to enhanced biodegradation.

*    Similar visual oil disappearance was also observed 10 days
     following application of the oleophilic fertilizer.  Again,
     no oil slicks were observed, and oil sampled from the rock
     surfaces when oil was beginning to disappear showed changes
     in composition indicating extensive biodegradation.

*    Laboratory studies with contaminated Prince William Sound
     beach material showed that the oleophilic fertilizer was not
     a chemical rock washer and that it enhanced the extent and
                               xvi

-------
rate of oil degradation as compared to untreated beach
material.

As observed in laboratory studies, enhanced oil
biodegradation from fertilizer addition was accompanied by
significant changes in the physical consistency of the oil.
A flaky, particulate material consisting of degraded oil,
degradation products, and microbial cells was produced.
This process occurs 1 to 2 weeks following incubation.

A more rapid and extensive disappearance of oil from Passage
Cove as compared to Snug Harbor suggested that physical
washing of the beaches spread a very thin layer of oil over
a large surface area of rock and gravel, facilitating
bacterial degradation.

Samples from untreated oil-contaminated beaches in Prince
William Sound showed 1,000 to 100,000 times more
oil-degrading bacteria than uncontaminated beaches.  Thus,
biodegradable carbon from the oil was sufficient to enrich
for high numbers of oil-degrading bacteria.  Because of
these naturally elevated bacterial numbers, it was difficult
to demonstrate significant increases in oil-degrading
bacteria resulting from fertilizer application.  The high
bacterial numbers are probably maintained due to an
equilibrium condition between the growth of bacteria on the
oil, protozoan predation, and physical sloughing.

Oil clean-up in the field was the direct result of enhanced
biodegradation coupled with washing of the degraded
materials from the rock surfaces by tidal action.

Chemical analysis of field samples over time showed that
changes in oil residue weight and oil composition were
greater in the fertilizer-treated beaches than in the
untreated reference beaches.  However, due to several
confounding factors, statistical verification of these
results has not been possible.  These factors include: (1)
surprisingly high rates of natural oil biodegradation due to
significant natural concentrations of nutrients in seawater
and freshwater; (2) high variability in oil concentration
and distribution in beach material; and (3) extensive
degradation of pristane and phytane, two branched chain
hydrocarbons normally used as conserved internal standards
to measure changes in oil composition.

Chemical analysis of oil from the two untreated reference
plots used in this study showed that on some shorelines
there were high rates of natural oil degradation that were
comparable to that observed in the treated plots.  However
there is no guarantee that other untreated beach areas would
respond in the same way (variable concentrations in nitrogen

                         xvii

-------
     and phosphorus).  The fact that oil still remains on some
     Alaskan shorelines is evidence that natural degradation
     rates are not uniformly high.  Therefore fertilizer addition
     assures oil degradation in a controlled and predictable
     manner during the short "time window" when water
     temperatures are high enough for optimal microbial activity.

*    Addition of fertilizer to oiled shorelines did not cause any
     increases in planktonic algae or bacteria or any measurable
     nutrient accumulation in adjacent embayments.

*    The concentration of oleophilic fertilizer and ammonia that
     is toxic to various marine species has been established.
     They were both mildly toxic.  Seawater collected directly
     over the beaches just treated with a combination of the
     oleophilic and water-soluble fertilizer (worst case
     situation) were toxic to the most sensitive marine species
     (oyster larvae).  A 50% dilution of this seawater, which
     would occur through tidal mixing within a few feet of the
     treated shoreline, reduced the concentrations to levels
     where toxicity is zero.

*    No oil was detected in tissues taken from mussels that had
     been placed (in flow-through plastic containers) just
     offshore of the fertilizer-treated beaches.

*    Samples taken from the fertilizer-treated beaches and tested
     in the standard Ames mutagenicity test showed that as the
     oil was biodegraded, the slight mutagenicity of fresh
     Prudhoe Bay crude was eliminated.  Thus, no mutagenic
     byproducts result from enhanced biodegradation.

     Overall, our findings indicate that fertilizer addition to
enhance biodegradation of the oil is effective and
environmentally safe.  Projects initiated this winter, in
conjunction with the Exxon Company USA were designed to continue
and complement the research effort.  Examples of projects
allowing more insight into the biodegradation of spilled oil
include the mechanism of action of the oleophilic fertilizer; and
identification of oil components that could act as internal
standards for evaluating the extent of degradation.  These
projects as well as several others are providing valuable
information for further, more effective scale-up bioremediation
this summer and for future oil spills.
                              xviii

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                         ACKNOWLEDGMENTS
     For any field work, the success of the study is dependent to
a great extent upon the support and dedication of the field crew.
We have been very fortunate to have an outstanding field team who
has consistently demonstrated its commitment to this project.
After many long and hard hours, and in some cases, work around
the clock, the team members have maintained their spirits and
brought this study to fruition.  Our sincere gratitude is
extended to John R. Baker, Martin Dillon, Wesley L. Kinney,
Dennis E. Miller, and Richard Wright.

     Deepest appreciation is expressed to the crew of the F/V
AUGUSTINE, Garth, Phyllis, and Jaris Tyler, Tyler Morgan, and
Vern Boyd, and to the crew of the F/V CARMEN ROSE, Brian King,
Dave Kuntz, Stuart Carter, and George Covel, for their support to
the onsite staff at Snug Harbor and Passage Cove, respectively;
to Dennis Thacker and Ken Lobe, pilots of the aircraft N756AF,
for their continued safe transport of personnel, equipment, and
samples; and to Don Carlson and Ken Broker, Norcon Inc.
carpenters, for demonstrating superior craftsmanship in the
construction of the two field laboratories.

     Gratitude is also extended to Russell Chianelli, Ph.D.;
Stephen M. Hinton, Ph.D.; and Roger C. Prince, Ph.D. at Exxon
Research and Engineering Co. in Annandale, New Jersey and to
Sara J. McMillan and Richard Requejo, Ph.D. at Exxon Production
Research Co. in Houston, Texas.  Their work on this project,
particularly in the Laboratory Biodegradation Screening
Evaluations was crucial.
                               XIX

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                            SECTION 1

                           INTRODUCTION
     On March 24, 1989, the EXXON VALDEZ went aground in Prince
William Sound, Alaska, releasing approximately 11 million gallons
of Prudhoe Bay crude oil.  After learning of the magnitude of the
spill, the EPA Assistant Administrator for the Office of Research
and Development  (ORD) convened a meeting of nationally and
internationally recognized scientists in the field of oil
biodegradation to evaluate the feasibility of using
bioremediation to assist in cleanup operations.  Members of the
Biosystems Scientific Steering Committee, a research group within
ORD that is developing the technology of bioremediation,
organized and implemented the meeting.  The meeting was chaired
by Dr. Hap Pritchard, a senior scientist in the ORD Environmental
Research Laboratory in Gulf Breeze, Florida.  After intensive
discussion, scientists at the meeting recommended that ORD plan
and conduct a field demonstration project to evaluate the use of
fertilizers for accelerating natural biodegradation of the
spilled oil.

     Specifically, the recommendation was based on the following
conclusions:

     The presence of readily degradable hydrocarbons from the
     spilled oil will enrich for naturally occurring oil-
     degrading bacteria.

•    Oil biodegradation in Prince William Sound waters is
     probably limited by the availability of nitrogen and
     phosphorus; therefore, fertilizing the beaches with these
     nutrients will enhance natural degradation of the oil.

•    Past studies have shown convincingly that the enhancement of
     oil biodegradation by nutrient addition readily occurs.
     Further verification of these studies by laboratory
     experiments are unnecessary.

•    Successful bioremediation will require consideration of the
     logistics and mechanics of long-term nutrient application
     and the physical agitation of oil.

     An oleophilic fertilizer, such as that produced by Elf
     Aquitaine Chemical Company, may be the only way to assure
     extended contact of the nutrients with the oil-contaminated
     beach material.

•    Bioremediation should be used as a finishing step for any
     cleanup program; that is, removing the bulk oil, regardless

-------
     of method, will allow bioremediation to clean up the
     residual.

 •    Treatment of the beaches with fertilizer will not
     necessarily remove the black oil residues  (i.e., visually
     unchanged) but will considerably reduce, if not eliminate,
     ecological availability of the oil.

 •    Inoculation of oil-contaminated beaches with hydrocarbon-
     degrading microorganisms enriched from Prince William Sound
     waters was not the best initial approach but should be
     considered in an experimental context or for future spills.

     Depending on the outcome of a demonstration project,
 recommendations for the use of bioremediation to help clean up
 oil-contaminated beaches on a larger scale would be made to
 Exxon.

     A detailed oil spill bioremediation research plan was then
 developed.  The major objectives of this plan were to:

 •    Examine the extent to which natural biodegradation of oil on
     the contaminated beaches was occurring.

     Determine if the rates of oil biodegradation on contaminated
     beaches could be enhanced by the addition of nutrients in
     the field.

 •    Develop methods for long-term application of nutrients to
     contaminated beaches.

 •    Establish methods for monitoring potential ecological
     effects resulting from nutrient addition.

     Develop information on the movement of nutrients in beach
     substrata (beach mechanics).

     Examine the possibility of inoculation as a means to enhance
     oil biodegradation.

     The research plan was based on the use of two types of
 fertilizers:  (l) a slow-release formulation in which nutrient
 leaching from point sources would be distributed over the
 contaminated beaches by tidal actions and (2) an oleophilic
 formulation in which nutrients would be "dissolved" into the oil
 on the surface of the beach substrata.  Biodegradation of the oil
would be followed through time using analytical chemistry and
microbiological techniques.  An ecological monitoring program
would be implemented to check for possible adverse ecological
effects resulting from the direct toxicity of the fertilizer or
 from eutrophication.  The bioenhancement (inoculation of
bacteria) part of the plan was designed as a small program to

-------
check feasibility in laboratory microcosm studies.  In addition,
development of a detailed quality assurance plan was also
initiated.

     The research plan was reviewed by a special committee of
EPA's Scientific Advisory Board.  The committee recommended that
the plan be implemented with minor modification.

     Following development of the research plan, a decision was
made to approach Exxon and propose a cooperative effort under the
Federal Technology Transfer Act of 1986.  On June 2, 1989, the
two parties reached an agreement, and the project was formally
initiated.  Exxon agreed to provide all logistical support
(transportation from Valdez to test sites, field laboratory
facilities, and subsistence) and $1.6 million for direct support
of the field demonstration project.  EPA provided $1.6 million
for management personnel, scientific expertise, quality
assurance, and operations technical support.

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                            SECTION 2

                            BACKGROUND
     The site of the Alaskan oil spill is a harsh and diverse
environment with poor accessibility.  The shoreline is
geologically young, is composed largely of metamorphic rock, and
ranges from vertical cliffs to boulder and pebble beaches.  High-
energy beaches are common, with tides that vary from +4 to -1 m.
In some areas, glacial and snow melt introduce large amounts of
fresh water to nearshore water of the Prince William Sound.
Prince William Sound has a considerable population of seals and
sea otters.  The area also has extensive herring spawning areas
and significant numbers of seabirds and shorebirds.  There is a
substantial migration of birds that feed at beaches and
intertidal areas.

     The spilled oil spread over an estimated 350 miles of
shoreline in Prince William Sound.  Major contaminated shoreline
areas include Knight Island, Eleanor Island, Smith Island, Green
Island, and Naked Island.  Knight Island, the largest and one of
the most heavily polluted of these islands, has restricted tidal
flushing action in some bays and coves.

     The oil settled into the beach gravel and on rock surfaces
and the faces of vertical cliffs.  Contamination occurred
primarily in the intertidal zone.

     Initial weathering of the oil resulted in a loss of
approximately 15% to 20% of the oil by volatilization.
Volatilized components included normal aliphatic hydrocarbons of
less than 12 carbon atoms and aromatic hydrocarbons such as
benzene, toluene, xylene, and some methyl-substituted
naphthalenes.  The resulting residue consisted of approximately
40% to 50% high-molecular-weight waxes and asphaltenes.  On most
beaches in Prince William Sound the weathered oil was black and
viscid rather than brown and mousse-like.

     Beaches were physically cleaned by a combination of flooding
and the application of water under high and low pressure and/or
high temperature.  Vacuum extraction and physical skimming were
used to remove the released oil from the water surface.  The
cleaning process partially removed oil from the surface of rocks
and beaches, particularly the pools of oil, but did not
effectively remove the oil trapped in and below the matrix of
gravel and cobble.  However, the washing process spread a thin
layer of oil over a much greater surface area of rock and gravel.
The extent of physical washing was dependent upon the degree of
contamination.

-------
     The biodegradation of oil has been extensively studied over
the last 20 years.  As a result, the fate and microbial
decomposition of oil in aquatic environments is well understood.
Studies have shown that oil degradation can occur in cold-water
environments.

     Oil degradation is commonly measured by extracting oil from
beach material and then analyzing oil composition using gas
chromatography.  A typical gas chromatogram of fresh and
weathered Prudhoe Bay crude oil is shown in Figure 2.1.  The
weathered Prudhoe Bay crude oil was taken from a Prince William
Sound beach (Northwest Bay) in the late spring.  The major peaks
represent detector responses for the normal alkanes; the
annotated numbers are the carbon lengths of the appropriate
alkane.  Normal aliphatic hydrocarbons of 12 carbons or less are
absent in the weathered oil.  Large quantities of biodegradable
hydrocarbons (C13-C28) remain.  Gas chromatograms for oil samples
fractionated into the aliphatic and aromatic components are shown
in Figures 2.2 and 2.3.  These fractionated samples of oil showed
the presence of small quantities of aromatic hydrocarbons in the
weathered oil, but hydrocarbons up to the methyl naphthalene were
absent.

     Pristane and phytane, branched alkanes, are slow to
biodegrade and have been used as conserved internal standards.
Therefore, changes in the ratios of hydrocarbon concentration for
the linear alkanes relative to the branched alkanes can be used
to indicate biodegradation.  Table 2.1 gives the calculated
ratios of C17 linear alkane to pristane and CIS linear alkane to
phytane for samples taken from Prince William Sound on April 4 to
May 2, 1989.  The sample from Disk Island (gravel) is the only
one with a significant difference in these ratios relative to
fresh Prudhoe Bay crude oil.  This suggests that natural
biodegradation was occurring at this beach.

-------
          10   11  12  13   14  15  16


                                    17

                                       18
                                         19
                                            20
                                               21
                                                 22
                                                   23
                                                      24 25      Fresh
                            13   14
                                     15
                        12
                   11
     10

KHWW)«»

                                16
                                            17

                                                18
                                                   19
                                                      20
                                                          21
                                                    22
Weathered
23
                                                             11
Figure 2.1.   Unfractionated Prudhoe Bay Crude Oil (Number Indicates
            Carbon Atoms of Alkane).

-------
            10   11  12  13  14  15 18
                                                            Fresh
                           15
        10    11
14

13


12

1
Aufc


u





a







*•

*
1







&
' 17







V«

^
18






A
19





/fJ
20 .




LlA
21




22




23 weati


^*
"-I6
"TT30
"M^flMrTw"**^*"1 IV *•»
Figure 2.2.   Prudhoe Bay Crude Oil, Aliphatic Fraction  (Number Indicates
            Carbon Atoms of Alkane).

-------
                                                       Fresh
                                                      Weathered
Figure 2.3  Prudhoe Bay Crude Oil, Aromatic Fraction.

-------
Table 2.1. Calculated Ratios of C17/Pristane and C18/Phytane
                                      N-C17/         N-C18/
SAMPLE                                Pristane       Phvtane
Fresh Prudhoe Bay Crude Oil
Eleanor Island
Northwest Bay
Surface
Surface Control*
6" Depth
6" Depth Control*
Seal Island
Smith Island
Disk Island
Gravel
Fresh Oiled Rock
Weathered Oiled Rock
1.7


1.5
<0.47
1.4
<0.45
1.6
1.5

0.8
1.4
1.8
2.0


1.9
—
1.7
—
2.1
1.9

1.0
1.7
2.0
* Sample taken from an uncontaminated beach area.
                                10

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                            SECTION 3

                      OVERVIEW OF ACTIVITIES
ORGANIZATION

     The organizational structure for the Oil Spill
Bioremediation Field Project is shown in Figure 3.1.  The
following organizations contributed their expertise to the
project:

•    EPA Environmental Research Laboratories - Gulf Breeze,
     Florida; Athens, Georgia; and Ada, Oklahoma

     EPA Risk Reduction Engineering Laboratory - Cincinnati, Ohio

•    EPA Environmental Monitoring Systems Laboratories - Las
     Vegas, Nevada, and Cincinnati, Ohio

     EPA Health Effects Research Laboratory - Research Triangle
     Park, North Carolina

     EPA Center for Environmental Research Information -
     Cincinnati, Ohio

     Exxon Research and Engineering - Annandale, New Jersey

•    Exxon Production Research - Houston, Texas

     Exxon Biomedical Services - East Millstone, New Jersey

     Principal scientists and support staff for the project are
listed in Table 3.1.

     Existing EPA contracts with several companies were expanded
to facilitate logistics, analytical services, and administrative
support.  The contractors are as follows:

•    Analytical support (oil and nutrient analysis):  Science
     Applications International Corporation (SAIC)

     Personnel and administrative support:  Technical Resources,
     Incorporated (TRI)

     Quality assurance planning:  Engineering Science Company
     (Lockheed)

     Data management and logistical support:  SAIC

•    Field team support and logistics:  Lockheed and SAIC


                                11

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                                                          Project Management
                                                 Project Manager
                                                      Costa
                                                    Principal Investigator
                                                         Pritchard
                                                                          Administration
                                                                      Administrative Assistant
                                              OA/QC
                                        Heggem. Papp, Pollard
                                        Stetzenbach. Beckert
Investigation
Areas
               Nutrient
               Addition
           VenosaVGIaser
Field

Laboratory

Microcosms
  Kremer
  Opatken
  Haines
Tabak
Opatken
Safferman
Gripe
                                  Ecological
                                  Monitoring
Clark/Claxton*
Parrish
Coffin
Cifuentes
Hod
Baraket
Macauley
Patrick
Stanley
Primrose
Heitmuller
                                                               Special Projects
                                                                   Sullivan
                                                                    Gray
                                                  Beach
                                                Hydraulics
                                                             Glaser*
                                                             Haines
                                                             McCutcheon
                                                             Opatken
                                                             Garland
                                                     Bloenhancement
Support  Areas
                                    Field Support
                                                Logistics
                                                       I Data Management
'Investigation Area Lender(s)
                                    Baker/Kinney"
                                    Kremer
                                    Miller
                                    Wright
                                    Dillon
                                    Wilson
              1 Support Area Manager(s)
                                                Shokes"
                                                Schmidt
                                                           Horn"
                                                           Gerlack
                                                           Pollard
                                                           Damiata
                                           Figure 3.1   Project Organization Chart.
                                                                           Rogers*
                                                                           Montgomery
                                                                           Shields
                                                                           Mueller
                                                                           Araujo
                                                               Sample Analysis
                                                                                          Microbiology
                                                                              Rogers"
                                                                              Araujo
                                                                              Montgomery
                                                                              Shields
                                                                              Bosworth
                                                                              Robinson
                                                                              Primrose
                                                                              Mueller
                                                                        I Hydrocarbon/
                                                                        I  Nutrient
                                                                           Payne**
                                                                           McNabb
                                                                           Sims
                                                                           McCabe
                                                                           Evans
                                                                           Stietvater
                                                                           Smiley
                                                                           Lopez
                                                                           Fogg

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              Table  3.1.  EPA Bioremediation Project Staff
Management


Headquarters
Special Projects
Administration
Nutrient Addition
Ecological
Monitoring
Name

Hap Pritchard
Chuck Costa

William Reilly
Erich Bretthauer
John Skinner
Dick
Valentinetti

Eulalie Sullivan
Ellen Gray

Valerie Furlong
Robin Shoemaker
Evelyn Clay
Wendy Barlow
Terry Morton

Al Venosa
John Glaser
Fran Kremer
John Haines
Ed Opatken
Henry Tabak
Steve Safferman
Rick Cripe

Jim Clark
Larry Claxton
Rod Parrish
Rick Coffin
Luis Cifuentes
John Macauley
Jerry Hoff
Sana Baraket
Jim Patrick
Roman Stanley
Ginny Primrose
Tom Heitmuller
Affiliation

EPA/ERL
EPA/EMSL

EPA/ORD
EPA/ORD
EPA/ORD
EPA/ORD
TRI
TRI

EPA/EMSL
EPA/EMSL
EPA/EMSL
EPA/EMSL
EPA/EMSL

EPA/RREL
EPA/RREL
EPA/CERI
EPA/RREL
EPA/RREL
EPA/RREL
EPA/RREL
EPA/ERL

EPA/ERL
EPA/HERL
EPA/ERL
TRI
TRI/Texas
A&M
EPA/ERL
TRI
TRI
EPA/ERL
EPA/ERL
TRI
Location

Gulf Breeze, FL
Las Vegas, NV

Washington, DC
Washington, DC
Washington, DC
Washington, DC
Rockville, MD
Seattle, WA

Las Vegas, NV
Las Vegas, NV
Las Vegas, NV
Las Vegas, NV
Las Vegas, NV

Cincinnati, OH
Cincinnati, OH
Cincinnati, OH
Cincinnati, OH
Cincinnati, OH
Cincinnati, OH
Cincinnati, OH
Gulf Breeze, FL

Gulf Breeze, FL
Res. Tri. Park, NC
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
                                   13

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                          Table 3.1.  Continued
B i oenhancement
QA/QC
Data Management
Field Support
Logistics
Microbiology
Analysis
Hydrocarbon/
Nutrient Analysis
Name

John Rogers
Stacy Montgomery
Rochelle Araujo
Jim Mueller
Malcolm Shields

Dan Heggem
Mike Papp
Linda
Stetzenbach
Jim Pollard
Werner Beckert

Wilson Horn
Brian Damiata
Bob Gerlack
Jim Pollard

John Baker
Wes Kinney
Fran Kremer
Dennis Miller
Rick Wright
Marty Dillon
John Wilson

Bob Shokes
Kurt Schmidt

John Rogers
Rochelle Araujo
Stacy Montgomery
Christy Robinson
Malcolm Shields
Lorna Bosworth
Ginny Primrose
Jim Mueller

Jim Payne
Dan McNabb
Rusty Sims
Mike McCabe
John Evans
Jeff Stiefvater
Elizabeth Smiley
Juliet Lopez
Tom Fogg
Affiliation

EPA/ERL
TRI
EPA/ERL
EPA/ERL
TRI

EPA/EMSL
Lockheed
EPA/UNLV
Lockheed
EPA/EMSL
SAIC
SAIC
Lockheed
Lockheed

Lockheed
EPA/EMSL
EPA/CERI
EPA/ERL
SAIC
SAIC
EPA/ERL

SAIC
SAIC

EPA/ERL
EPA/ERL
TRI
TRI
TRI
TRI
TRI
EPA/ERL

SAIC
SAIC
SAIC
SAIC
SAIC
SAIC
SAIC
SAIC
SAIC
Location

Athens, GA
Gulf Breeze, FL
Athens, GA
Gulf Breeze, FL
Gulf Breeze, FL

Las Vegas, NV
Las Vegas, NV
Las Vegas, NV
Las Vegas, NV
Las Vegas, NV
San Diego, CA
Golden, CO
Las Vegas, NV
Las Vegas, NV

Las Vegas, NV
Las Vegas, NV
Cincinnati, OH
Ada, OK
San Diego, CA
Valdez, AK
Ada, OK

San Diego, CA
San Diego, CA

Athens, GA
Athens, GA
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Gulf Breeze, FL
Cincinnati, OH

San Diego, CA
San Diego, CA
San Diego, CA
San Diego, CA
San Diego, CA
Seattle, WA
Anchorage, AK
San Diego, CA
San Diego, CA
                                    14

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     Beach hydraulics:  Battelle Northwest Laboratories

•    Toxicity testing:  Battelle and E.V.S. Consultants

CHRONOLOGY OF EVENTS

     After finalizing the Research Plan, an implementation
strategy was developed.  It was imperative to initiate the field
demonstration as quickly as possible to provide enough time
during the summer for large-scale application if results were
favorable.  Considering the extensive logistical problems that
were faced, a target date for field application of the nutrients
was set for June 6, 1989.  The following is an overview, in
approximate chronological order, of the different stages in the
project.

May 15 -    A command center for the Bioremediation Project was
            established in Valdez through the dedicated help of a
            local citizen, Bill Wyatt.  The center was used by
            both EPA and Exxon personnel.

May 16 -    A search for information on the characteristics of
            different fertilizer preparations that might be
            applied at the test sites was initiated.  Laboratory
            studies to determine nutrient release rates were also
            begun.  These studies are described in Section 5.

May 19 -    A beach survey team was assembled to select
            appropriate test sites.  Snug Harbor (southeast
            Knight Island) was selected as the initial test site.
            Efforts were initiated to establish test plots and
            set up a staging operation.  Description of this site
            is provided in Section 4.  Staging was greatly
            facilitated by the 93-foot fishing vessel (F/V)
            AUGUSTINE (under Exxon contract), which provided
            space for microcosms, microbiological and nutrient
            analyses, and sample-handling facilities as well as
            berthing space for field personnel.

May 20 -    Studies to provide information on the movement of
            groundwater and nutrients in the beaches were
            initiated once the staging operation was in place.
            Results from these studies are provided in Section 9.

May 23 -    Methods for adequately sampling contaminated beach
            material were developed at this time.  A sampling
            design was established, and methods for application
            of the fertilizers to the beaches were finalized.
            Information on these aspects is given in Section 6.
                                15

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May 25 -    Background information on the extent of oil
            contamination on the test beaches in Snug Harbor was
            collected, and chemical analyses were subsequently
            conducted.  These results are given in Section 7.

Jun 2  -    Extensive efforts were undertaken to develop an
            ecological monitoring program to look for potential
            toxicological and eutrophication effects.  Collection
            of background data from the site was started. These
            activities are summarized in Section 7 of this
            report.

Jun 4  -    Microcosm studies were designed to model the nutrient
            treatments on the test plots.  These studies had the
            best potential for determining weight changes in the
            oil as a result of biodegradation and nutrient
            enhancement.  The design and construction of
            microcosms that could be accommodated aboard the F/V
            AUGUSTINE was initiated in the first week of June.  A
            summary of the microcosm studies is given in Section
            9.

Jun 4  -    Because of a greater than expected time requirement
            in establishing a microbiology laboratory, only one
            type of method could be established to measure the
            number of oil degraders.  Microbiological media to
            perform these tests were prepared at the EPA
            Environmental Research Laboratory in Athens, Georgia,
            and shipped to Valdez for the first sampling.
            Results from the microbiology analyses studies are
            given in Section 7 of this report.

Jun 8  -    Following delays due to complications with logistics
            and weather, field application of the nutrients was
            begun.  Details of this application are given in
            Section 6 of this report.

Jun 12 -    A major rainstorm washed much of the oleophilic
            fertilizer off the beache.s.   This fertilizer was
            reapplied.

Jun 15 -    A workshop on "Beach and Nearshore Hydraulics" was
            held in Seattle, Washington, to determine the effects
            of tides, waves, and groundwater flow on the
            transport and transformation of nutrients in the
            porus, steep beaches in Prince William Sound.
                                16

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Jun 20 -    Visual loss of oil from the rock surfaces of the
            cobble beaches treated with oleophilic fertilizer was
            noticed.  The clearing remained apparent for
            approximately 3 weeks, at which time reoiling
            occurred reducing the contrast with untreated areas.
            The untreated control plots had not changed.

Jul 1  -    The first status report on the field demonstration
            project was submitted to Exxon.

Jul 18 -    A recommendation for large-scale application of
            fertilizers to beaches in Prince William Sound was
            submitted to Exxon.

Jul 24 -    A site at Passage Cove, on the north end of Knight
            Island, was selected for additional nutrient and
            beach hydraulics investigations on a beach that had
            been physically cleaned of spilled oil.

Jul 25 -    Application of the Oleophilic and slow release
            granular fertilizers was performed on Tern Beach.
            The sprinkling system on Kittiwake was also set into
            operation.

Jul 27 -    Wells were dug and the well equipment installed on
            the Kittiwake Beach plot in Passage Cove.

Aug 10 -    Passage Cove beaches treated with oleophilic and
            slow- release fertilizer showed marked removal of
            black oil residues relative to the reference beaches.

Aug 18 -    Clearing of oil from the beach at Passage Cove
            treated with fertilizer solution from a sprinkler
            system was evident.

Sep 4  -    Final sampling at Passage Cove.

Sep 8  -    Final sampling at Snug Harbor.

Sep 15 -    September 15 Addendum to the July 1 Status Report.

Nov 8  -    Workshop held in Gulf Breeze, FL, to discuss data
            analysis, interpretations, and significant findings.

Dec 22 -    Followup workshop held in Washington, D.C., in which
            the outline for a winter research plan was
            established.
                                17

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                            SECTION 4

                SITE SELECTION AND CHARACTERISTICS
     Two test sites were selected for the field demonstration
project, Snug Harbor and Passage Cove.  Snug Harbor was selected
to serve as a beach with oil contamination that approximated the
degree of contamination remaining after a heavily oiled beach had
been physically washed.  Physical washing was the major cleanup
procedure used by Exxon.  Physically washed beaches were not
available for testing early in the summer.  In July, a second
site was selected that had been physically washed by the Exxon
operations.  This site, Passage Cove, served as the main
reference beach for the large-scale application of fertilizers
and as a means to evaluate a sprayer system for fertilizer
application.

     Criteria for the selection of the test sites were based on
the following:

•    Typical shoreline of Prince William Sound; i.e., mixed sand
     and gravel and cobblestone beaches

     Sufficient area with fairly uniform distribution of sand,
     gravel, and cobble for the test plots

•    Protected embayment with adequate staging areas and
     sufficient size to support several test and control plots

     Uniform oil contamination

     Minimal impact from freshwater inputs

•    Shoreline with a gradual vertical rise

SNUG HARBOR PROJECT SITE

     Snug Harbor is located on the southeastern side of Knight
Island.  The shoreline utilized for the demonstration is located
on the western side of this harbor (Figure 4.1).  The area is
surrounded by mountains, reaching an elevation of approximately
2,000 feet, with steep vertical ascents.  Major sources of
freshwater runoff are from precipitation and snowmelt,  which is
typical of islands in Prince William Sound.  Although other
shorelines in Snug Harbor were heavily contaminated with oil, it
appeared that little oil was being released to the water,  thus
minimizing the prospect of reoiling on the beaches chosen for
treatment and reference plots.
                                19

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Ecological Monitoring Stations

D  10.0m from mean low tide
A  1.0m from mean low tide
O  caged mussels
0  control sites
      beach plots
                                                                Cobble Control .
                                                                      Prince
                                                                      William
                                                                      Sound
 Cobble Oleophilic



Cobble Water Soluble
               Gravel Water Soluble


                    Gravel Oleophilic
                         Gravel Control
                                    stream
  Figure 4.1.   Sampling Locations at Snug Harbor, Knight Island, in
                Prince William  Sound, Alaska.
                                     20

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     Table 4.1 identifies the beach types, dimensions, and
treatment.  Each plot was divided into 21 blocks, with 7 blocks
in 3 tidal zones:  high, intermediate, and low tidal areas.

     Oil contamination in the test area represented a continuous
band along the length of the beach.  This band was approximately
15 to 20 meters wide and corresponded roughly with the average
boundaries of the high and low tides observed in Snug Harbor.  To
determine the approximate distribution of oil on the beach,
samples of beach material from one of the designated mixed sand
and gravel plots were taken on May 25, 1989.  The samples were
extracted, and the oil weight and chemical composition were
determined.  Methods for the sampling and analysis are given in
Section 6.  The oil residue weights and ratios of C17/pristane
and C18/phytane at two different depths are shown in Table 4.2.
It is clear that oil concentrations varied considerably, ranging
from a high of 67,000 mg/kg of beach material to a low of 37
mg/kg of beach material.  In general, higher concentrations were
found in the top 10 cm of the beach.  Changes in the ratios,
relative to fresh Prudhoe Bay crude, were also apparent in some
samples, indicating biodegradation of the oil.  Changes were
quite variable, but it does appear that biodegradation was
occurring at the lower depths.

PASSAGE COVE PROJECT SITE

     Passage Cove is located on the northwestern side of Knight
Island.  This site was originally heavily contaminated with oil
and was subjected to physical washing by Exxon.  Even after
physical washing, considerable amounts of oil remained at this
site, mostly spread uniformly over the surface of rocks and in
the beach material below the rocks.  Pools of oil and mousse-like
material were minimal on the surface.  Contamination was apparent
to about 50 cm below the beach surface.  The shoreline area and
the designated beaches in Passage Cove are shown in Figure 4.2.
All of the beach areas tested were cobblestone set on a mixed
sand and gravel base.  Table 4.3 lists Passage Cove beach
designations and plot dimensions and fertilizer treatments.
                               21

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   Table 4.1. Description of Fertilizer Treatment Demonstration
   Plots at Snug Harbor
Beach Name


Eagle


Otter


Otter


Seal


Seal


Seal
Beach Type
Sand, gravel


Sand, gravel


Cobble


Cobble


Cobble
Fertilizer
Treatment
Length fm)
Sand, gravel     None-reference    21
Oleophilic        21
fertilizer

Water-soluble     35
fertilizer

Water-soluble     28
fertilizer

Oleophilic        28
fertilizer

None-reference    21
Depth (m)


   12


   12


   12


   12


   12
                                    22

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Table 4.2.  Analysis of Oil Extracted from Mixed Sand and Gravel Samples
Taken from Otter Beach on May 28, 1989, Two Weeks Prior to Fertilizer
Application*
                     TOP fO-10 cm)
                                           BOTTOM (10-20 cm)
Block No.

   1
   2

   3
   4
   5
   6
   7
   8
   9
  10
  11
  12
  13
  14
  15
  16
  17
  18
  19
  20
  21

Mean
Std Dev
Residue
wt. fma/ka)
100
29,000
30,100

6,070
2,030
6,600
1,440
1,030
7,600




9,820
658
67,200







C17/
Pristane"
0.8
1.6
1.5

1.5
1.2
1.2
1.1
0.8
1.4




1.5
1.5
1.6







CIS/
Phytane
1.0
1.9
1.7

1.8
1.5
1.7
1.4
1.1
1.7




1.8
1.9
1.8







Residue
wt. (ma/ka)
253
18,300

296
2,600
37




97
365
469
412
512
8
9,280
9,620
8,100
45
538
80
622
125
C17/
Pristane
0.9
1.6

1.0
1.5
0.8




1.1
0.8
0.9
0.9
1.2
1.0
1.6
1.5 .
1.6
0.9
1.3
0.9
1.1
0.9
C18/
Phytane
0.8
2.0

1.2
1.9
1.0




1.3
1.1
1.1
1.1
1.5
1.2
1.8
1.8
1.9
0.9
1.6
1.3
1.4
1.3
    12,242
+/- 18,556
                 1.0
    1.3
+/- 0.3
               1.3
    1.6
+/- 0.3
    1.790

    2,169
+/- 4,842
    1.1
+/- 0.3
    1.4
+/- 0.3
a The Otter Beach plot was divided into three equal zones lengthwise across
  the beach to represent high, mid, and low tide areas.  Each zone was
  divided into 7 equal blocks and blocks were numbered from left to right
  consecutively, starting with the high tide zone.
b C/17 Pristane and C18/Phytane ratios in fresh Prudhoe Bay crude oil are
  approximately 1.7 and 2.0, respectively.
                                     23

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                                 Prince William Sound

Ecological Monitoring Stations

a 0.5m, 5.0m from mean low tide
A 0.5m from mean low tide
O caged mussels
0 control site
       beach plots
                                                              bluff
                               Tern
                    (oleophilic fertilizer
                  + water soluble fertilizer)
   1   Kittiwake
(water soluble fertilizer)
     Figure 4.2.    Sampling Locations at Passage Cove, Knight Island, in
                     Prince William Sound, Alaska.
                                          24

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Table 4.3.  Description of Fertilizer Treatment Demonstration Plots at
Passage Cove
Beach Name
Beach Type
Nutrient
Applications
Length fm)  Depth
Raven
Cobble over mixed
sand and gravel
None-reference
     28
21
Tern
Kittiwake
Cobble over mixed
sand and gravel
granules

Cobble over mixed
sand and gravel
Oleophilic and      35
slow-release
Nutrient solution   28
sprinkler system
             21
             21
Guillemot
Mixed sand and
gravel with patchy
cobble
Oleophilic and
granules
slow-release
     21
                                    25

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                             SECTION 5

              FERTILIZER SELECTION AND  CHARACTERISTICS
 BACKGROUND

      An important aspect of this project was the selection of
 fertilizers for the field test.  The goal was to find fertilizer
 formulations which would release nitrogen and phosphorous
 nutrients over extended time periods or would keep nutrients in
 contact with surface microbial communities over extended time
 periods.  In addition, consideration was given to formulations
 that were amenable to practical and inexpensive application to
 contaminated shorelines, keeping in mind the possibility of
^Large-scale applications in the future.  Three types of
 fertilizer were selected:

 1)   Solid, slow-release fertilizer, in which nutrients would be
      released slowly from a point source and tidal action would
      distribute the nutrients over the beach surface.

 2)   Liquid oleophilic fertilizer, in which nutrients would
      "dissolve" into the oil covering the rock and gravel
      surfaces.  Nutrient distribution over the beach material
      would be accomplished in the original fertilizer
      application.

 3)   Fertilizer solutions, in which inorganic nitrogen and
      phosphorus would be dissolved in seawater and distributed
      via fixed sprinkler systems.

      Several commercially available fertilizer formulations that
 satisfied these requirements were selected and their nutrient-
 release characteristics determined.  A small study was also
 conducted to see how specific solid fertilizer formulations
 physically behaved under field conditions.
 SELECTED FERTILIZER FORMULATIONS

      The fertilizers selected are described below.

      An oleophilic fertilizer was incorporated for testing under
 recommendation by participants in the original April workshop
 held to evaluate the feasibility of bioremediation.  It was
 suggested that the oleophilic application might be suited for
 application to cobble and rocky beaches where the application of
 soluble nutrients might prove difficult or ineffective.
                                27

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"Woodace" Briquettes

     This fertilizer formulation contains isobutylidene diurea
(IBDU),e a chemical that spontaneously hydrolyzes into isobutyl
aldehyde and urea when released from the briquette matrix into
water.  This process is responsible for the slow release of
nitrogen that is characteristic of the product.  Hydrolysis is
temperature dependent, being slower at lower temperatures but
still  significant.  The source of phosphorus is Linstar, a citric
acid soluble phosphatic fertilizer developed by Mitcubichi
Chemical Corp.  Each briquette weighs approximately 17 grams and
has a  specific gravity of 1.5 to 1.8.  This fertilizer has a
N:P:K  ratio of 14:3:3.

IBDU Granules

     This fertilizer is a granular 24:4:12 (N:P:K) fertilizer
formulated to give an immediate and sustained release of
nutrients.  The product used was Par Ex, produced by Estech, Inc.
All of the nitrogen is derived from ammonium phosphate, urea, and
IBDU.  A minimum of 45% of the nitrogen is derived from the IBDU.
The available phosphorus is derived from potassium magnesium
phosphate.  Iron is also present as ferrous sulfate.  The
granules have a specific gravity of 1.3.

"Osmocote" Briquettes

     Manufactured by Sierra Chemicals, this fertilizer contains
urea formaldehyde as the nitrogen source in a slow release
formulation created by thermoplastic resin encapsulation.  The
urea formaldehyde released from the briquettes must be
biologically hydrolyzed to produce ammonia.  This fertilizer has
an N:P:K ratio of 20:10:5.  Phosphorus is present as calcium
phosphate.  Iron is also present as ferrous sulfate.  Each
briquette weighs 21 grams.
     'isobutylidene  Diurea  (IBDU)  is  a  condensation  product of
urea and iso-butyraldehyde.  The reaction can be carried out both
in aqueous solution and between solid urea and liquid aldehyde as
follows:
CH3
CH3
   > CHCHO +  2 NH2CONH2 =
     CH3             NHCONH2
          > CHCH <           + H20
     CH3             NHCONH2
                                28

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"MAGAMP"

     This fertilizer formulation contains magnesium ammonium
phosphate (MAGAMP), which is sparingly soluble in water.  It is
made by Martin Marietta Magnesia Specialties.  The chemical
congeals when wetted and can then be cast into different shapes
and dried into solid forms (granules, briquettes, bricks) of
varying densities, which will slowly release ammonia and
phosphate when submersed.  This fertilizer has a N:P:K ratio of
9.2:27:0.  This fertilizer is available in briquettes that weigh
209 gm each and bricks of 8 or 40 Ibs.

"Sierra Chemical" Granules

     This fertilizer formulation consists of inorganic nutrient
sources (ammonium nitrate, calcium phosphate and ammonium
phosphate) contained in a vegetable oil coating  (polymerized by
reaction with a diene).  The coating gives the fertilizer its
slow-release characteristic.   The N:P:K ratio is 28:8:0.  The
granules have a specific gravity of 1.8.

Oleophilic Fertilizer

     The only oleophilic fertilizer that was available in
sufficient amounts to use in a scaled-up operation was the Elf
Aquitaine (France) product, Inipol EAP 22.  This is a mixture of
nutrients encapsulated by oleic acid (the external phase).  Oleic
acid and surfactants in the fertilizer formulation cause the
nutrients to become sequestered to the oil phase, preventing
rapid release of the nutrients into the aqueous phase and
subsequent washout.  Inipol EAP 22 is a clear liquid with a
specific gravity of 0.996, a viscosity of 250 cst, a pour point
of 11°C, and a flash point of >100°C.  The N:P:K ratio is
7.3:2.8:0.

     The main ingredients in Inipol EAP 22 are oleic acid and
urea, along with chemicals to maintain them in a microeroulsion.
The chemical composition of Inipol is given in Table 5.1.  The
product is designed to initially stimulate oleic acid degrading
bacteria.   The quantity of nitrogen and phosphorus present is
sufficient to allow the natura,! oleic acid degraders found in the
receiving environment to consume all of the oleic acid carbon
present in the Inipol.  Once the added oleic acid is consumed,
and the numbers of oleic acid degraders have increased
substantially, oil biodegradation is thought to commence.  It is
not exactly clear why degradation of oil hydrocarbons occurs in
this way,  but many oleic acid-degrading bacteria are known to
                                29

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   Table 5.1.  Inipol EAP 22 Chemical Composition
CHEMICAL

Oleic acid
CHEMICAL FORMULA

CH3 (CH2) 7CH=CH (CH2) 7COOH
Lauryl Phosphate


2-Butoxy-l Ethanol


Urea

Water
NH2-C-NH2

H20
PURPOSE

Oleophilic Phase
(Continuous)

Primary Carbon
Nutrient

Phosphate Nutrient
Surfactant

Co-Surfactant
Emulsion Stabilizer

Nitrogen Nutrient

Hydrophilic Phase
                                   30

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degrade petroleum hydrocarbons.  Elf Aguitaine representatives
have suggested that the oleic acid degrading microorganisms may
die once they reach a certain density, creating a natural
recycling of nutrients through mineralization of this dead
biomass.

     Oleophilic fertilizer has been shown to work well on sandy
beaches, but similar testing has not been done with rock and
cobble beaches.  Therefore, its use on the rock and cobble
beaches found in Prince William Sound represents a new
application.

NUTRIENT RELEASE CHARACTERISTICS - METHODS

Static Tests

     A specific weight of slow-release soluble fertilizer was
placed in cloth bags in a beaker and covered with 1400 mis of
artificial seawater (Instant Ocean, supplied by Aquarium Systems,
Inc.)  Granules were contained in cloth bags.  The beakers were
incubated at 15"C without mixing.  According to an established
schedule, water was decanted out of the beaker and replaced with
fresh seawater.  The amounts of ammonia (EPA method 350.1),
nitrate (EPA method 353.1), total phosphorus (EPA method 365.4),
and total Kjeldahl nitrogen (TKN) (EPA method 365.4) were
measured in the decanted water.

     Flask studies, to determine the effect of microbial
activities on the release of ammonia from the TKN leached out of
the IBDU briquettes, were conducted by covering briquettes with
three types of water:  deionized, sterile (filter sterilized -
0.22/i) seawater, and non-sterile seawater.  The amount of ammonia
released over time was determined.  The experiments were also run
at two different temperatures (9' and 21°C).

     The oleophilic fertilizer was applied to the surface of oil-
 contaminated rocks obtained from Prince William Sound.  The
rocks were then covered with seawater.  Varying amounts of
fertilizer were applied to determine .the best application rate
for retention of Inipol on the oiled rocks.   Excess fertilizer,
when not adsorbed to the oiled surfaces, loses its solution
properties on contact with water and releases urea very rapidly.

Intermittent Submersion Tests

     A rocker table equipped with a 19" long, 4" wide pipette
tray was used to simulate tidal action and the intermittent
submersion of the different fertilizer types.  A nutrient bag or
nutrient briquettes were buried in clean Alaskan beach material
at one end of the tray, or the oleophilic fertilizer was sprayed
directly on beach material and artificial seawater added.  The
device was maintained in a cold room at a temperature of 15*C.

                               31

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The rocker table was started and samples were taken after 5, 15,
30, and 60 minutes.  The table was then stopped, the water
drained, and the beach material allowed to remain undisturbed for
4 hours.  The rocks were covered again with water, the table was
operated for another hour, and sampling was repeated.

Field Tests

     To test nutrient release characteristics of MAGAMP under
field conditions, 8 Ib and 40 Ib MAGAMP bricks, were placed on a
sand and gravel beach at Snug Harbor and samples were taken at
different locations down the beach from the bricks.  Sampling
points were placed to measure the downward and lateral spread of
nutrients released from the bricks during tidal cycle.  The
weight of the bricks was expected to minimize their movement.
Nutrient samples were collected 12, 24, and 96 hours after
placing the bricks on the beach.

NUTRIENT RELEASE CHARACTERISTICS - TEST RESULTS

"Woodace" IBDU Briquettes

     The cumulative nutrient release pattern for ammonia, total
phosphorus, and TKN from static tests with the IBDU briquettes is
shown in Figure 5.1.  Nitrate was formulated into the briquettes.
Release rates per day for each nutrient are shown in Figure 5.2.
Rates for ammonia release were generally constant, excluding the
initial rapid release.  Release rates for TKN dropped
dramatically over the 60-day test period.  Release rates for
total phosphorus, although somewhat variable, were constant over
time.  The variability was probably associated with analytical
error.  Overall, release rates were slightly increased for all
nutrients if the frequency of water exchanges rates was increased
5% (data not shown).

     It is apparent that small amounts of ammonia and phosphorus
are released with each 24-hour soaking of the briquettes.  The
average amount of ammonia released per day is approximately 100-
fold higher than background levels in Snug Harbor waters (see
Section 8:  Nutrients).  However, considering the rapid dilution
of ammonia that will occur in the field following release from
briquettes, it would be unlikely to measure any increased
concentrations of ammonia (discarding contributions from the TKN)
in the field as a result of fertilizer application.  The total
amount of ammonia released is only a small fraction of the total
nitrogen available in the formulation.

     Large amounts of TKN were released, accounting for as much
as 17% of the total available nitrogen after 17 days, 31% after
60-days, and 45% after 140 days.  The TKN is probably urea since
the IBDU will rapidly hydrolyze on release from the briquettes.
Urea, in the absence of bacteria would not be hydrolyzed to NH4.

                                32

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U)

OJ
       100
        90 -
    <£  80
    TJ
    0)

    w  70 -


    15
    oc

    «-  60
    c
    0)


    3  50 •
|  40

'5
    *••
    o
        30
        20 -
        10 —
                                                                    TKN
                                                                    Total Phosphorus
                                                                    Ammonia
                                                                           AAAAAA
                                         60        80


                                          Time (Days)
                                                        100
120
140
            Figure 5.1. Cumulative Release of Ammonia, Total Kjeldahl Nitrogen (TKN),
                       and Total Phosphorus from Woodace IBDU Briquettes in Static
                       Flask Experiments.

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X
Z
                                                                    160
                                                                 — 140
                                                                 — 120
                                                                 — 100
                                                                    80
                                                                 — 60
                                                                    40
                                                                 — 20
                                                                          (Q
                 10
60
                               Time (Days)
 Figure 5.2 Daily Nutrient Release Rate of Ammonia (NH4), Total Phosphorus
           (TP),  and Total Kjeldahl Nitrogen (TKN) from IBDU Briquettes.

-------
     To determine if the TKN was a source of ammonia under
natural conditions, briquettes were soaked for 3 successive
1-hour periods (water was changed for each period) using 2
different temperatures and 3 different sources of water.  The
results are shown in Figure 5.3.  Significant amounts of ammonium
were released under all conditions.  The lowest amount of ammonia
released occurred with filtered seawater (containing no
microorganisms).  Since this was less than that released in the
presence of deionized water, it suggests that there was an ionic
strength effect on the ammonia release.  Temperature had little
effect on release rates in these media.  However, with unfiltered
seawater, considerably more ammonia was released, particularly at
the higher temperatures.  This indicated a possible biological
effect on ammonia release, presumably through the microbial
breakdown of the TKN fraction.

     Nutrient release from the IBDU briquettes was also tested
using the rocker table system.  In general, concentrations of the
nutrients and TKN released were similar to those observed in the
static tests.

     Under all conditions, the physical integrity of the IBDU
briquettes was excellent with very little change in shape and
consistency occurring after one month of submersion in water.
A simple freeze/thaw experiment was also conducted on the Woodace
briquettes.  The experiment consisted of alternately freezing and
thawing submerged and non-submerged briquettes, then weighing and
visually observing changes.  Preliminary results indicated good
durability.  The briquettes appeared to be a good choice for
field application.

     Studies were also performed on the movement of briquettes
broadcast on the beach.  Results showed that unconfined
briquettes will not retain this position distributed after
several tide cycles.  Greater redistribution occurred on sand and
gravel beaches as compared with cobble beaches.  Due to the rapid
redistribution of briquettes on sand and gravel beaches, this
form of fertilizer application is best applied in containers
which will hold the briquettes in place.  Unconfined briquettes
may be of some limited use on sheltered cobble beaches, where
wave action may have less influence on the beach.

IBDU Granules

     The cumulative nutrient release pattern for ammonia,
phosphate, and TKN from IBDU granules in bags using static tests
is shown in Figure 5.4.  Typically high amounts of nutrient were
released initially, followed by a very slow release.  If the
experiment was repeated with the granules layered on the bottom
of the beaker (i.e., no bag to contain the granules),  a slower
and more gradual nutrient release pattern was observed.  This is
shown in Figure 5.5.  The greater initial release when the

                                35

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          6)

          o

          I
          o
          O

          .2
          n
          o
          O
350



300



250



200



150



100



 50
              350
              300
              250
          5   200
150


100



 50
                      9°C
                     21°C
Deionized Water
Filtered Seawater
Unfiltered Seawater
                                 1          2

                                  Soaking Time (Hours)
                                  1          2

                                 Soaking Time (Hours)
Figure 5.3.   Ammonia Release From IBDU Briquettes at 9 and 21 Degrees
             Centigrade in 3 Different Water Sources.
                                   36

-------
Co
         T3
         0)
         CO
         (0
         
-------
          100
OJ
OO
           10-
                                                 * • *
                                                         *  *«*
                                                                    Total  Phosphorus
                                                                    TKN
                                                                    Ammonia
                    i    i    i    i    r    i    i     i    i
                            20            40           60

                                              Time (Days)
\     \    I     I    I     I

        80           100
          Figure  5.5. Cumulative Release of Ammonia, Total Kjeldahl Nitrogen (TKN), and
                    Total Phosphorus from IBDU Granules in Static Flask Experiments.

-------
granules were contained in bags was probably due to either the
methods of exchanging water in these experiments (unbagged IBDU
granules were drained through a fine mesh, whereas bagged
granules were drained through very fine mesh cloth) or increased
water contact experienced by the unbagged granules.  In the later
case, unbagged granules were loosely packed in the bottom of the
flasks, and the water was stirred both before and after each
water exchange.  Granules in bags were tightly packed, and the
water was not stirred when it was exchanged.

     Additional studies also showed that if the granule bag
volume is reduced relative to the bag surface area, slightly more
nutrient release occurred (Table 5.2).  Thus, the more water
passing over the granules, the higher the release rate.

"Osmocote" Briquettes

     The cumulative nutrient release pattern for ammonia,
phosphate, and TKN for static tests is shown in Figure 5.6.
After 2 months of testing, approximately 25% of the available
nitrogen was released, primarily as TKN, where as almost 60% of
the phosphate was released over this time period.  The physical
form of the briquette was unstable, flaking soon after initial
submersion and further decomposing over time.  A dye within the
briquette turned the water green with each water exchange.  These
briquettes, despite their good nutrient release characteristics,
appeared unsuitable for long-term use in the field.

MAGAMP Briquettes

     When high-density and low-density MAGAMP briquettes were
tested, the low-density briquette  (about half the weight of the
high density) disintegrated almost immediately upon submersion in
the defined nutrient medium, and consequently was not tested.
Release of ammonia, TKN, and phosphate from the high-density
briquettes was slow and constant (Figure 5.7).  After 10 days,
only 1.5% of the available nitrogen was released.  At 75 days,
approximately 5% to 6% had been released.  The release rate
appeared to be independent of the number and volume of water
exchanges.  The high-density briquettes appeared to be very
durable.  When MAGAMP powder was tested, it congealed to a putty-
like consistency soon after the experiment was started.
Accumulative release of ammonia was about the same as the
briquettes.

     Results from the intermittent submersion tests showed the
same low release rates.  A high burst of ammonia seen within the
first half hour of the test was explained by initial flaking of
the briquettes.  Flaking at later times did not occur.
                                39

-------
Table 5.2.  Total Kjeldahl Nitrogen  (TKN) Released from IBDU Granular
Fertilizer in Bags, Static Water Conditions
Bag Volume/
Surface Area
(cubic cm/square cm)
Fertilizer
Weight fen
Seawater
Volume (ml)
% of Cumulative
Available Nitrogen
(TKN) Released in:
24 hours  45 Days
1.8
1.3
1.3a
0.6
893
256
258
32
4,800
1,400
1,400
450
34
36
39
41
38
42
46
49
"Prince  William Sound beach material.
                                    40

-------
   100
    90 -
^  80 -
0)   7H

-------
   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

-------
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)

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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

-------
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

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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

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     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

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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

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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

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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

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                            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

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      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

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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

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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)
O
C
O
O
          BLOCK=15
         ta
            17 IB
BLOCK = 9
1.4

U
IJ
U
0*
04
u
u
K
/
/
/
/
/
/
(-"

fv
r\

h\
r

i
17 IB
BLOCK=16
14
U
OJ
a;
u
u
»4
«J
eu
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
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                            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
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                                              BLOCK=5
                BLOCK = 6
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                                              17 18
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                                           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
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                                                                                         17 18
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                                                                                                    BLOCK=14
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                                17 18
                                                17 18
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                                                                                17 18
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                                             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
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                                    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.

-------
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                                        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
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                                   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
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                                                                               BLOCK=5
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                                             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

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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

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 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

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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

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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

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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

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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

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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

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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

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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

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                            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

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                         SNUG HARBOR
li
  !••
  II
  1C
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                                      t"  u
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                                        u
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                        S = \ 3
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                         Figure 7.22 (Continued)
                              119

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                               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
                                              
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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

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                             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

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                               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

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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

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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

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                            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

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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

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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
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0>
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     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.

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     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
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                                     n-Alkanes (n-C12  to n-C32)
                                                                      17 18
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
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-------
             Passage  Cove - Cobble Surface  - Water Soluble  Fertilizer - 3  Weeks After Application
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                                           n-Alkanes (n-C12 to n-C32)
     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
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             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.

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                       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.

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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.

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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

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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

-------
     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

-------
                                 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

-------
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

-------
                               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

-------
                          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

-------
                 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

-------
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

-------
 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

-------
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

-------
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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, 	 I
1
.flrin.rL - -
          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

-------
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                                   6 weeks
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                                             A.  Acenaphthene
                                             B.  Acenaphthylene
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                                             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

-------
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 -
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Nutrient « -
Medium % j
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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 .
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0-

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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

-------
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

-------
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

-------
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

-------
      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

-------
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

-------
                                                                 mean low tide
Figure 9.13.  Location of Wells for Beach Hydraulics Experiment at Passage Cove
                                     194

-------
           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

-------
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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                            REFERENCES
Caron, D.A.,  1983.  Technique  for Enumeration of Heterotrophic
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).
                               224

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