EPA-600/3-C4-071
June 1984
A SURVEY OF THE TOXICITY
AND CHEMICAL COMPOSITION
OF USED DRILLING MUDS
by
Fdgerton Research Laboratory
New England Aquarium
Boston, Massachusetts 02110
Project CR806776
Project Officer
Thomas W. Duke
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FL 32561
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing!
1. REPORT NO.
EPA-600/3-84-071
2.
3. RECIPIENT'S ACCESSION NO,
PBR A 207AM
4. T.TL6 ANO SUBTITLE
A SURVEY OF THE TOXICITY AND CHEMICAL COMPOSITION
OF USED DRILLING MUDS
5. REPORT DATE
June 1984
6. PERFORMING ORGANIZATION CODE
7. AUTHORlS)
Edgerton Research Laboratory
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
New England Aquarium
10. PROGRAM ELEMENT NO.
Boston, Massachusetts 02110
11. CONTRACT/GRANT NO.
CR 806776
12. SPONSORING AGENCV NAME ANO ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research and Development
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/04
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Chanical characterization and toxicity of oil drilling fluids were
investigated by the Edgerton Research Laboratory from 1 October 1979 to
August 1983 as part of a comprehensive research program sponsored by the
U.S. Environmental Protection Agency (EPA) to determine fate and effects of
such fluids in the^narine environment. Drilling muds used in the research
were supplied by the EPA, the Petroleum Equipment Suppliers Association
(PESA), and the American Petroleum Institute (API). The drilling muds were
designated "May 15," "May 29," "Sept. 4," "Exxon," "Gilson," "Mobile Bay,"
"Jay Field," and "PESA." Investigations during the first year centered on
the chemical composition and the acute toxicity of drilling muds, and the
effects of drilling muds on the recruitment of benthic organisms. In the
second year, studies focused on toxicity testing with planktonic copepods,
chanical characterization of the toxicity test phases, bioaccumulation
studies, and the effects of muds onllarval and adult benthic organisms.
Investigations during the ,third and fourth year examined sublethal effects
of drilling fluids on clanv^larvae, trace metal and organic constituents in
both drilling fluids and toxicity test-phases, and the preliminary development
of a drilling fluid solid phase toxicity test.
1 7.
KEY WORDS AND DOCUMENT ANALYSIS
a DESCRIPTORS
b. IDENTiFIERS/OpEN ENDED TERMS
c. COS AT I I icki/Group
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY Class (This Report!
unclassified
21. NO. OF PAGES
\ 124
20. SECURITY CLASS (This page)
unclassified
22. PRICE
EPA Form 2220-1 (R«v. 4-77) drevious eo tion 5 obsolete
i
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DISCLAIMER
The information in this document has been funded wholly or in part
by the U.S. Environmental Protection Agency under cooperative agreement
number CR 806776 to Edgerton Research Laboratory, New England Aquarium,
Boston, Massachusetts. It has been subject to the Agency's peer and
administrative review and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or reconmendation
for use.
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FOREWORD
The protection of our estuarine and coastal areas from damage caused
by toxic organic pollutants requires that regulations restricting the intro
duction of these compounds into the environment be formulated on a sound
scientific basis. Accurate information describing dose-response
relationships for organisms and ecosystems under varying conditions is
required. The Environmental Research Laboratory, Gulf Breeze, contributes
to this information through research programs aimed at determining:
. the effects of toxic organic pollutants on individual species
and communities of organisms.
. the effects of toxic organics on ecosystems processes and
components.
. the significance of chemical carcinogens in the estuarine and
marine environments.
This report summarizes findings on the impact of drilling fluids (muds)
on selected marine organisms and the chemical composition of several fluids.
These data provide needed information on the effects of used drilling fluids
on the clam, Mercenaria mercenaria, and other marine organisms and relates,
where possible, effects to components of the drilling fluid. Results of
this research will provide the regulatory arm of the Agency, and others,
an additional data base on the fate and effects of drilling fluids that
can be applied to the permitting process.
Di rector
Environmental Research Laboratory
Gulf Breeze, Florida
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ABSTRACT
Chemical characterization and toxicity of oil drilling fluids were
investigated by the Edgerton Research Laboratory from 1 October 1979 to
August 1983 as part of a comprehensive research program sponsored by the
U.S. Environmental Protection Agency (EPA) to determine fate and effects of
such fluids in the marine environment. Drilling muds used in the research
were supplied by the EPA, the Petroleum Equipment Suppliers Association
(PESA), and the American Petroleum Institute (API). The drilling muds were
designated "May 15," "May 29," "Sept. 4," "Exxon," "Gilson," "Mobile Bay,"
"Jay Field," and "PESA." Investigations during the first year centered on
the chemical composition and the acute toxicity of drilling muds, and the
effects of drilling muds on the recruitment of benthic organisms. In the
second year, studies focused on toxicity testing with planktonic copepods,
chemical characterization of the toxicity test phases, bioaccunrulation
studies, and the effects of muds on larval and adult benthic organisms.
Investigations during the third and fourth year examined sublethal effects
of drilling fluids on clam larvae, trace metal and organic constituents in
both drilling fluids and toxicity test-phases, and the preliminary development
of a drilling fluid solid phase toxicity test. Toxic components of the used
drilling muds tested were present as dissolved components or associated with
very slowly settling particles. Some used drilling muds contained
lipophilic fractions that were similar to hydrocarbons found in #2 fuel oil
in the liquid fraction and suspended particulates fraction and contained
#2 fuel oil in whole muds. Muds that contained those components were more
toxic than those that did not. Juvenile copepods (Acartia tonsa) were not
more sensitive to toxic drilling mud solutions than adults of this species.
In general, Cancer irroratus larvae appeared to exhibit toxicity responses
to drilling muds that were similar to the copepods tested. Arrested shell
development induced by exposure to drilling muds appeared to be a sensitive
indicator of stress in bivalve larvae. Total chromium concentration showed
no correlation to toxicity in the drilling muds that were tested; however,
the highest concentrations of Cr(VI), the most biologically toxic form of
chromium, occurred in the test phases that exhibited the greatest toxicity
to Mercenaria mercenaria larvae. The muds designated "May 15" and "Sept.
4" appeared to be relatively non-toxic to Pseudopleuronectes americanus and
to Menidia menidia, although the "May 15" mud was toxic to Neomysis americana
and to Acartia tonsa. A study of the effects of drilling mud on invertebrate
recolonization of defaunated sediment showed that recolonization decreased
in drilling mud layered on top of sediment when the muds were mixed with
sediments. Capitella capitata was much more numerous in recolonization
sediments that contained drilling mud. Test results showed that the methods
used to prepare drilling mud test media affect the apparent toxicity of the
muds.
iv
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TABLE OF CONTENTS
Page
ABSTRACT iv
LIST OF FIGURES vii
LIST OF TABLES V111
LIST OF CONTRIBUTORS X
OVERVIEW CONCLUSIONS
INTRODUCTION: PREVIOUS DRILLING FLUID STUDIES AT THE EDGERTON
RESEARCH LABORATORY, NEW ENGLAND AQUARIUM i
I. EFFECTS OF USED DRILLING FLUIDS ON THE EMBRYONIC
DEVELOPMENT OF THE HARD CLAM Me rcenari a me rcena ri a (L) II
1. 1 Background II
1.2 Materials and Methods 13
1.2.1 Conditioning and Holding of Brood Stock 13
1.2.2 Spawning and Rearing 15
1.2.3 Exoerimental Procedure 16
1.2.4 Statistical Analysis 19
1.3 Results 19
1.3.1 Year-round Toxicological Testing 19
1.3.2 One-Hour Fertilized Egg Toxicity Test 20
1.4 Discussion 25
II. TRACE METALS IN DRILLING FLUID/SEA WATER TOXICITY
TEST PHASES 33
2. 1 Background 33
2.2 Materials and Methods 35
2.2.1 ^articulate and Dissolved Metals 35
2.2.2 Free Metal 35
2.2.3 Chromium Speciation 37
2.2.4 Trace Metal Analysis 37
2.3. Results and Discussion 38
2.3.1 Preliminary Studies 38
2.3.2 Test Phase Results 40
2.3.3 Barium 46
2.3.4 Chromium 47
2.3.5 Free ChromiumC111) vs ChromiumCVI) 48
v
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TABLE OF CONTENTS (Continued)
Page
2.3.6 Copper 49
2.3.7 Manganese 51
2.3.8 Zinc 51
2.3.9 Conclusions 52
III. ORGANIC CONSTITUENTS IN WHOLE DRILLING FLUID AND
DRILLING FLUID SEA WATER TOXICITY TEST PHASES 54
3. 1 Background 54
3.2 Materials and Methods 55
3.2.1 Whole Drilling Fluid Analyses 55
3.2.2 Drilling Fluid/Sea Water Test Phases 56
3.3 Results and Discussion 57
IV. SOLID PHASE RECOLONIZATION STUDIES 64
4.1 Materials and Methods 64
4.2 Results 72
4.2.1 Laboratory-Based Experiment 74
4.2.1.1 Two Week Samples 74
4.2.1.2 Four Wee* Samples 79
4.2.1.3 Comparison of Recruitment Periods .... 80
4.2.2 Field-Based Experiment 82
4.2.2.1 Two Week Samples 82
4.2.2.2 Four Week Samples 83
4.2.2.3 Six Week Samples 87
4.2.2.4 Comparison of Recruitment Periods .... 88
4.3. Discussion 91
4.4. Evaluation of Methodology for Solic Phase
Recolonization Tests 98
V. LITERATURE CITED 100
vi
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LIST OF FIGURES
FIGURE Page
1. Unit for testing effects of drilling fluids on clam embryos.. 17
2. EC50 values for M_;_ mercenaria larvae after a 48 hour exposure
to drilling fluid 23
3. EC50 values for mercenaria larvae after a 48 hour exposure
to drilling fluid 24
4. Laboratory-based Experimental System 67
5. Arrangement of Test Containers for Labcratory-based
Experiment 68
6. Platform Used for One Recruitment Period in Field-based
Experiment 69
7. Arrangement of Test Containers for Field-based
Experiment 70
8. Number of Individuals Collected after Two-, Four-, and
Six Week Recruitment °er iods for Control, Homogeneous
and Surface Test Treatments in Laooratory- and Field-
based Experiments 73
9. Laboratory-based Experiment: Total Number of Individuals
for Important Taxa in Control, Homogeneous and Surface
Test Treatments over Two Recruitment Periods 81
10. Field-based Experiment: Total Number of Individuals for
Important Taxa in Control, Homogeneous and Surface
Test Treatments over Three Recruitment Periods 89
vii
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list of tables
TABLE Page
I -A. 48 hour drilling fluid toxicity test: suspended solids phase ... 21
I-B. 48 hour drilling fluid toxicity test: liquid phase 22
II. Chemical and physical characteristics of the used drilling
fluids employed in the Mercenaria mercenaria larval
toxicity tests 26
III. Concentration and speciation of barium in drilling fluid/
sea water test phases 41
IV. Concentration and speciation of chromium in drilling fluid/
sea water test phases 42
V. Concentration and speciation of copper in drilling fluid/
sea water test phases 43
VI. Concentration and speciation of manganese in drilling fluid/
sea water test phases 44
VII. Concentration and speciation of zinc in drilling fluid/
sea water test phases 45
VIII. Free chromium (III) and chromium (VI) from Donnan dialysis of
drilling fluid/sea water liquid phases 53
IX. Bulk characteristics of whole drilling 'luids 58
X. Analyses of drilling fluids for #2 fuel oil 60
XI. Concentration of #2 fuel oil-like hydrocarbons in drilling
fluid/sea water test phases 61
XII. Total number of Individuals (N), soecies (S), and phyla (P)
per treatment and for all treatments combined 75
XIII. Numcer of individuals (N), number of species (S), and diversity
index (S/N), for control, homogeneous and surface test treatments
in the laboratory- and field-based experiments 76
XIV. Laboratory-based experiement: Faunal distribution by phylum
for control, homogeneous and surface test treatments over
two recruitment periods 77
XV. Laboratory-based experiment: List of predominant species for
control, homogeneous and surface test treatments over two
recruitment periods 78
viii
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LIST OF TABLES (Cont'd)
T ABLE Page
XVI. Field-based experiement: Faunal distribution by phylum
for control, homogeneous and surface test treatments over
three recruitment periods 84
XVII. Field-based experiment: List of predominant species for
control, homogeneous and surface test treatments over three
recruitment periods 85
ix
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CONTRIBUTORS TO THIS REPORT:
Albert 3. Barker
Elke Bergholz
Paul 3. Boyle
Thomas J. Coffey
Thomas R. Gilbert
Joseph P. KakareKa
Beth A. Penney
William E. Robinson
Oavid K. Ryan
Kathleen Smith
Lisa A. Urry
Oavid A. Wayne
George A. Zoto
Elgerton Research Laboratory
New England Aquarium
Central Wharf
Boston, MA 02110
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OVERVIEW - CONCLUSIONS
Drilling muds used in this research were supplied by the Gulf
Breeze Environmental Research Laboratory (EPA) and the Petroleum
Equipment Suppliers Association (PESA), and by the American Petrolejm
Institute (API). The muds were designated as follows: muds from
Mobile Bay were called "May 15", "May 29", and "Sept. 4"; additional
muds from API and PESA were labelled "Exxon", "Gilson", "Mobile Bay",
"Jay rield", and "PESA". The conclusions listed below are meant to
provide general overview statements concerning the findings of a
series of research projects. Due to the complexity and the diversity
of the tests that were conducted, the final report and progress
reports should be consulted for the specific criteria and conditions
of each test.
1. Toxic compcnents of the muds that were tested were present as
very slowly settling species. For most of the elements analyzed
(8a, Cd, Cr, Cu, re, Hg, Mn, Ni , Pb, Zn) suspended/dissolved
concentrations following slow speed centrifugation (600 x gravity
(G) for 15 min) and six days of further settling were greater
than those observed in samples that were centrifuged for 15 min
at 30,000 x G.
2. Both high and low speed centrifugation of drilling mud
suspensions yielded supernatants with barium concentrations in
excess of those expected based on the solubility of BaS34 in
sea water; barium concentrations significantly above background
occurred in mixtures (1 ppm mud concentration) of tne medium
density lignosulfonate muds tested ("Sept. 4" and "Gilson").
Sea water suspensions (10 mL mud in 990 mL sea water) of tne
three muds labelled "May 15", "May 29", and "Sept. 4" contained
no detectable amounts of cadmium, mercury, nickel, lead, or
aluminum. Results from the measurements of trace metals in
drilling fluid - sea water mixtures snowed tKat the average
concentrations of the detectable elements decreased in the order
8a>Cr>Mn»Zn>Cu (in drilling fluids obtained from EPA, API, and
PESA that were labelled: "AN31"," MIBLKA51", "SV76", "PI", "P2",
"P3" "?4". "P5". "P6". "P7" . "P8". and "Sept. 4".).
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3. Early comparisons of drilling mud toxicity data with the total
chromium content of tne muds revealed no clear correlation
relationship. However, metal spsciation is an important factor
in bioavailability and toxicity. An analysis of cnromijm
speciation showed that the liquid phase of the mud designated
SV-76 had the highest concentration of Cr(VI), the most
biologically toxic form of chromium. This pnase was also one of
the most toxic to Mercenaria mercenaria larvae.
4. The drilling muds laoelled "Gilson", "May 15", "May 29", and
"Sept. 4" contained lipophilic fractions that were similar to #2
fuel oil. The "Seat. 4" mud contained approximately 1.15 "rig of
hydrocarbons per gram of mud that were similar to #2 fuel oil
hydrocarbons. Drilling muds that contained organic components
similar to #2 fuel oil were more toxic than those that did not.
In acute toxicity tests with the ccpepod Acartia tonsa, "Exxon"
was the least toxic mud while "May 15" was the most toxic.
Toxicity of the muds to A_;_ tonsa showed an inverse relationship
with the amount of time that the test suspensions were allowed to
settle prior to the assay. Filtration of the test suspensions
greatly decreased the toxicity of the "May 29" mud but not the
"Sept. A" mud, suggesting that the former contained toxic agents
that were associated with the drilling mud particulates while the
latter apparently contained dissolved or colloidal toxicants.
Lignosulfonates did not by themselves appear to be the principal
toxicants to A_;_ tonsa. Centropages typicus was equally as
sensitive as Acartia tonsa to "May 15" and "Sept. A" test
suspensions that had settled for 3 days, although typ icus was
more sensitive to filtered "Sept. 4" mud and to water soluble
components of #2 fuel oil than was A^ tonsa¦
Test results indicated that juvenile A^ tonsa were not more
sensitive to toxic drilling mud solutions than adults of the same
species, and that decreased fecundity occured among adult A.
tonsa at concentrations of drilling muds which were only slightly
below those that caused mortality.
6. A 48 n exposure to as little as 1 mL/L of the "Sept. 4" mud
produced a significant mortality of sea scallop larvae
(Placopecten magellanicus); exposure to 3 mL/L of this mud cajsed
100% mortality of these larvae. The "Exxon" mud had no
measurable effect on survival or shell development of P.
magellanicus larvae. Shell development was arrested in sea
scallop larvae that were exposed for 96 h to: >0.1 mL/L of the
"Sept. 4" mud; 0.3 mL/L of the "May 29" mud; or >1 mL/L of
the "May 15" mud. Arrested shell development appeared to be a
sensitive indicator of toxicity in bivalve larvae induced by
exposure to drilling muds.
7. In "liquid phase" (settled 72 h prior to use) toxicity tests witn
larvae of the quahog Mercenaria mercenaria, no fertilized eggs
developed to the earliest shelled larval stage ("straight-
hinge") when exposed for 48 h to concentrations of 500 mL/L or
greater of the "Sept 4" mud, while at a concentration of 150 mL/L
of this mud 68% of the fertilized eggs developed to shelled
larvae).
xii
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8. In studies on the toxicity of the drilling muds to crab larvae
(Cancer irroratus) , no significant differences in mortality or
number of molts was evident between any of the treatments and the
controls, even at mud concentrations that produced abnormal shell
development in scallcp larvae magellanlcus). Exposure of
crab larvae to the "Sept. 4" mud at a concentration of 100 uL per
liter of sea water temporarily inhibited feeding. In general,
crab larvae appeared to exhibit the same general toxicity
responses as the copepods.
9. Toxicity tests showed that none of the four muds labelled
"Exxon", "Gilson", "May 15", and "Sept. 4" exhibited acute
toxicity to young flounder (Pseudopleuronectes americanus) when
8.7 ml mud was mixed with 1 L sea water, allowed to settle 30
min, decanted and the supernatant mixed with sea water to yield
test suspensions of 30, 10, 3, and 1% supernatant. No flounder
died during the 48 h test or during a 48 h recovery period. In
addition, exposure of P^_ americanus eggs to "Exxon", "Gilson",
and "Sept. 4" mud suspensions had no detectable effect on
fertilization, although the drilling -nuds appeared to have an
agglutinating effect on the sperm.
10. In additional toxicity tests, the "Sept. 4" and the "May 15" muds
were relatively non-toxic to the Atlantic silverside minnow,
Henidia menidia¦ All minnows survived when exposed to the
undiluted, settled test suspensions of these muds. However, the
"May 15" mud was toxic to the mysid shrimp Neomysis americana
(96 h LC5Q 0.81 mL/L), and the coDepod Acartia tonsa
(96 h LC5Q 0.39 mL mud/L).
11. In studies to analyze the effects of drilling muds on the
recolonization of defaunated sediments, the presence of drilling
mud either layered on top or mixed with reference sediment
inhibited recolonization. The presence of drilling mud also
affected the distribution of species that successfully
recolonized the mud/sediment test phases. In the recolonization
studies, Capite1la capitata was much more numerous in sediments
that contained drilling mud.
In general, data showed that a used PESA arilling mud decreased
recolonization when layered (0.4 cm) on top of defaunated
reference sediment (3.6 cm), but not when mixed (1:4) witn it.
The deposition of a new layer of detrital material on top of
drilling mud seemed to reduce or reverse these effects; after
four to six weeks exposure of defaunated test sediments in the
field, the effects were no longer obvious. Greater numbers cf
animals occurred in field recolonization experiments than in a
lab-based, flow-through recolonization set-up. Recolonization
studies, although lenqthy. ^ere generally found to be an irnrove^ent over
traditional solid-phase toxicity tests as anethod for neasurini the impact
cf ccntam'nated sedinent on the benthic environment.
xiii
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12. Results showed that the methods used for preparing drilling mud
test media affect the apparent toxicity of the muds. In "liquid
phases" of drilling muds (i.e., mixtures of mud and sea water
that were allowed to settle for some period of time) toxicity was
generally found to decrease with increased settling time.
Filtration further decreased the toxicity of the "May 29" and the
"Gilson" muds. Settling for as little as 1 hour reduced the
toxicity of the "May 15" mud, while settling was found to yield
only slight reductions in the toxicity of the "Sept. 4" mud.
xiv
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INTRODUCTION
DRILLING FLUID STUDIES AT THE
EDGERTON RESEARCH LABORATORY, NEW ENGLAND AQUARIUM
Several studies have been conducted in our laboratory to investigate
the chemistry and marine toxicology of a number of drilling fluids,
including "Mobile Bay" ("May 15," "May 29," and "Sept. 4"), "Jay Field,"
"Gilson," "Exxon," and "PESA" ("AN31", "MIBLKA51," "SV76," "PI," "P2,"
"P3," "P4," "P5," "P6," "P7," and "P8"). A brief discussion of the
results of the early studies is appropriate at the start of this report
to allow a synthesis of both the chronology and the evolution of our
research program on the toxicity and chemical composition of used oil
drilling fluids. The terms "drilling fluid" and "drilling mud" are used
interchangeably throughout this report, as are the labels ppm (parts per
million) and pL/L (microliters per liter). The names of the drilling
muds used in this research program are introduced here in quotations to
indicate their use as code names for specific specimens provided by EPA,
API, or PESA; throughout the remainder of this report, quotations are
used only where their omission could lead to confusion.
Studies conducted from the fall of 1979 to the summer of 1980 dealt
with: (1) the physical and chemical composition of Mobile Bay, Exxon,
and Gilson drilling muds; (2) acute toxicity testing using a number of
different marine animal species; (3) fertilization efficiency, egg and
larval development of flounder; and (4) benthic recruitment studies (New
England Aquarium, 1980).
1
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The results cf our trace metal characterization studies indicated
that drilling mud suspensions prepared by slow centrifugaticn (600 x
gravity (G); 15 min) to simulate actual oceanic discharges, had higher
metal concentrations than samples prepared by high speed
centrifugation (30,000 x G; 30 min). In particular, suspensions of
medium density mud at a concentration of 1 ppm yielded barium
concentrations that were significantly above background. Organic
analysis indicated that four of the drilling muds which we tested
contained lipophilic fractions that were similar in composition to #2
fuel oil.
Acute toxicity studies using the estuarine copepod, Acartia tonsa,
showed that three of the Mobile Bay muds (Sept. 4, May 15 and May 29)
oroduced toxic effects, and that the techniques used in the
preparation of mud ohases affected their toxicity (i.e., time of
settling after mixing; 'iltered vs. non-fi ltered; filtration with
extraction; and dilute phase preparation method). The May 15 mud was
tested under only two treatment conditions, ncn-settled and settled
for 1 hour (h), with respective 96 r. L350 values of 0.03 and 0.39 ml
mud/L sea water. LC50 (96 h) values ror the May 29 mud ranged from
0.09 to 25.0 mL mud/L with the highest toxicity related to the
presence of solids that initially were in tne suspension. The 96 h
LC50 values for the Sept. 4 mud (0.60 tc 1.74 tL/l) indicated a
possible effect from dissolved or colloidal soecies. Further analysis
indicated that lignosulfonate-solubilized hydrocarbons could be a
possible toxic agent in drilling muos.
Toxicity studies were conducted also on the mysid shrimp, Neomysis
aner icana, and the Atlantic silverside, Menjrjia menidia. The results
of these tests inaicated that both the Sept. 4 and May 15 muds were
2
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relatively r,on-toxic to M. meniciia, although mortality was observed
with the mysid, N. americana, for which a 9 6 h LC50 value of
0.81 mL mud/L was obtained. In additional tests, juvenile winter
flounder, Pseudopleuronectes americanus, showed no acute toxicity
following a 96-h exposure to the May 29 drilling mud at 8.7 mL mud/L
(mixed 30 min, settled 1 h).
Tests were conducted to measure the effects of drilling muds on
the fertilization efficiency of P. americanus. Exposure to the liquid
and the suspended solid phases (8.7 mL mud/L) of Exxon, Gilson, and
Mobile Bay May 15 muds showed no apparent adverse effects on
fertilization efficiency. However, microscopical observations
indicated that sperm motility seemed to be affected after exposure to
drilling muds at a concentration of 1.0 mL mud/L for all of the muds
that were tested. The exposure of fertilized eggs to the liquid phase
and the suspended solid phase (1.0 mL mud/L) indicated that apoarently
normal development occurred through the gastrulation stage. Further
testing on egg develooment was prevented by the ending of the P.
americanus spawning season.
Similar fertilization efficiency studies were attempted by using
the yellowtail flounder, Limanda ferruqinea, and tne gray sole,
Glyptocephalus cymnoqlossus. Species availability during spawning
season and the constraints associated with snipboard experiments
oroved insurmountable for extensive testing with both of these
soecies. Ultimately, further experimentation with gametes and larvae
of fish w3s terminated in favor of the more promising area of
invertebrate toxicolcgical assessment.
A field study of recruitment and recolonization was conducted
using layered fractions of Exxon drilling muds over natural sediment
3
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in a sheltered embayment, Preliminary results, although inconclusive,
indicated a suppression of overall population size and a general
decrease in species diversity in the treatment with drilling mud,
compared to the recolonization and recruitment seen in the control
sediment.
Research from the summer of 1980 to the spring of 1981 centered on
further investigations of the chemistry and toxicity of spent drilling
fluids, including: (1) toxicity testing with two species of copepods;
(2) chemical characterization of the test-phase preparations used in
toxicity bioassays; (3) bioaccumulation of trace elements in organisms
exposed to drilling muds; (4) distribution of trace elements in
sediments and water; (5) effects of drilling muds on larval
development of sea scallops (Placopecten Maqel lanlcus) and rock crabs
(Cancer irroratus); and (6) effects of drilling muds on colonization
by benthic organisms (New England Aqjarium, 1981).
Acute toxicity tests were continued by using Ac a rtia tonsa and a
second copepod species, Centrooaqes typicus, which is common on
Georges Bank. As found in the earlier studies, the method of test
phase oreparation affected the observed toxicity. Among the phases
prepared from the Hay 15 and the May 29 Mobile Bay mud, toxicity to A.
tonsa decreased with increased mud settling time. In addition, the
Sept. 4 Mopile Bay mud displayed greater toxicity when the individual
concentrations in the test series were prepared separately, as opposed
to the preparation of the same concentration series by sequentially
diluting a primary stock suspension. Tne otner scecies of copepod
tested (C. typicus) was as sensitive as A. tonsa (0.49 - 1.5 mL mud/L
= 96 h LC50 value) to phases of the Sept. 4 ana May 15 mud suspensions
which were allowed to settle for three days.
4
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Results of fecundity and juvenile exposure studies on A. tonsa
indicate that decreased fecundity occured among adults at exposure
concentrations only just below those which caused mortality, and that
juveniles exhibited responses to drilling muds that were similar to
adults of the same soecies.
A study was initiated to determine which chemicals may
contribute to the toxicity of some drilling muds. Test phases used
in the toxicity tests were analyzed for both trace metal and organic
components. Chromium was targeted as a possible toxic component
because high concentrations of this metal were found in sea water
suspensions of Mobile Say drilling mud. Early results from toxicity
tests with both of the copepod species indicated that chromium did
not appear to play a dominant role in determining drilling mud
toxicity. However, metal speciation is an important factor in
bioavailability and toxicity, and further investigation of chromium
speciation showed that the liquid phase of the mud labelled SV-76 had
the highest concentration of Cr(VI), the most ciologically toxic form
of chromium. This phase was also one of the most toxic to Mercenaria
mercenaria larvae. Further analyses are needed to determine the
speciation, and metal-binding properties of drilling mud components.
The measurement of sea water-soluble and acid-soluble elements in the
ten drilling muds that were tested showed detectaole concentrations
aDove background for barium and chromium, as well as the release of
aluminum, iron and manganese. Aluminum, iron and manganese occurred
at concentrations likely to be minimal in impact considering
dispersion and dilution factors. Organic analysis indicated that all
test ohases contain tritutyl phosphate, alky1-substituted catechols
and polycyclic aromatic hydrocarbons. In addition, the test phase
5
-------
Produced by tne Sept. U mud contained a naphtnalene-oaseo ketone.
A study cf trace element concentrations was conducted on sampies
cf the drilling mud test phases taken curing the course of 96-n
toxicity tests conducted at tne National Marine fisheries Service,
Sandy Hook laboratory with P £ S A drilling muds. Results of these
studies indicated tnat, although the concentrations of sight trace
elements were very low in the sea w3ter test media, the settled
drilling mud ocntinjsd to release metals when re-mixed with sea water.
Analyses were conoucted to measure the 0ioaccumulation of trace
metals Oy red-winged oysters (species unknown), scalloos (Placopecten
magellanicus) , ana sea jrcnir, (species unknown) spines exposed to Jay
Field drilling mud suspensions. In the soft tissue of the scallop
and oyster, only oariurn concentrations increased witn increased
exposure to drilling muds in the 10 cpm and 100 ppm range. Following
even one week cf depuration in clean water, animals exposed previously
to 100 ppro drilling muds exhibited elevated concentrations of Ba
compared tc cackgrcurd. Analysis cf oyster shells snowed an increase
i-1 zinc over cackgrour.d in animals exposed to 10 ppm and 100 pom
drilling muc. For coth the oyster shells and the sea urchin spines,
C a r i u m concentrations were lower in the controls than in animals
exposed tc 13 ana 130 opm drilling fluid, cut were higher in the
controls tran in animals exposed to 1 pom drilling fluid. The
resulting data gave no clear explanation for this unusual
phenomenon.
A ruTiDer of toxicity studies were conducted oy using the larvae
of the sea scallop, =lacopscten mage 1 lan icus. The results indicated
that with tne Sect. 4 muc as little as 1 xL mud oer L produced
significant mortality amc-g larvae exposed for 48 hours. Arrested
shell development was ooserved after 12 hours among the test
6
-------
populations exposed to all concentrations of Sept. 4 mud and to the
highest concentrations of May 15 and May 29 muds. A 96 n exposure to
either 1 mL/L of the May 15 mud or 0.3 mL/L of the May 29 mud
significantly inhioited shell formation. The Sept. 4 mud was
considerably more toxic; it almost completely arrested shell
formation at 0.1 mL/L.
Test populations held for 6 days in clean sea water following a
96 h exposure to Sept. 4 mud exhibited an inaoility to recover from
arrested shell formation. None of the organisms exposed tc 3 mL/L or
10 mL/L filtered phases of this mud survived. Organisms exposed to
liquid phases of the two May muds were also unable to recover over a
6-day period in clean sea water following exposure to tne Sept. 4
mud.
The results of the 96 h exposures indicate that the Gilson mud
was as toxic to the larvae as the May muds from Mobile Bay, although
it was less toxic than the Sept. 4 mud. The filtered test phase of
the Exxon mud had no measurable effect on survival or on shell
development.
Arrested shell formation in bivalve larvae appears to be a
sensitive indicator of stress induced by drilling muds. The resuits
of 96 h tests in which a variety of marine fauna were exposed to
filtered mud suspensions indicated that copepods were the most
sensitive of the species tested. uC50 values (96 h) for Ac art ia
tonsa exposed to filtered ohases of the Seot. 4 dilling T.ud were
between 1 and 2 mL/L. However, considering sub-lethal effects, a 96
h exposure to 0.1 mL/L arrested shell development in sea scallop
larvae.
7
-------
Twenty-day larval development experiments were conducted witn
the brachyuran crab, Cancer irroratus. The larvae were exoqsed for
24, 48, or 72 hrs to 5, 10, and 100 uL/l concentrations of Sept. 4
dilling mud. Both mortality and the number of molts were recorded
over the 20-day test period. Results indicated no significant
differences between any of the treatments and the control for both
mortality and number of molts, even at concentrations that produced
abnormal shell development in the sea scallop.
Feeding experiments with the crab larvae indicated that exposure
to a 100 uL/L test phase of Sept. 4 drilling mud can temporarily
inhibit their feeding. However, the effect did not persist once
the exposure ended, and tnere did not appear to be any long-term
effect on crab larval growtn.
More concentrated suspensions of the Sept. 4 drilling nud were
acutely toxic to 5-day-old crab larvae. The 96 h LC50 value was 1.02
mi_ of settled, decanted liquid phase per liter of sea water. This
result suggests that the larvae were as sensitive to toxic drilling
¦nuds as the copepods that we have tested.
The recruitment and recolonization studies centered on the
testing of a variety oF laboratory designs to develop a functional
year-round experimental system. Two systems that were evaluated
included a longitudinal trough design ard 3 system utilizing a
circular tank. After several modifications, the circular tank design
provided a reliable laboratory system with a uniform water
circulation pattern. In addition, a field-based system was designed
to augment the laboratory studies with several objectives in mi no.
The first cDjective was to document any effects of drilling mud on
either the rate of colonization of defaurated sediment or the species
0
-------
composition of the recolonizing population. Secondly, we intended to
determine what time period was most critical during the
recolonization process. Finally, we compared results of concurrent
laboratory- and field-based experiments. For a more detailed
description of these studies, refer to New England Aquarium Progress
Report #2 (1981), and the results of these studies which are
contained in Part IV of this report.
Research conducted between May 1981 and February 1982 dealt with
two major areas of interest. The first was the construction and
development of a mariculture facility to allow year-round larval
production of the hard clam, Mercenaria mercenaria, for drilling mud
tests. The second dealt with the results of both laboratory and
fie la studies on the effects of drilling fluids on benthic
recolonization (New England Aquarium, 1982).
The operation of the mariculture system over a four-month period
proved tnat the system is capable of maintaining and conditioning
adult M. me rcenari a for year-round soawning. Procedures developed
for liquid phase testing of larval M. mercenaria represented a
feasible standard protocol. Preliminary results showed that when
1-h-old larvae are exposed for 48 h to the liquid phase of the Mcbile
Bay Sept. 4 mud (0.5, 1.5, 5.0 and 15 ppt), failed to form shells,
while control animals developed normal shells. There was no
significant difference in shell formation between fed and unfed
animals in these treatments. Observations were also made on the
effects of these liquid phases on larval embryogenesis. Growth
inhibition was found to be directly related to concentration.
Results of the drilling mud recolonization studies are contained
in the Dart IV of this report. As a method for measuring the impact
9
-------
of contaminated sediment on the benthic environment, the recruitment
studies were generally found to be an improvement over traditional
solid phase toxicity tests that have been used for assessing the
impact of dredged materials. However, the time required for both
testing and data processing in recruitment studies is too lengthy for
the efficient evaluation of whole drilling muds.
The following report outlines studies conducted by the Edgerton
Research Laboratory concerning the toxicity and chemical composition
of spent PESA drilling fluids.
10
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I. EFFECTS OF USED DRILLING FLUIDS ON THE EMBRYONIC DEVELOPMENT OF
1HL hard CLAM, Mercsnaria mercenaria (L.)
1.1 Background
Drilling fluids, or drilling muds, are vital to the offshore
exploration for oil and gas because they fulfill the requirements for
drill bit lubrication, bore hole stabilization to prevent cave-ins
and blow-outs, and for removing rock chips (cuttings) from the
cutting surface of the drill bit. These and other tasks are
performed by circulation of the barite-rich (approximately 94 percent
BaSO^) mud dcwn the oore hole to the drill bit from which cuttings
are carried to the surface for removal and subsequent discharge into
the ocean. The composition of a discharged drilling mud is altered
from its original state by the drilling conditions (geological
formation, temperature, and pressure) (IMCO Services, 1978;
Perricone, 1980) and thus the complex mixture of organics (Strosher,
1980) and trace metals (Liss e_t a_l. , 1990) in the mud can vary. The
composition of the mud is changed in response to the drilling
conditions and, consequently, the subsequent effects of discnarged
muds on marine organisms may also change over the duration of a
drilling operation. This potential for changes in mud toxicity as a
function of drilling conditions was recently reported in laboratory
toxicity tests by Tornberg e_t aK ( 1980) and Conklin e_t a_l. (1933)
who showed that mud toxicity increased with drilling depth.
The toxicity of used drilling muds to marine fauna is well
established (Sprague and Logan, 1979; Houghton, Beyer, and Thielk,
1980; Thornberg s_t , 1980; Neff et_ a_i. , 1980; Thompson and Bright,
1980; Conklin et al. , 1980; Carr, Reitsema, and Neff, 1980; Gerber e_t
11
-------
a I. , 1930; Gerber et a_l. , 1980, Neff et al_. , 1980; Carr e_t a_l. , 1980;
Crawford and Gates, 1981; Chaffee and Spies, 1982). The discharge of
cuttings and muds into the pelagic zone and their subsequent impact
on the early life stages of the seasonal plankton has only begun to
be addressed. Most studies on drilling mud toxicity have
concentrated on adult animals from the benthic community. Although
it has been demonstrated that adults can often survive high
concentrations of drilling fluids, the ultimate survival of an
imoactsd population in succeeding generations depends to a great
degree on the tolerance of the planktonic early life stages.
Althougn juveniles of a few fish species have been shown to exhibit
greater sensitivity to metals as they age (Chapman, 1978; Blaster,
1977), the generally greater sensitivity of early life stages is well
documented in Doth the olant and animal kingdom for a number of
environmental and anthropogenic stresses (Odum, 1962; Portmann, 1970;
Buikema and Benfield, 1979; Neff et a_l. , 198G; Carr et_ a_l. , 1980).
Tnis study was initiated in response to concern over the impact
of offshore drilling activities on commercially important bivalve
molluscs and the lack of information concerning the effects of
drilling muds on molluscan larvae. A diverse collection of used
drilling muds was used to evaluate the sensitivity o* the hard clam,
Mercenaria mercenarla, fertilized eggs (i-h post fertilization).
M. mercenaria was chosen because of its wide climatic range in the
coastal zone of the United States and because adult orood stcck can
be held for year-round spawning and subsequent toxicologica 1 testing
without gonad resorption (Loosanoff and Davis, 1950). Dilute
12
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sea water suspensions were used in all the tests with the goal of
approximating "real-world" test conditions.
1. 2 Materials and Methods
The natural sea water jsed for maintaining marine tanks at the
New England Aquarium was used to culture M. mercenaria and its algal
food. The water for all tests was filtered through a series of
cotton filter cartridges to remove oarticles greater than 0.45 urn.
Tne salinity of the sea water was 30-320/qo; pH was 7.9 + 0.2,
and dissolved oxygen content was at least 83% of saturation
throughout the analysis.
1.2.1 Conditioning and Holding of Brood Stock.
Adult M. tnsrcenaria (aoproximately 50-60 g) were collected from
the mouth of the Marstons Mills River in Cctuit (Cape Cod),
Massachusetts, at various times of the year. The clams were
transported immediately to the lab in a styrofoam cooler at ambient
temperature and then slowly acclimated to the conditioning (15°C)
or holding (11°C) tank temperature.
During the conditioning period (minimum of 6-8 weeks), no more
than 80 (50-60 g size) brood stock animals were maintained at
15.0 — 1.0°C in two deep trays (76x91x25 cm) containing 8-10 cm
of sand from the area in which the animals were collected. Before
tne clams were introduced into the conditioning tray, the date was
marked on the shell with an indelible ink marker, and the clans were
placed on the sand and allowed to dig into the substrate. The
conditioning trays received aporoximately 200 L/day of cultured algae
13
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and approximately 1000 L/day of filtered (1 urn) sea water, amounting
to approximately 6-8 water changes/day. Depending on the cell
densities of the algal cultures, the adult animal conditioning trays
contained from 0.4 to 1.0 x 105 algal cells/mL at all times.
Feeding was provided by the continuous harvest of 360 I,
fioerglass-tube cultures (Solar storage tube, Kalwall Corporation,
Manchester, N.H.) of mixed cr unialgal cultures cf the diatom
Thalassiosira pseudonana (3H strain) and the flagellated chrysophyte
Isochrysis galbana (Tahitian strain). Algal harvesting was carried
out with a small diaphragm pump. The mass culturing of microalgae
was initiated after the sea water was chlorinated overnight (60 ml
chlorine/360 L), and then dechlorinated with sodium thiosulfate
(10 g/360 L). Eight to twelve one-liter algal stock cultures were
added after the water was supplemented with Guillard's F/2 nutrient
supplement (Guillard, 1974).
Newly collected animals which could not be accommodated in the
conditioning trays were acclimated to the 11 — 1.0°C holding
temoerature and held until they were needed for conditioning. These
animals either received continuous feeding from the overflow of the
conditioning trays, wnich contained an excess of algae (greater than
4x10^ cells/mL), or they were fed 15-20 L batch quantities from the
algal tube cultures daily. This cold-water holding system consisted
of two 350-L insulated trays maintaining 200 adult clams. The sea
water flowed into the holding trays at a minimum rate of one turnover
per day.
14
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1.2.2 Spawning and Rearing.
Conditioned brood stock animals were spawned by the methods
described by Loosanoff and Davis (1963). Fertilization was achieved
when the pooled sperm and eggs were mixed and allowed to remain in
contact for 1 h. Depending on the sperm concentration, approximately
6 mL of sperm were mixed with 1 liter of egg suspension to yield
^ 7
between 10"" and 10 sperm/mL. Lower sperm concentrations can
result in incomplete fertilization, while higher levels can yield
larval deformities, probably from polyspermy (Culliney e_t a 1. ,
1975). After a 1 h fertilization period, the fertilized eggs were
sieved onto a 63 um Nitex screen to remove sperm, and were
resuspended in a one liter beaker containing 0.45 um filtered sea
water. The egg suspension was mixed gently with a perforated Teflon
plunger to yield a uniform suspension before a sample was taken with
a microoipette *or the quantification of eggs at 25x under a
dissecting microscooe. Eggs were counted in triplicate 100 uL
aliquots and the mean was multiplied by 10 to give the number of eggs
per mt. To reduce variaoility in sampling fertilized eggs, two
separate aliquots of the uniformly mixed egg suspension were
inoculated into each tube to yield a final density of 10-15
embryos/mL.
Test vessels consisted of round-bottom, 60 mL Pyrex test tubes
containing 50 mL of the drilling mud test phases. The test vessels
were held at 27°C -±1°C in a constant temperature bath. Each
test vessel was aerated for 60 min prior to inoculation with
fertilized eggs to increase the dissolved oxygen concentration.
A specially designed water bath was made to contain the test
vessels at 27°C il°C for the 48 n incubation period (Fig. 1).
15
-------
This unit consisted of the following components: (1) a large
styrofoam cooler (75x35 cm) with holes (2.5 cm diameter) bored into
the cover to hold the test vessels; (2) a Neslab-Endocol constant
temperature circulator to provide a flow of water through the cooler
for bath temperature maintenance; and (3) an air manifold system to
ensure uniform bubbling within each test tube. The air was filtered
through an oil separator to remove any residual oil and pioed by a
manifold through 0.22 mm (ID) teflon tubing to each test vessel.
1.2.3 Experimental Procedure.
Drilling muds were provided by the Petroleum Equipment Suppliers
Association (PESA) and distributed by the Environmental Protection
Agency, Gulf Breeze, Florida. These muds were collected from various
drilling sites, identified by an EPA code and categorized according
to mud type.
Test ohases were orepared with the intent that they would
closely approximate the actual composition of drilling mud
suspensions released into the ocean. Dilute drilling mud/sea water
mixtures were prepared by the addition of from 0.15 to 3 mL of used
drilling mud into a liter of 0.45 urn filtered sea water. Two phases
were prepared from each drilling mud to test the toxicity of both the
water soluble components alone (liquid phase), and the suspended
solids plus water soluole components (suspended-solids phase).
Two-liter glass bottles were washed first with acetone and then with
6N HC1 to remove adsorbed organics and metals, and were then rinsed
three times with distilled water. The liquid and suspended-solids
test phases were prepared by adding a volume of the drilling mud to
two liters of 0.45 um filtered sea water. These dilute suspensions
16
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a
2:
Air filter
Constant
temperature
water bath
Water
inflow
Water
outflow
Test tube
Air supply
1
o
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en
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W
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c
-------
of drilling fiuics were stirred for 30 min with a Teflon-coated
magnetic stirrer and allowed to settle at room temperature for either
1 h, to produce a suspended-solids phase, or for 72 h, to produce a
liquid phase. After the aoorooriate settling period, the test ohases
were collected by siphoning for use in the bioassay procedure.
Particles with the density of clay and dimensions of 0.5 um or
greater in diamete r sett led out of the test phases after 72 h. This
procedure was preferred over membrane filtration oecause it achieved
the same results without the problems of degassing ard adsorotion of
toxicants to the membrane filters. In addition, the settling method
more closely resembles conditions found at sites where drilling muds
are released.
In order to allow the testing of several muds at different times
of the year, the use of several different adult brocd animals was
required for spawning Larvae, and this necessitated the inclusion of
a standard toxic control mud to allow comoarison between tests. For
this ourpose we chose a used saltwater 1ignosu1fonate mud identified
as Mobile 3ay - Sept. 4, collected off the coast of Alabama,
in 1979. Prior to testing, each test vessel was acid washed (6N
Hwl), rinsed with distilled water, and placed in a 550°C oven for
60 min.
Drilling fluid concentrations were tested in triplicate and each
test vessel was placed randomly into the larval bioassay test
apoaratus along with triplicate controls. The eggs were accec to the
liquid pnase within one hour after fertilization. Dissolved oxygen,
pH, and salinity were measured at the beginrirg and end c f each
test. Temperatjre was monitored throughout eac~ 43-n test oeriod.
18
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The number of D-stage, shelled prodissoccmch-I (straight-hinge,
or D-stage) larvae that developed with normal shells in the control
containers was compared to the number of normal-shelled, D-stage
larvae that developed from the embryos in the test containers to
develop an index of toxicity in these tests.
After the 48-h static testing period, 1 nl_ of 10% phosphate-
buffered formalin and 2.5 mL of 0.5 % Rose Bengal (both in 0.45 um
filtered sea water) were added to each test vessel and the preserved
samples were stored in a refrigerator at 4°C —2°C. The
contents of each vessel were filtered through a 25-um Nitex screen to
retain the larvae. The screen was washed three times and all
organisms were transferred to a Petri dish. A binocular dissecting
microscope was used to count the larvae.
1.2.4 Statistical Analysis.
The results of these tests were expressed as 48-h EC 50 values
related to the proportion of the test population which failed to
develop into straight-hinge, orodissochonch-I larvae (D-stage).
These values and their 95% confidence limits were calculated by using
probit analysis (Finney, 1971).
1. 3 Results
1.3.1 Year-round Toxicological Testing.
Year-round toxicological testing of used drilling muds continued
throughout the 15-month analysis period. A temperature of 15 —
1.0°C was optimal for the year-round conditioning of Mercenaria
19
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mercenaria brood stock. Higher temperatures caused the accidental
spawning of ripe broodstock and lower temperatures reduced feeding
activity and lengthened the conditioning period.
Our oDjective in the mass-scale culturing of microalgae was to
provide the clam brood stock with a reliable supply of nutritious
food which was free of toxicants and pathogens. This was easily
accomplished by using 360 L fiberglass tube cultures of Thalassiosira
pseudonana (3H strain) and/or Isochrysis galbana (Tahitian strain) at
a density of 1 to 4x10^ cells/mL. Sufficient food was supplied by
one 360-t culture to sustain the nutritional requirements of the
brood stock animals in the flow-through conditioning trays for 2-3
days. The algal mass cultures were pumped continuously into the
conditioning trays to yield a constant supoly of algae (approx. 104
cells/mL) to the brood animals. Three mass-culture tubes were ample
for culturing these algae as food for the year-round conditioning of
brood stock. The cultures attained a harvest density of 1-4 x 10^
cells/mL approximately 4-5 days after it received its F/2 r.jtrient
supplement (Guillard, 1974) and algal inoculum.
1.3.2 One-hour Fertilized Egg Toxicity Test.
A summary of the data f rom 12 used drilling muds is presented in
Tables I-A and 1-8, and Figures 2 and 3. The toxicity is presented
for both liquid (72 h settled) and suspended-solid phases of for each
fiud . The suspended-solids phases of muds PI, P2, and P3 (which also
includes the water soluble fraction) were found to be more toxic than
the water-soluble fractions alone. The remaining muds did not show
an appreciable difference in toxicity between the two mud phases.
20
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TABLE I-A: 48 HOUR DRILLING FLUID TOXICITY TEST;
SUSPENDED S0LID5 PHASE
Continuous exposure (48 h) of 1 hour old, fertilized eggs of
Mercenaria mercenaria to the suspended solids phases of various
drilling fluids showing percent of each test group
(n = 625 + 125 eggs) that developed into normal, "D" stage larvae.
Drilling
Fluid
Test Date
EC50
Confidence
LCL*
Limits
UCL*
Co ntrol
% "D" Staqe
AN31
5/20/82
1,771
ppm**
1,710
1,831
93
MISLKA51
1/26/82
>3,000
ppm
--
--
95
S V 76
5/20/82
117
ppm
115
119
93
P-l
9/20/82
122
ppm
89
151
99
P-2
9/20/82
156
ppm
149
162
99
P-3
9/20/82
64
ppm
32
96
99
P-4
9/20/82
347
ppm
330
364
99
P-5
9/20/82
382
ppm
370
395
99
P - 6
11/22/82
>3,000
ppm
93
P-l
11/22/82
2,779
ppm
2,667
2,899
93
P-9
11/22/82
212
ppm
200
223
93
Sept. 4
5/20/82
125
ppm
120
130
93
(Mobile
Bay)
9/20/82
97
ppm
94
101
99
11/22/82
119
ppm
111
128
93
* LCL = lower confidence limit; * UCL = upper confidence limit; {95%
confidence limits
** Vol/Vol mixture of a 1 hour-settled drilling mud suspension and 0.45
um-filtered natural sea water to yield the indicated concentration of
drilling mud suspended solids phase in sea water.
21
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TABLE I-B; 48 HOUR DRILLING FLUID TOXICITY TEST:
LIQUID PHASE
Continuous exposure (48 h) of 1 hour old, fertilized eggs of
Mercenaria mercenaria to the liquid phase of various
drilling fluids showing percent of each test group
(n = 625 +_ 125 eggs) that develop into normal, "O" stage larvae.
LIQUID PHASE
Drilling Control
Fluid
Test Date
EC50
LCL*
UCL*
% "D" Staqe
AN31
3/15/82
2,427
ppm**
2, 390
2,463
88
MIBLKA51
1/26/82
>3,000
ppm
95
SV 76
3/15/82
85
ppm
31
38
88
P-l
6/14/82
712
ppm
690
734
97
P-2
6/14/82
318
ppm
308
328
97
P-3
7/26/82
683
ppm
665
702
98
P-4
7/26/82
334
ppm
324
345
98
P-5
7/26/82
385
ppm
371
399
98
P-6
8/16/82
o
o
o
r\
A
ppm
--
--
97
P-7
8/16/82
>3,000
ppm
--
--
97
P-8
11/22/82
269
ppm
257
280
93
Sept. 4
3/15/82
134
ppm
126
141
88
(Mobile
Bay)
6/14/82
112
ppm
91
122
97
7/26/82
176
ppm
168
193
9B
8/16/82
187
pom
170
212
97
* LCL = lower confidence limit; UCL = upper confidence limit;
(95% confidence limits)
** Vol/Vol mixture of a 72 hour-settled drilling mud suspension and 0.45
urn-filtered natural sea water to yield the indicated concentration of
drilling mud "liquid phase" in sea water.
22
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PIGt'RE 2
ECS0 VALUES FOR M. MERCENARIA LARVAE AFTER a 48 HOUR
EXPOSURE TO DRILLING FLUID
I clq
•In
t=
LIQUID PHASE
P: SUSPENDED SOLI
rs PHASE
u
rl-
SV-76
P-8
P-2 P-4
FLUID TYPE
23
P-S
P-3
SEPT 4
* Bars indicate 05% confidence intervals
-------
r i 'j " r e 3
rh
t
EC50 VALUES FOR M. MERCENARIA LARVAE AFTER A 48 HOUR
EXPOSURE TO DRILLING FLUIDS
P-1
>3000
AN31
>3000
P-7 MIHLKA51
FLUID TYPE
Mrs indicate 95i Confidence Intervals,
,;.js LIQUID PHASE
3=SUPENDED SOLID
j PHASE
P-6
SEPT 4
&
-------
The range of the 48 h EC50's for the liquid phase of 8 of the 12
muds was from 35-712 opm and the range of EC50's for the suspended
solids phase of these muds was from 64-382 pom. The EC50's for the
remaining muds exceeded 2,000 opm (v/v mixture cf whole mud/sea water
prior to settling). More than 88 percent of the control animals
achieved D-stage in all of the tests in this study.
1.4 Discussion
The results of these tests showed that fertilized eggs of M.
mercenaria were very sensitive to both the liquid and the
suspended-solids phases of a diverse assortment of used drilling
muds. Tne mud types (Table II) included a "lime" mud (P3), a sea
water potassium polymer Tud (P8), a low solids non-dispersed mud (Pf
and various chrome lignosulfonate types (AN31, MIBLK, Mobile Bay •
Seot. 4, SV76, PI, P2, P4, P5, and P7).
The wide range of 48 h EC50 values (64-3000 ppm) indicates large
variations in the chemical composition of used drilling tiuds. As was
found by other investigators who also employed early life stages for
testing (Crawford and Gates, 1981; Chaffee and Spies, 1982), the 1-h
fertilized eggs of M. mercenaria showed the lowest 48-h EC50 values
when exposed to drilling muds. EC50 values for most muds were in the
64-800 ppm range. The sensitivity of molluscan early-life stages has
also been demonstrated for a number of other toxicants (Calabrese,
1972; Caiabrese e_t a_l. , 1973; Calabrese and Nelson, 1974; Maclnness
and Calabrese, 1979; Coglianese and Martin, 1981; Watling, 1982).
25
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TABLE II. CHEMICAL AND PHYSICAL CHARACTERISTICS OF THE USED DRILLING FLUIDS
EMPLOYED IN THE Mercenaria mercenaria LARVAL TOXICITY TESTS
Depth of
Drillinq FLuid Code
Mud Type
Well (m)
Density
% Water
AN31
Sea water lignosulfonate
3576
1.50
49.9
MIBLKA51
Sea water lignosulfonate
2277
1.26
68.5
SV76
Sea water lignosulfonate
NI
2.17
27.3
Mobile Bay (9/4/79)
Sea water lignosulfonate
6236
1.55
54.2
PI
Lightly-treated lignosulfonate
4311
1.93
33.8
P2
Fresh water lignosulfonate
4153
2.02
30.0
P3
Lime mud
5241
2.19
26.8
P4
Fresh water lignosulfonate
3645
1.93
33.4
P5
Fresh water/sea water lignosulfonate
3945
2.20
26.3
P6
Low-solids, nondispersed mud
NI
1.22
75.1
P7
Lightly-treated lignosulfonate
3747
1.37
57.0
P8
Heavily-treated lignosulfonate
3760
2.17
27.3
NI= no information available
-------
Other investigations reporting on sensitive toxicity tests
include those of Crawford and Gates (1981) on unfertilized sand
dollar (Echinorachnius parma) eggs and Conklin et_ a_l. (1980, 1983) on
grass shrimp (Paleomonetes puqio) molting stages. Crawford and Gates
(1981) exoosed unfertilized sand dollar eggs for a 15 min pulse
exposure to drilling muds and found that fertilization was affected
at concentrations between 100 ppm (88 percent fertilization) and
1,000 ppm (6 percent fertilization). Conklin e_t al_. (1980)
identified the molting of grass shrimp as a sensitive stage for use
in toxicity tests since they showed a 96-h LC5Q value range from 363
to 739 ppm for 5 drilling muds.
The Mobile Bay (Seat. 4) mud was used as the standard toxic
control throughout the 15 month testing period. It was intended to
serve as a reference between groups of larvae tnat were spawned at
different times. During this study, the toxicity of this reference
mud (designated "toxic control" or "toxic reference mud") was
150 — 37 opm for the liquid phase and 111 — 14 ppm for the
suspended phase. This indicated good reproducibility between tests
and spawn groups. These results suggest that the response of the
different test populations was sufficiently uniform to allow direct
comparison of results from tests performed at different times.
Our toxic reference mud (Mobile Bay - Sept. 4) was alsc used by
Conklin e_t a_l • (1983) who called it mud XVII for their 96-h test with
Palaemonetes puglo. Our analyses with this mud verified the extreme
sensitivity of the M. mercenaria fertilized egg test. A comparison
27
-------
of the sensitivity of these two organisms to this mud shows that the
150 ppm 48 h EC50 for M. mercenaria fertilized eggs was considerably
lower than the 740 ppm 96 h LC50 seen in tests of effects on
Palaemonentes puqio molting, especially when considering that the M.
mercenaria EC50 is for a 48 h test as opposed to the 96 h pugio
test. A similar test was performed with this same Mobile Bay mud in
our lab on the fertilized eggs of the sea scallop (Placopecten
magellanicus) (0. Wayne, New England Aquarium, unpublished data). In
this study, the Mobile Bay mud elicited toxicity to sea scallop eggs
and also inhibited development at a low concentration (100 ppm) in a
48-h EC50 bioassay. Therefore, fertilized eggs of M. mercenaria and
Placopecten magellanicus seemed to be equally sensitive to the same
toxic mud. The developing embryos and larvae of both mollusc species
appeared to be more sensitive to the muds tested in this study than
molting grass shrimp.
Davis (1963) and Davis and Hidu (1969) identified the importance
of turbidity to the survival of M. mercenaria eggs and larvae. Eggs
exposed to silt were found to have an approximate EC50 value of
1.5-2.0 g/L while eggs exoosed to kaolin clay had an EC50 of
approximately 0.5 g/L (Davis and Hidu, 1969). Drilling muds contain
high concentrations of clay and barium sulfate particles (Perricone,
1980) and the drilling muds used in our studies contained between 0
and 55.6% clay, and (for those that had a known barite content),
between 75 3nd 8 7% barite; at the 0.02-0.855 g/L concentrations of
suspended solids typically found in the plume of an oil well
discharging aporoximately 1,000 bbl/hour of used drilling fluid
(Ayers et a_l. , 1980), it is quite likely that fertilized eggs could
28
-------
be severely impacted by turbidity effects alone. However, the
maximum drilling mud concentrations (3 ml mud oer L of sea water)
used in our test phases were relatively low by comparison, and it is
unlikely that turbidity effects could have contributed to toxicity in
any of the suspended-solids phases that were tested in this study.
The 72 h settling time used to produce the liquid phase more
closely aoproximated natural environmental conditions than previous
methods which utilized 0.45 um membrane filtration (Neff e_t a 1. ,
1980). Membrane filtration probably removes metals (Truitt and
Weber, 1979) and organic comocnents by adsorption (Bates e_t al. ,
1983). However, the 72 h (settled) liquid ohase of the drilling
fluid SV76 was found to contain less water-soluble fuel oil
components than the newly prepared suspended-solids phase of this mud
which was centrifuged at 1,000 X G for 3 min (Table X, section III).
The diminished water-soluble fuel oil content of the 72 h (settled)
liquid phase could oe attributed to the lengthy incubation period
which may have permitted microbiological decomposition of some
organic components. The bactericidal ingredients in drilling muds at
the 15-3,000 ppm testing concentrations (v/v, whole mudrsea water)
m3y not have oeen sufficient to prevent biodegradation during the
tests on diluted muds . In addition, more volatile components could
have been driven off when the test phases were vigorously aerated
before inoculation with the fertilized eggs.
The suspended-solids phases contained the largest quantitites of
fuel oil-like substances, and these were the most toxic phases for 8
of the 12 muds. However, there was no direct correlation between
toxicity and the concentration of the fjel oil-like components of the
drilling muds. Vet, studies on the toxicity of drilling muds by
29
-------
Conklin et aj.. ( 1983) on shrimp and by Miller e_t aj.. (1580) on
vascular land plants identified diesel fuel as the single mcs t toxic
drilling mud component when it is oresent. Until similar standard
experiments can be performed, it will be difficult to separate the
effects of biodegradation and adsorption on drilling mud toxicity.
Consideration of the 'nigh surface area of the mud oarticulates
that is available for adsorption suggests that mac toxicity may oe a
function of the particulate surface area. Thus, in the presence cf a
given concentration of a toxic hyarccarbon, a high-particulate,
hign-surface-area mud, may oe less toxic than a mud with less surface
area for toxicant adsorption.
Trace metal analyses (Tables III, V11) identified bariun and
chromium as the most common metal comocnents in the 12 drilling mud
samoles. In agreement with Ccnklin e_t a_l. ( 1983), the toxicity of
these muds showed no correlation with the content of cnromium.
Barium is commonly emoloyed as a weighting agent and has been shown
to be biologically inert (Cabrera, 1971; George, 1975). while
chromijm as Cr(VI) is generally Known to ce the most toxic species cf
this metal (Meams et_ a_l. , 1976), drilling-mud chromium usually
occurs as Cr(III) in the form of cnrcmiuT, 1; gnc su 1 f onate chelates or
as insoluble chromium hydroxide. This trivale~c (crelateo :r
insoluble) chromium is a stable soecies arc may ce ciologically
unavailable to the nonfilter-feeding 1 i c e stages that precede the
prodissochoncn I, straicnt-hinge stage (Mertz, 1969). Cnrcnium (III)
has been shown to impair the ciliary mechanism cf tne gill in adults
of the mussel M y t i 1 j s e d u 1 i s and the soft-s^el'. ti=n M^_a arenarla
(Capuzzo, 1974; Chipma->, 1966; Cshida s_t aj.. , !9Si;. Altrc^ch, i ->
30
-------
other investigations, Cr(III) was innocuous at concentrations as high
as 5 0,400 ug/1 (Oshida e_t aj.. , 1981).
In view of these studies, the hard clam egg toxicity test should
serve as a useful tool for identifying the potential imp act of
drilling muds on the survival of commercially important oivalve
mollusc species in the offshore environment. As demonstrated in this
study, very law concentrations of drilling muds can adversely affect
the survival of newly fertilized eggs. In addition, this information
should be useful in determining the maximum oermissabie mud discharge
rates in coastal zones that serve as seasonal nurseries for
commercially important off-shore species such as the sea scallop
Placopecten mage 1lanicus (Posgay and Norman, 1958; Naidu, 1970), the
ocean quahog flrctica islandica (Loosanoff, 1953; Jones, 1981) and the
surf clam Spisula solidissima (Ropes, 1968, Jones, 1981). Unlike
offshore species such as the sea scallop P. mage 1lanicus and the sea
clam A. islandica which require a minimum of 4-8 days for the
developing larvae to reach D-stage (Culliney, 1974 and Lutz et al. ,
1982), M. mercenaria fertilized eggs develop to this stage in 24-48
h, a distinct advantage in using this species for marine toxicity
testing.
The nature and extent of the impact of oil drilling fluid
discharges on molluscs and other marine organisms would depend on the
time of year, the quantity and the frequency of the discharge. In
addition, the hydrodynamics of the release site would be important in
assessing the potential impact of drilling mud releases. Adverse
effects associated with the long-term discharge of muds from several
drilling operations in a small area could be greater than expected,
since the sensitive, early life stages of many marine organisms can
31
-------
remain planKtcnic for at least several days (Culliney, 1974;
Goldberg, 1980; Lutz et al., 1982), thus prolonging tneir potential
exposure.
In conclusion, while the hard clam fertilized egg stage ias been
shown to be sensitive to a variety of used drilling muds, it has not
been possible to correlate toxicity conclusively with any specific
mud components. Further toxicologica1 studies are required to
deliniate the effects of turbidity and particulate loading (barium
and clay), the effects of adsorption of toxicant to particulates, and
the role of microorganisms in the biodegration of the various
drilling fluid components.
32
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II. TRACE METALS IN DRILLING FLUID/SEA WATER TOXICITY TEST PHASES
2.1 Background
Trace metal analysis of the drilling fluid/sea water toxicity
test phases was conducted in an attempt to identify inorganic
components that may be toxic. The metals of interest include barium,
cadmium, chromium, copper, manganese, nickel, lead and zinc. These
metals can be present in tne marine environment in several forms
which will affect their availability and ootential toxicity to marine
organisms. For this reason a scheme was developed to "speciate" or
determine the various farms of the trace elements listed above as
well as their total concentrations in drilling fluid/sea water
mixtures.
Three general types of metal species were targeted in the
speciation scheme to be described. Tnese were: free ionic forms
including inorganic complexes (i.e., chloro, hydroxy, etc.),
organically bound metals, and particulate associated metals. These
classes of trace elements are considered to be the major types
present and have been the subject of numerous speciation studies
(Bender, 1982; Florence, 1982; Hart, 1982). Total concentrations
give no information about a metal's availability and frequently show
no significant correlation with toxicity (Allen e_t a_l. , 1980).
Metals adsorbed, occluded or in some way associated with
colloidal-sized particulate matter may not be readily available to
many types of marine organisms and are often assumed to be non-toxic
(Florence, 1982). Metals complexed by organic ligands are known to
be less toxic than free metal ions (Allen et a_l. , 1980; Luoma, 1983;
Zamada and Sunda, 1982).
33
-------
In addition to the types of species discussed above, chromium
can exist in two oxidation states in sea water (Cranston and Murray,
1978; Nakayama e_t a_l. , 1981). Chromium(VI) is oresent as CrO^~
and is considered highly tcxic while Cr(III) as CrCOl-O^.A^O
is much less toxic (Carr et al_. , 1982; Mayer and Schick, 1981;
Florence, 1982). Determination of the oxidation state and species of
Cr was also included as part of the speciation scheme.
In order to clearly interpret results from the speciation of
drilling mud/sea water mixtures, a knowledge of drilling mud
components and additives is essential. Tne 0a in drilling muds is
added in large amounts as barite (BaSO^) which is insoluble.
Barium is, therefore, expected to be mainly in a particulate form
with a very small amount present as soluble 3a. Chrome or
ferrochrome ligncsul^onates are widely used as defioculants and are
the source of Cr in drilling fluids (Perricone, 1980). It has also
been reported that Ct(VI), as dichromate, is sometimes added to a
drilling mud to regain lost thinning action or for corrosion control
(Moseley, 1980). Lead may be oresent in drilling fluids as a
contaminant from pipe dope used for drill strings (Liss e_t a_l. , 1980;
Kalil, 1980). Many of the other metals may be introduced into
drilling fluids as impurities in clay, barite or other additives
(Macdonald, 1982). Manganese for example, is often associated with
clay minerals in significant quantities.
Drilling fluids also contain additives that can chelate trace
metals. «!_ignosulfonates are polymeric lignin derivatives containing
large numbers of sulfonic acid, carboxylic acid and phenolic groups
that can bind metal ions. Although Cr(III) and Fe are probably
34
-------
principally associated with lignosulfonates, it is unlikely that the
binding capacity of this ligand is completely expended in typical mud
mixtures. Metals such as Cd, Cu, Mn, Ni, Pb and Zn are probably
complexed to some extent when present in drilling muds. Lignite is
another additive that can have metal complexing components. Lignite
contains humic acid and related compounds (Perricone, 1980) that are
well known naturally occurring metal chelators (Mantoura et a 1.,
1978).
2. 2 Materials and Methods
2.2.1 Particulate and Dissolved Metals.
To obtain information on particulate, dissolved, and free metal
ion concentrations, individual aliquots of both the liquid (72 h
settled) and suspended solids (1 h settled) test phases where taken
and treated separately. For each drilling nud a range of
concentrations was prepared for toxicity testing, but only the most
concentrated sample of each type of test phase (typically 3 mL
drilling mud/L sea water) was sampled for trace metal analysis. Four
5 mL portions were pipetted from each test phase. Two of the 5 mL
aliquots were centrifuged for 1C min at approximately 300G x G.
Analysis cf the uncentrifuged sample gave total metal concentrations
while the centrifuged samples were used to measure dissolved metal
concentrations. The difference between the two was considered
particulate metal concentration.
2.2.2 Free Meta 1.
Free metal ion concentrations in the drilling fluid/sea water
mixtures were determined by performing equilibrium dialysis (ED)
separations prior to trace metal analysis. Dialysis experiments were
35
-------
performed in duplicate on 200 or 250 mL aliquots by using two tyoss
of membranes. Nominal 1,000 molecular weight cut-off (MWC0) tubing
(Spectrum Medical Industries, Spectra/Por 6) was cleaned by the
method of Truitt and Weber (1981a) and sealed at the ends with
plastic closures to form dialysis bags. Ion exchange membranes (RAI
Research Corp, Millioore R-1010) were prepared according to 31aedel
and Kissel (1972) and used with dialysis cells made from polyethylene
bottles approximately 1.8 cm in diameter. After removing the
bottoms, the bottles were fitted with memoranes held in place with
rubber "0" rings. The dialysis cells or bags were filled with 15 mL
of 0.45 um filtered sea water used as the toxicity test control.
Oialysis was performed in 250 mL polyethylene centrifuge bottles
containing the drilling fluid/sea water test phases. The bottles
were placed on a rotary shaker at low speed (75 rev/min). After a
predetermined amount of time, the dialysis cells or oags were removed
and sampled for trace metal analysis. Additional samples were
checked for UV absorbance at 275 r.m to detect the presence of
lignosulfonate or other UV-absorbing organic compounds. In addition
to analyzing the internal dialysis solution, the test phase external
to the cell was analyzed. Prior to sampling, the external solution
was centrifuged for 20 minutes at aoproximately 2,500 x G. The
internal solution gave free metal concentration while the external
solution, after centrifugation, gave total dissolved metal
concentrations. The difference between the two was considered bound
or complexed metal concentration.
Initially ED experiments were conducted on aqueous (free) metal
ion solutions alone. These experiments were designed to determine
36
-------
the amount of time needed to attain equilibrium across the membranes
in the sea water medium.
2.2.3 Chromium Speciation.
Additional experiments were needed to determine what fraction,
if any, of the free Cr measured in ED experiments was present as
Cr(III). To achieve this, Qcnnan dialysis (DO) was employed. The
ion exchange membranes aescribed for ED were also used in these
experiments but the dialysis cells were loaded witn 15.0 ml of 1 M
HNO to promote DD. Initial DD experiments were done in triplicate
witn Cr(III) or Cr(IV) only, at three concentrations. Sample
aliquots were taken 2, 3, A or 6 h after the start of dialysis. The
desired result was to maximize transport of Cr(IIl) across the
membranes while minimizing Cr(VI) transport. Once optimum conditions
were determined, experiments were conducted with selected drilling
fluid/sea water phases.
2.2.4 Trace Metal Analysis.
Ail solutions for trace metal analysis were stored and
transferred by using acid-cleaned plasticware. The samples for
particulate and dissolved metal analysis were analyzed within two
hours of sampling. The uncentrifuged samples were shaken
periodically prior to analysis to keep particles susoended. All
other sample aliquots (i.e., from dialysis) were preserved with 5
uL/mL of redistilled HNO
Eight elements (9a, Cd, Cr, Cu, Mr,, Ni, Pb and Zn) were
determined simultaneously by direct current plasma emission
spectrometry (DCP) with a Spectrametrics Spectraspan I1IB. Sample
37
-------
emission intensities were compared with a two-point calibration curve
of standards in acidified and 0.45 urn filtered sea water. Data
aquisition and calculations, including instrument drift correction,
were performed by a dedicated minicomputer (Charles River Data
Systems, model MF-211).
2.3 Results and Discussion
2.3.1 Preliminary Studies.
It was necessary to test certain aspects of the speciation
procedure on laboratory-orepared solutions before application to
drilling fluids. The 1,000 MWCQ dialysis membranes have been used in
speciation studies in freshwater and were demonstrated to adequately
separate trace metals and certain natural ligands via equilibrium
dialysis (ED) (Truitt and Weber, 1981a, 1981b; Rainville and Weber,
1982). These membranes have not been used in a sea water medium or
for all the metals studied here. Initial experiments demonstrated
that equilibrium was reached between 18 and 24 hours after t^s start
of dialysis for all eight metals tested. This included both Cr(III)
and Cr(V I).
The ion exchange membranes were used in two modes, for ED and
Donnan dialysis (DD). For ED the sample was dialyzed against a
similar medium (0.45 um filtered sea water) and equilibrium was
reacned slowly. The ion exchange membranes contain sulfonic acid
groups (-SO-jH) that are dissociated and negatively charged at the
pH of sea water. The porous nature of the membranes allow small ions
to pass under ED conditions (i.e., same medium on both sides), but
large anionic molecules such as 1ignosulfonate or lignite are
repelled by the sulfonate groups. It was determined that
38
-------
approximately 100 hours were necessary for equilibrium to be reached
with metal ions alone. ChromiumCVI), however, did not reach
equilibrium over this time period because of its anionic form. For
this reason, the ion exchange membranes could not be used for E0 of
Cr(VI). Dialysis of ferrochrome lignosulfonate (Q-Broxin, Baroid
Corp.) demonstrated that 90 to 100% of the 1ignosuifonate was
rejected by the membranes. Lignosulfonate was quantified by
measuring UV absorbance of the oeak at 275-280 nm (Alberts, 1982,
Almgren et, al., 1975). This done for the dialysis of test phases
as well. Only 0 to 7% of the UV-absorbing species were able to pass
the membranes.
Donnan dialysis is a relatively rapid orocess compared to ED and
it is possible to achieve an enrichment of the analyte in the
dialysis cell (Cox and Twardowski, 1980). In DD the sample is
dialyzed against a high concentration of a particular cation.
Experiments with Cr
-------
of Cr(III) and 0.008 ppm of Cr(VI) in the dialysis cell. It was
concluded that these results were adequate for the technique to be
useful for speciation of Cr in drilling muds.
2.3.2 Test Pnase Results.
The results of trace metal analysis and speciation of liquid and
susoended-solids test phases are listed in Tables III through VII for
Ba, Cr, Cu, Mn and Zn. When comparing results for different drilling
fluids, the sea water dilution factors (test phase concentration)
must be considered. The concentrations of metal are listed under
three categories. Total metal includes all forms: particulate, free
and organically bound. Solution phase metals are dissolved forms,
both free and bound, from analyses of centrifuged samples. Free
metal is the concentration that can pass the ED membranes and is
assumed to oe small inorganic species.
The concentrations of Cd, Ni and Pb in the muds diluted with sea
water were undetectable. Detection limits for the DCP system were
0.01, 0.02 and 0.20 ppm for Ni, Cd and Pb resoectively. Tne results
for the Sept. 4 Mobile Bay mud that was used as a quality control
toxicity standard are shown at the end of each table (III—VII).
These data give a realistic idea of the day-to-day precision of
preparation and s'ampling of the test phases.
40
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Table III. CONCENTRATION AND SPECIATIQN OF BARIUM IN
DRILLING FLUID/SEAWATER TEST PHASES
Drilling
Fluid
AN31
MI9LKA51
SV 76
PI
P2
P3
P4
P5
P6
P 7
P8
Sept. 4
(Mobile
Bay)
Type of
Phase3
Liquid
Suspended
Liquid
Suspended
Liquid
Suspended
A v g . L i q. (2)
Suspended
Avg .Liq.(2)
Suspended
Liquid
Liquid
Susoended
Liquid
Suspended
Liquid
Suspended
Liquid
Avg . Sus.(2 )
Liquid
Avg.Sus.(2)
Avg . '_iq . (2 )
Avg.Su s.(2)
Avg.Liq.(3)
Avg.Sus.(4)
Phase
Cone.b
(mL/L)
3.0
2.5
3.0
5.0
3.0
0.15
3.0
1.0
3.
0.
2,
3.
1,
3,
0.
3,
0,
0
5
,0
0
0
.0
5
,0
5
3.0
3.0
3.0
3.0
3.0
3.0
1.0
0.5
Total
0.2 5 ±0.01
3.1 ±0.5
0.093±0.004
1.8 ±0.3
0.7 ±0.4
2.61 ±0.07
Concentration of Barium (mq/L)
0.4
5.5
±0.2
±0 • 1
0.34 ±0.05
5.3 ±0.1
0.85 ±0.06
1.23 ±0.09
9.0 ±0.9
0.43 ±0.02
13.5 ±0.9
1.16 ±0.03
6.4 ±0.8
0.58 ±0.003
0.4 ±0.3
0.152±0.009
5 ±3
0.7
23
±0. 1
±14
0.4 ±0-2
8 ±3
Solution
0.104±0.002
0.120±0.002
0.082±0.002
0.071±0.002
0.13 ±0.01
0.130±0.01
0.087±0 .002
0.034±0.003
0.124±0.004
0.07 ±0.001
0.153±0.008
0.28 ±0.01
0.27 ±0.07
0.29 ±0.02
0.87 ±0.01
0.158±0.003
0.119±0.00 5
0.052±0.002
0.05 0±0.003
0.109±0.001
0.12 ±0.01
0.13 3±0.006
0.4 ±0.3
0.03 3±0.003
0.028±0.011
Free
0.098±0.002
0.0 78±0.002
0.117±0.003
0.09 ±0.02
0.067±0. 009c
0.112±0.005
0.049±0.002
0.092±0 .002c
0.2 ±0.2C
0.092±0.002
0.2 ±0.02c
0.098±0.006
0.09 ±0.01
0.067±0.003°
0.06 ±0.01c
0.I3l±0.005c
0.14 ±0.04c
0.15 ±0.02c
0.20 ±0.01
0.04 ±0.02=
0.10 ±0.08c
a Replicate phases are expressed as an average with number of replicates
in parentheses.
b Concentrations are mL of whole mud per L of 0.45 jm filtered seawater.
c There was no significant difference between free and solution phase
barium in these experiments.
41
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Table IV.
CONCENTRATION AND SPECIATION OF CHROMIUM IN
DRILLING FLUID/SEAWATER TEST PHASES
Phase
Drilling
Type of
Cone.D
Concentra
tion of Chromium
(mg/L)
Fluid
Phase3
(mL/L)
Total
Solution
Free
AN31
Liquid
3.0
0.03810.002
0.03410.002
0.01710.002C
Suspended
2.5
0.10510.009
0.03610.004
MIBLKA51
Li quid
3.0
0.01010.002
0.01010.002
0.00510.002C
Suspended
5.0
0.076±0.005
0.01610.003
SV 76
Liquid
3.0
0.59 10.02
0.54310.004
0.21410.007=
Suspended
0 . 15
0.13510.008
0.05110.003
PI
Avg.Liq. (2)
3.0
0.15510.004
0.15 10.02
0.06710.002C
Suspended
1.0
0.11010.003
0.03710.003
P2
Avg-Liq.(2)
3.0
0.04410.005
0.04110.003
0.02510.002C
Suspended
0.5
0.08310.003
0.01610.003
P3
Liquid
2.0
0.11010.006
0.09310.003
Liquid
3.0
0.12 +0.02
0.11 +0.01
d
Suspended
1.0
0.11510.009
0.03710.004
P 4
Liquid
3.0
0.3 3910.008
0.34110.005
d
Suspended
0.5
C.13410.005
0.05610.004
P5
Liqui d
3.0
0.03910.003
0.03310.003
d
Suspended
0.5
0.01410.003
0.008
P6
Liqu id
3.0
0.004
0.004
d
Avg.Sus.(2)
3.0
0.004
0.004
P7
Liquid
3.0
0.00710.002
0.00610.002
d
Avg.Sus.(2)
3.0
0.09 10.03
0.00910.002
P8
Avg .Liq.(2)
3.0
0.27 1 0.02
0.26410.008
0.03110.006=
Avg.Sus.(2)
3.0
0.6 10.2
0.2 7 510.003
Sept. 4
Avg .Liq.(3 )
1.0
0.9 10.2
0.8 10.2
0.07610.002
(Mobile
Avg.Sus.(4)
0.5
1.3 10.5
0.6 10.2
0.11 10.03
Bay)
a Replicate phases are expressed as an average with number of replicate
in parentheses.
b Concentrations are mL of whole mud per L of 0.45 um filtered seawater.
c Used 1000 MWCO memDranes for one test phase in duplicate; see text
for discussion,
d Not determined.
42
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Table V. CONCENTRATION AND SPECIATiON OF COPPER IN
DRILLING FLUID/SEAWATER TEST PHASES
Phase
Drilling
Type of
Cone
Concentration of Copper
(mg/L)
Fluid
Phase3
(mL/L)
Total
Solution
Free
AN31
Liquid
3.0
0.00410 .002
0.00410.002
0.00310.002
Suspende d
2.5
0.007
0.007
MI3LKA51
Liquid
3.0
0.00 3±0.001
0.00210.001
0.00210.002
Suspended
5.0
0.01310.004
0. 002
SV76
Liquid
3.0
0.04 310.002
0.04110.002
0.01010.002
Suspended
0.15
0.007
0.007
PI
Avg.Liq.(2)
3.0
0.009±0.002
0.00710.003
0.00510.002
Su spended
1.0
0.004±0.001
0.00310.001
0.006
P2
Avg.Liq.(2)
3.0
0.00210.001
0.00210.001
0.00310.002
Suspended
0.5
0.006
0.004
0.006
P3
Liqu id
2.0
0.01110.003
0.00910.003
0.004
Liquid
3.0
0.01510.002
0.01410.002
Suspended
1 .0
0.01010.003
0.00510.003
0.006
P4
Liquid
3.0
0.004
0.004
0.004
Suspended
0.5
0.006
0.006
0.006
P 5
Liquid
3.0
0.01210.003
0.01310.003
0.004
Suspended
0.5
0.005
0.005
0.005
P 6
Liquid
3.0
0.004
0.004
0.004
Avg.Su s.(2 )
3.0
0.004
0.004
0.004
P7
Liquid
3.0
0 .004
0.004
0.004
Avg.Sus.(2)
3.0
0.00410.002
0.004
P8
Avg.Liq.(2)
3.0
0.00610.002
0.00610.002
0.00310.002
Avg.Sus.(2)
3.0
0.01610.008
0.00410.004
0.004
Sept. 4
Avg .Liq.(3)
1.0
0.01310.002
0.01110.003c
0.004
(Mobile
Avg . Sus.(4)
0.5
0.01310.003
0.00510.002C
0.005
Bay)
a Replicate phases are expressed as an average with number of replicates
in parentheses.
b Concentrations are mL of whole mud per L of 0.45 urn filtered seawater,
c Average was computed using only the values above the detection limit.
43
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Table VI. CONCENTRATION AND SPECIATION OF MANGANESE IN
DRILLING FLUID/SEAWATER TEST PHASES
Phase
Drilling
Type of
Cone.
Concentration
of Manganese
(mg/L)
Fluid
Phase3
(mL/L)
Total
Solution
Free
AN31
Liquid
3.0
0.03 ±0.01
0.027±0.007
0.0 36±0.007
Su spended
2.5
0.02910.005
0.014±0.005
MIBLKA51
Liquid
3.0
0.01 ±0.01
0.01 ±0.01
0.02
Suspended
5.0
0.014+0.006
0.012
SV76
Liquid
3.0
0.27 ±0.01
0.27 ±0.01
0.23 ±0.01
Suspended
0. 15
0.06 ±0.01
0.021±0.009
PI
A v g . L i q. (2 )
3.0
0.04 ±0.01
0.04 ±0.01
0.03 ±0.01
Suspended
1.0
0.018±0.005
0.010±0.004
0.02
P2
Avg.Liq.(2)
3.0
0.02
0.02
0.02
Suspended
0.5
0.012±0.005
0.01
0.02
P3
Liquid
2.0
0.12 ±0.02
0.114±0.009
0.104±0.007
Liquid
3.0
0.15 ±0.02
0.15 ±0.02
Suspended
1.0
0.060±0.007
0.02 3±0.004
0.08 ±0.01c
P4
Liquid
3.0
0.2 3 ±0.01
0.22 ±0.01
0.19 ±0.01
Suspended
0.5
0.02 8±0.005
0.017±0.005
0.05 ±0.01c
P5
Liquid
3.0
0.10 ±0.01
0.Q93±0.008
0.084±Q.008
Suspended
0.5
0.026±0.006
3.007±0.005
0.02
P 6
Liquid
3.0
0.041±0.003
0.037±0.007
0.04 ±0.01
Avg . Sus.(2)
3.0
0.026±0.008
0.021±0.000
0.0 3 ±0.0Id
P7
Liquid
3.0
0.008
0.008
0.002d
Avg . Sus.(2)
3.0
0.0 3 ±0.01
0.011±0.003
P8
Avg.Liq.(2)
3.0
0.11 ±0.02
0.10 ±0.01
0.106±0.008d
Avg.Sus.(2)
3.0
0.10 ±0.04
0.055±0.008
0.047±0.006d
Sept. 4
Avg.Liq.(3 )
1.0
0.03 ±0.01
0.021±0.008
0.015±0.0 0 7 d
(Mobile
Avg.Sus.(4 )
0.5
0.07 ±0.02
0.020±0.008
0.024±0.009
Bay)
a Replicate pnases are expressed as an average with number of replicate
in parentheses
b Concentrations are mL cf whole mud per L of 0.45 urn filtered seawater
c Slight contamination but no difference between free or solution phase
manganese.
d One test phase analyzed in duplicate
44
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Table VII. CONCENTRATION AND SPEC I AT I ON OF ZINC IN
DRILLING FLUID/SEAWATER TEST PHASES
Phase
Drilling
Type of
Cone .b
Concentration of Zinc
(mq/L)
Fluid
Phase3
(mL/L)
Total
Solution
F ree
AN31
Liquid
3.0
0.008
0.008
0.006
Suspended
2.5
0.01510.004
0.008
MIBLKA51
Liquid
3.0
0.004±0.003
0.006
0.003
Suspended
5.0
0.019±0.005
0.010
SV76
Liquid
3.0
0.02210.003
0.01010.003
0.017±0.003
Suspended
0. 15
0.02710.006
0.010
PI
Avg.L iq .(2 ) 3.0
Suspended 1.0
0.00510.003
0.01910.002
0.00510.003
0.00710.002
0 . 005 + 0•003
0.01710.007C
P2
Avg.Li q.(2)
Suspended
3.0
0.5
0.008
0-03410.008
0.008
0.004
0 .008
0.01310.007C
P3
Liquid
L iqu id
Su spended
2.0
3.0
1.0
0.00910.004
0.008
0.02710.004
0.00810.003
0.008
0.00710.002
0.01410.003°
0.01110.006
P4
Liquid
Suspended
3.0
0. 5
0.01710.004
0.04210.004
0.01310.004
0.00710.004
0.03910.004C
0.01910.004°
P 5
Liquid
Suspended
3.0
0.5
0.008
0.006
0.008
0.008
0.01910.006°
0.01310.004°
P6
Liqu i d
Avg.Sus.(2)
3.0
0.01810.003
0.01310.004
0.01710.006
0.01310.005
0.04310.008C
P7
Liquid
Avg.Sus.(2)
3.0
0.006
0.01210.002
0.006
0.006
0.22 10.05^
P8
Avg.Liq.(2)
Avg.Sus. (2 )
3.0
3.0
0.00710.003
0.06 10.03
0.00810.005
0.01110.003
0.00510.003
0.21 I0.0ic,d
Sept. 4
(Mobi le
Avg.Liq.(3 )
Avg.Sus.(4 )
1.0
0.5
0.07210.008
0.23 10-09
0.04010.005
0.05 10.02
0.02 10.01
0.04 10.02
Bay)
a Replicate phases are expressed as an average with number of replicates
parentheses.
b Concentrations are mL of whole mud per L of 0.45 um filtered seawater.
c Slight contamination but no difference between free and solution phase
zinc in these experiments,
d One test phase analyzed in duplicate.
-------
2.3.3 3arium.
The concentrations of 3a in the test ohases (Table III) were the
highest of any element determined. Liquid phase results show that
even after 72 hours of settling time, Ba suspended in the water
column was still significantly higher than dissolved Ba. Total
concentrations were higher than solution concentrations in every
case. This was probably due to colloidal BaSQ^.
The high and variable total concentrations were expected for
susoended-solids phase Ba because a great deal of particulate BaSQ^
was still suspended after 1 hour of settling. Rough calculations
Dased on the Stoke's Law settling velocities of quartz spheres
indicate that only oarticles less than 10 um in diameter would remain
suspended in these phases. This is an upper limit for BaSO^
because its density is almost twice that of quartz. The poor
instrumental precision obtained for Ba in susoended-solids phases may
oe due to BaSO^ crystallizing to a limited extent in the instrument
nebulizer. The accuracy of the measurement for these particles is
probably still good. It has been demonstrated that particles up to
14 um are atomized with virtually 100% efficiency by using the DCP
system (Saba et_ al_. , 1981).
Solution phase Ba numbers were similar for liquid and
suspended-solids phases of a given mud even though for some muds the
two phases were of different concentration. These values varied
somewhat from mud to mud and were significantly higher than the
published sea water solubility limit for BaSO^ (Chow and Goldberg,
i960) with the possible exceptions of the P6 and Sept. 4 muds.
Barium can exist in solution above its sea water solubility because
46
-------
certain mud components in these complex mixtures can alter the
solubility equilibria involved.
Data for free Ba concentrations were very similar to solution
phase values, but were slightly lower in some cases. This indicates
that the predominant form of this element in solution was Ba, but
a small amount could have been soluble as ion pairs of BaSO^.
2.3.4 Chromium.
Particulate Cr was a significant portion of the total
concentrations only for the suspended-solids phase, with total values
ranging from three to five times the solution concentrations (Table
IV). The only exception was the P7 suspended solids phase which had
a particulate Cr concentration that was ninefold higher than the
solution concentration of Cr. Total Cr values for the liquid phases
were essentially the same as solution Cr values. Solution
concentrations were similar for both types of test phases when phase
concentration is considered.
As mentioned above, the ion exchange membranes were
unsatisfactory for ED of Cr(VI). The 1000 MWCO membrane results were
the only data used to obtain free Cr values. For this reason, the
data in the "free" column of Table IV are limited. A significant
difficulty with these membranes, however, is that tney are not as
good as the ion exchange membranes for preventing the passage of the
organic ligands. In experiments with the 1C00 MWCO membranes, an
average of 40% of the UV absorbing material from the test phases
passed through the membranes with the free ions. This means that
some bound Cr may pass the membranes so the results could
overestimate the free ion concentrations. Although this possibility
47
-------
exists, it is not likely that this overestimate is very large.
Results for Ba, Cu, Mn and Zn showed agreement between membranes.
This is possible even in light of the UV data because these
measurements are not specific for the organic ligands and may measure
other UV absorbing components of the phases that do not bind metal
ions.
2.3.5 Free Chromium(III) vs Chromium(VI) .
Donnan dialysis (DD) of selected test phases separated free
Cr(III) from free Cr(VI) and bound Cr. The results are shown in
Table VIII. Free Cr(Vl) was calculated by difference from the total
free Cr data (Table IV) and the free Cr(III) values. In most cases
the Cr(VI) values were very low or undetectable; however, for the
SV76 and PI test phases, the Cr(Vl) concentration was significant.
To confirm the dialysis results, Cr(VI) in the SV76 and PI test
phases was also determined by differential oulse oolarography (DPP).
The value determined for SV76 was 0.1 opm, but Cr(VI) in the PI test
phase (3.0 mL/L) was below the detection limit of approximately 0.02
ppm for the conditions used. Both of the values determined by DPP
were lower than the corresponding DD results. The reason for this is
the overestimate of free Cr discussed above (Section 2.3.4). This
overestimation may be by a factor of two, judging from this limited
data. DPP results are not without potential interferences that could
affect accuracy especially in a high organic matter matrix like
drilling muds (Jacobson and Lindseth, 1976). However, results from
these two independent methods indicate that Cr(VI) was definitely
present in the SV76 mud and possibly in PI as well. Information
48
-------
concerning the composition and history of the drilling muds used in
this study indicate that Cr(VI) as dichromate was added to the 5V76
mud at a substantial level (0.2 lbs/bbl) during drilling operations.
Since Cr(VI) has been demonstrated to be the most toxic form of Cr to
marine organisms (Carr et_ a_l. , 1982; Mayer and Schick, 1981), and to
certain larvae (Bookhout e_t a_l. , 1982), it is likely that the
concentrations of Cr(VI) in these two phases contribute to their
toxicity. Of the phases that were tested, the SV76 liquid phase was
found to be the most toxic to M. mercenaria larvae (Table I, Section
1), and the PI mud was the most toxic to mysid shrimp (personal
communication, Thomas W. Duke, EPA Gulf Breeze).
2.3.6 Copper.
The total Cu concentrations in all test phases were extremely
low and were below detection limits in many cases (Table V). The
SV7 6 test phases had the highest amount of Cu, with approximately
0.04 mg/L. Solution concentrations of Cu were similar to total
values in general, with a few exceptions for suspended solids phases
(MIBLKA51, P8, and Sept. 4 muds). Free Cu concentrations were less
than Q.Q06 mg/L for all but the SV76 liquid phase. These data
indicate that Cu occurs principally in a bound form in these muds.
This is what would be expected, since Cu is known to form strong
associations with organic ligands (Ryan and Weber, 1982; Mantoura e_t
al., 1978).
49
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TABLE VIII. FREE CHROMIUM (III) AND CHROMIUM (VI)
FROM DONNAN DIALYSIS OF DRILLING FLUID/SEAWATER LIQUID PHASES3
Drilling
Fluid
Concentrations of Chromium (mg/L)
Free Cr13
Free Cr(III)
Cr(VI) by difference0
AN31
MIBLKA51
SV 76
PI
P2
P8
0.01710.002
0.00510.002
0.21410.007
0.067±0.002
0.02510.002
0.03110.006
0.02210.006
0.02610.006
0.02510.007
0.02010.004
0.01410.007
0.02210.009
0
0
0.19 10.01
0.04710.004
0.01110.007
0.00910.009
a All phases were 3.0 mL of drilling fluid per L of 0.45 urn filtered
seawa ter.
b From Table iv.
c Cr(VI) concentrations are the difference between free Cr and Cr(III)
values.
50
-------
2.3.7 Manganese.
The results for Mn were quite consistent among total, solution
and free values for the liquid ohase (Table VI). The suspended-solids
phase exhibited a degree of particulate Mn with solution and free
values similar in most cases. These results show that in addition to
the low particulate quantities, most of the Mn is present in the free
form. Manganese, however, is not known to be highly toxic. Even
tnough the concentrations of Mn reported he re are substantially above
the concentrations measured for coastal waters, there is probably
little danger to marine organisms from Mn in drilling fluid
discharges.
2.3.8 Zinc.
A small fraction of the total Zn measured for suspended-solids
phases was present as a particulate form. 'he remainder of the Zn
was present in a free form in these phases and the liquid phases
(Table VII) .
Difficulties were encountered with contamination of Zn in the
dialysis experiments. Zinc is prevalent in urban environments and
t n e source of the problem is believed to be the 0.45 urn filtered sea
water which is pumped from Boston Harbor. The contamination,
however, does not preclude the conclusion that Zn is primarily free
in the drilling mjds that were analyzed. The justification for this
is that the contaminant Zn was found to be between zero and three
times the original concentration. Typically, any Zn present will
partition to some extent, and may associate with particles or organic
ligands. The dialysis experiment allows measurement of the final
51
-------
equilibrijm state attained. Since all of the solution phase Zn was
free, this is assumed to be the predominant form of the original Zn.
2.3.9 Conclusions
Results from the measurement of trace metals in drilling
fluid-sea water mixtures showed that the average concentrations of
the aetectabie elements decreased in the order 8a>Cr>Mn»Zn>Cu.
The concentrations of Cd, Ni and Pb were below the detection limits
of the measurement system (0.02, 0.01 and 0.2 mg/L respectively).
All metals exhibited some oarticle association in 1 h settled phases
(suspended solids) with Ba being present principally in the
Darticulate fern. Chromium and Cu were bound, probably as
lignosulfonate complexes, but Mn and Zn were primarily in free
forms. A significant portion of the Cr was present as highly toxic
Cr(Vl) in two cf six muds analyzed for this f orm of Cr.
The potential threat of metal toxicity, bioaccumulation and food
chain biomagnification with respect to marine organisms is greater
from Cr than other elements tested. Although most of the Cr is
probably present as Cr(lII) complexes of lignosulfonate, its
concentration is relatively high. Tne lack of toxicity usually
observed for Cr(III) nas been attributed to its low solubility (Carr
et a 1., 1982). Lignosulfonate complexes Cr(III) and increases its
solubility, thus increasing the potential threat of this form of Cr
(Liss et a_l. , 1980; Knox, 1978). In addition Cr(VI) is often the
form of Cr added to lignosulfonate to prepare chrome or ferrochrome
lignosulfonate (Knox, 1978). Chromium(VI) salts are sometimes added
to drilling muds as well (Moseley, 1980; Section 2.3.5). Considering
52
-------
the large quantities that are used, it is very likely that some
Cr(VI) will remain unreactsd in the mud where pH conditions are
unfavorable for Cr(Vl) reduction (Moseley, 1980). In oxygenated sea
water, the stable form of dissolved Cr is Cr(VI). It has been
demonstrated that Cr(III) is present, but slow oxidation occurs in
the presence of and is catalyzed by manganese oxide (Van der
Weijden and Reith, 1982). This suggests that any Cr inputs to the
ocean, regardless of the form, are potentially hamful.
53
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III. ORGANIC CONSTITUENTS IN WHOLE DRILLING FLUID AND DRILLING
FlUID/SEA water toxicity test phases
3.1 Background
Since there is a oaucity of general information in the
literature or, drilling fluids and their specific chemical
constituents (Sprague and Logan, 1979), it was considered necessary
to perform qualitative/quantitative analysis of organic
constituents. The possibility that the organic constituents of the
used drilling fluids may contribute to the various toxicological
responses of marine organisms is of important consideration.
Development of methodologies for organic analyses of the used whole
drilling muds and the drilling mud test ohase solutions was conducted
by modifying accepted organic analysis techniques (IERL-RTP Procedure
Manual: Level 1; Environmental Assessment, U.S. EPA, 1978). These
procedures were employed to provide a satisfactory characterization
of the whole mud and test phases by identifying the major classes of
organic compounds and their concentrations.
Qualitative/quantitative analyses were directed specifically
toward the analysis of petroleum hydrocarbons in drilling fluids.
The presence of hydrocarbons indicates either their intentional
addition to drilling fluids to aid in the drilling process or their
natural occurrence in strata penetrated by the drill (Grahl-Nelson e_t
a 1¦, 1980; Neff, 1981). There is an abundance of data on
physiological responses of marine organisms to petroleum
hydrocarbons; knowledge of the typical addition of diesel oils (#2
fuel oil) to drilling fluids warranted the analysis and
quantification of petroleum hydrocarbons in drilling fluids.
54
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3.2 Materials and Methods
3.2.1 Whole Drilling Fluid Analyses.
Quantitative and qualitative determinations of organic compounds
(i.e., #2 fuel oil components) in whole drilling muds were made by
following standard modified methodologies (previously referenced,
Sec. 3.1). All glassware was precleaned by either solvent rinsing or
heating at 500°C. Solvents used in extraction and chromatography
procedures were of "distilled-in-glass" purity, and the chemical
adsorbents (magnesium sulfate and silica gel) were pre-cleaned by
either solvent extraction or heating at 500cC.
Initially, a 25.0 g aliquot of drilling mud was weighed in a
beaker, and the pH of the sample was adjusted to 2.0 _+ 0.5 with
hydrochloric acid. After 0.5 h equilibration in an ice bath,
magnesium sulfate was added with mixing to adsorb the water and
orovide a homogeneous mixture for extraction. The mixture was then
extracted in a Soxhlet-extraction apparatus for 24 h by using 250 mL
of methylene chloride. Next, the methylene chloride extract was
concentrated to 5.0 mL in a Kuderna-Danish concentrator on a steam
bath, and a 2.0 mL portion of the concentrated extract was pioetted
into a glass chromatographic column (30C mm x 10.5 mm) packed with a
6.0 g portion of freshly activated silica gel in 50% v/v methylene
chloride/pentane. The sample was eluted with 50 m'_ methylene
chloride/pentane (50% v/v) at a flow rate of 1-2 mL/min to elute
petroleum hydrocarbon aliphatic and aromatic fractions. The eluent
was concentrated to 5 mL by using steam or rotary evaporation and
then adjusted to 5.0 mL for gas chromatograohic/mass soectrcmetric
analysis.
5 5
-------
Gas chromatographic/mass spectometric (GC/MS) analyses were
performed by using a Hewlett-Packard 5992A GC/MS with data system.
Chromatographic separations were carried out by using 18G cm x 2 mm
i.d. glass columns packed with 10% SP-2100 on Supelcoport 100/120
mesh, and with temperature programming. The mass spectrometer was
operated in the electron-impact mode, and low resolution mass spectra
were obtained by continuous scanning under control of the dat3
system. Quantification was based on the measurements of external
standards (i.e., API #2 fuel oil).
3.2.2 Drilling Fluid/Sea water Test Phases.
One-liter aliquots of the drilling fluid-sea water test phases
were sampled, and 100 mL of methylene chloride was added to each
sample for preservation of sample integrity until extraction. The
1iajid sample was transferred to a 2 L separatory funnel and was
shaken vigorously for approximately 2 min. The methylene chloride
extract was drawn off. A second 100 mL volume of methylene chloride
was added to the sample for an additional 2 min. extraction. The
combined methylene chloride extracts were dried over anhydrous sodium
sulfate and concentrated to 10 mL by rotary evaporation under reduced
pressure or by steam. After transfer to calibrated sample tubes, the
samole volume was further reduced to 100-500 uL by air blow-dcwn.
Various aliquots of tns samole concentrate were analyzed by
previously described GC/MS procedures.
Bulk characteristics of the whole drilling muds were determined
by a variety of procedures. Percentage water i% H2o) was
determined by overnight drying of a whole muo subsample at 105°C.
56
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Tne percentage weight loss was determined gravimetrically. Tne pH of
each sample was measured by mixing a 1:5 ratio, whole mud
sample:distilled water, and determining the pH potentiometrically.
Density (g/mL) was determined by the weight determination of a
specific volume of whole mud. Organic volatiles (mg/g) were
determined by combusting dried whole mud samples at 550°C for 1 h.
The weight loss was determined gravimetrically.
3.3 Results and Discussion
For organic constituent analyses, the whole drilling muds
containing 25-70% water (Table IX) could not be treated as aqueous
solutions. Extraction techniques (i.e. mixing the drilling fluid
with a polar extraction solvent like methylene chloride) produced an
emulsion which could not be separated satisfactorily. An alternative
approach was to freeze dry the drilling fluid sample and to extract
the residue with solvents to isolate the organic constituents.
However, freeze drying caused the loss of volatile organic components
through co-distillation processes and proved this method to be
unsatisfactory. Finally, a more satisfactory method was used that
involved the addition of an excess of anhydrous magnesium sulfate
(MgSO^) to dehydrate the drilling fluid, giving a oowdery mixture
from which organic constituents we re readily extracted with methylene
chloride.
Silica gel column chromatography proved suitable for sample
clean-up and separation. Gas chromatography/mass spectrometry
analysis (GC/MS) was performed on these fractions to identify the
principal organic-extractable constituents of the whole drilling
57
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TABLE IX.
BULK
CHARACTER1STI
CS OF WHOLr
DRILLING
FLUID
Drilling
Dens ity
Volatiles
Fluid
fiH
%rl20
q/mL
lb/gal
(dry
weight, mg/c
AN31
10.5
49.9
1.50
12.5
50.8
MI5LKA51
10.0
69. 5
1.26
10. 5
38.5
SV76
10. 5
27.3
2.17
18.1
31.4
PI
11.9
33.8
1.93
16. 1
37.6
P2
12.1
30.0
2.02
16. 9
37.6
P 3
11.2
26.8
2 . 19
18.3
34. 5
P4
8.0
33.5
1.93
16. 1
20.5
P5
11.5
26.3
2.20
18.4
16.7
P6
8.8
71. 5
1 .22
10 .2
26.6
P 7
11.4
57.0
1.37
11.4
44.2
P8
10.4
27 .3
2.17
18.1
21.0
Sept. 4
(Mobile
9.7
Bay)
54.2
1 .55
12.9
49.9
58
-------
fluid. GC/MS scans of the whole drilling fluid extracts closely
resemDied those of fuel or diesel oils. These results are presented
in Table X as #2 fuel oil (mg/g). Presence of an unresolved complex
mixture (UCM) and an n-alkane homologous series of _ Cj2
together indicate the hydrocarbons were of petrogenic origin. It
should be noted that some of the lower molecular weight (earlier
eluting) components were of lower concentration than found in #2 fuel
oil. Also, the jnresolved mixture shifted to longer retention times.
Additional information detailing bulk characteristics of the
whole drilling fluids are presented in Table IX. Percentage water,
p'H, density, and organic volatiles were determined for the whole
muds. These data detail various physical characteristics of the
drilling fluids.
The drilling mud-sea water test phases (liquid and suspended
solids) were more easily analyzed than the whole drilling fluids for
organic constituents. Toxicity test solutions were extracted with
polar solvents to isolate organic-extractable components. After
concentration of the extracts, GC/MS analyses presented
qualititative/quantitative results (Table X). It should be noted
that these test solutions were neither filtered nor centrifuged prior
to extraction and analyses; therefore, total organic constituents
(i.e., in solution and adsorbed to Darticulate matter) were measured
for each test phase. This procedure was followed because the
toxicity test phases were prepared in a similar manner with test
organisms being exposed to similar constituents either adsorbed to
the particles or in solution. It should be noted that there was a
fivefold decrease in #2 fuel oil-like hydrocarbons in the
59
-------
TABlE X. ANALYSES OF DRILLING FLUIDS FOR #2 FUEL OIL
Concentrations are in mg/g whole mud (wet weight)
Drilling Fluid No.2 Fuel Oil
AN31 1.18
MIBLKA51 0.19
SV76 3.59
Pi 9.43
P2 2.14
P3 3.98
P4 0.67
P 5 1.41
P6 0.10
P 7 0.50
P8 0.56
Sept. 4 2.34
(Mobile Bay)
60
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Table XI. CONCENTRATIONS OF #2 FUEL OIL-LIKE HYDROCARBONS
IN DRILLING FLUID/SEAWATER TEST PHASES
Drilling
Fluid
Type of
Phase
Phase
Concentration
(mL/L)£
Concentrati
(mg/L,ppm)
AN31
Liquid
3.0
0.05
Suspended
3.0
0.61
MIBLKA51
Liquid
5.0
n.d.
Suspended
5.0
0.09
SV76
Liquid
5.0
0.07
Suspended
3.0
2.10
Sus.(Centrifuge)
3.0
0.41
PI
Liquid
3.0
0.04
Suspended
1.0
2.55
P2
Liquid
3.0
0.01
Suspended
0.5
0.24
P 3
Liquid
3.0
0.03
Suspended
1.0
1.23
P4
i_ i q u i d
3.0
0.01
Suspended
0.5
0.10
P5
Liquid
3.0
0.02
Suspended
0.5
0.13
P6
Liquid
3.0
n.d.
Suspended
3.0
n.d.
P7
Liquid
3.0
n.d.
Suspended
3.0
0.07
P8
Liquid
3.0
0.06
Suspended
3.0
0. 36
Sept. 4
Liquid
1.0
C.01
(Mobile
Suspended
0.5
1.67
Bay)
Liquid
1.0
0.04
Suspended
0.5
0.87
Suspended
3.0
0.43
a Concentrations are mL of whole mud per L of 0.45 urn filtered
seawater.
n.d. non-detectable.
61
-------
centrifuged, susoended SV76 sample (continuous centrifugation at
1,000 x G rpm; flow rate 1.0 m_/min) compared to the suspended
(non-centrifuged) phase of the same mjd (Table XI). Therefore, a
major portion of t'ne hydrocarbons in the muds were adsorbed to
particles.
GC/MS scans of tne susoended phases (1 h settlement) also
resembled those of standard API #2 fuel oil. Therefore, #2 fuel oil
was used as a standard for quantification of the samole extracts.
However, GC/MS scans of tne liquid phases (72 h settlment) did not
resemble #2 fuel oil. Tnese samples were quantified by using
naphthalene as an external standard. GC/MS data from tne 1 h settled
solution (highest ccncentation) show various aromatic organic
hydrocarbons in tne solution. These compounds ranged from 1 to 3
aromatic ring sjostituted and non-substituted hydrocarbons.
In the 72 n settled solutions, most of the lower molecular
weight aromatic cot,pcuncs were not present. Also, the higher
molecular weight alkanes (C ^ q - C2g r a n g 9 ) were acsent.
Adsorption, microbial degradation, and volatilization most probably
are important factors influencing this decrease of organic
constituents over the 72 h settling period.
In order to determine the cause of tnese Decreases, changes in
the GC/MS scans of similarly prepared samples or 10 opm (v/v) it2 fuel
oil solution were monitored with time. T^e licuid samples showed 73%
loss of methylene chloride extractable co~ocunds ever a 72 n period.
The Boston Harbor sea water (0.45 urn filtereo; used in these
experiments may have contained hydroca:con-uti 1 i2ing bacteria which
contricuted to loss of compounds in tne 72 ^ liquid test phases.
62
-------
Results from the drilling mud-sea water test phase organic
analyses have proven to be difficult to interpret since there are
many unidentifiable peaks. Adsorption, microbial degradation, and
volatilization are three phenomena that may complicate the spectra.
Also, tributyl phosphate and acetovanillin (degradation product of
lignin) were found in some samples at ppb concentrations.
These previously described phenomena may also contribute to the
disparity of concentrations of #2 fuel oil in the liquid phase
drilling fluid test solutions compared to concentrations in the whole
mud samples. Higher concentrations of hydrocarbons would be expected
in the liquid phase because of their relatively high concentrations
in the whole mud samples. In particular, the low molecular weight
hydrocarbons (earlier eluting compounds) appear to have decreased in
concentration in some samples. Exact determination of the carbon
chain length of these hydrocarbons was not obtained.
The gas chromatograms of liquid phases of the MIBLKA51, D6, anc
P7 drilling muds did not show hydrocarbon profiles or other
ex tract able components. This is consistent with the suspended-solids
phase data which show low #2 fuel oil concentations for these muds.
63
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IV. SOLID PHftSE RECOLONIZftTION STUDIES
The recolonization study described in Progress Report #2
(MEA, 1981) was designed to test the effects of a used drilling
mud on the recruitment of benthic organisms in defaunated
sediment. Data from this study, which involved both a
laboratory-based and a field-tased experiment, nave been
analyzed; results were reported in Progress Report #3 (NEA,
1982) and are Presented below in their entirety.
4.1 Materials and Methods
The matrices of the laboratory- and field-based
experiments were identical and included 15 samples each of
three treatments of sediment: a fine-grained reference
sediment (Control); a drilling mud mixed with reference
sediment (Homogeneous test); and drilling mud deposited on the
surface of reference sediment (Surface test). Five replicate
samples o^ each treatment were removed after two weeks, four
weeks, and six weeks.
Reference mud consisted of a fine-grained sediment
collected from Buzzards Bay, Massachusetts. The reference mud
was defaunated by sieving through a 0.5-mm screen and
refrigerated until needed. A preoetermined weight of reference
sediment was added to each container to provide the required
volume. The drilling fluid was a medium density lignosulfonate
drilling mud supplied by PESA. It was washed by nixing
1 part whole mud with 9 parts sea water. This mixture was
thoroughly stirred once an hour for six hours, then allowed to
settle for two weeks; the liauid phase was discarded. The
64
-------
volume of the remaining slurry was greater than that of the
original whole drilling mud by a factor of 2.4. This factor
was used to calculate the volume of washed slurry necessary to
provide the required volume of whole drilling mud in each test
treatment.
Plastic freezer storage boxes measuring 15.5 cm x 15.5 cm
x 10.5 cm high were used as sample containers. Each control
sample contained 4 cm of reference sediment. Each homogeneous
test sample contained 3.2 cm of reference sediment thorougnly
mixed with 0.8 cm of whole drilling mud. Each surface test
sample haa 0.4 cm of whole drilling fluid deposited on 3.6 cm
of reference mud. These two volume ratios were used because
they provided a 1:4 ratio of drilling mud to reference sediment
in the top 2 cm of substrate, the region in which Wocdin (1974)
found preferential occupation oy five families of polycnaetes
in a natural mud flat.
For the laboratory-based study, a circular tank 1.9 nn in
diameter and 0.3 m deep was installed in the laboratory of
Northeastern University's Marine Science Institute in Nahant,
Massachusetts. This system operated on the principle of
passive overflow drainage. Unfiltered sea water, pumped from
Massachusetts Bay to holding tanks, was gravity-fed into the
center of the tank 21 cm below the surface of the water. A
trough attacned around the entire circumference of the tank
allowed water to drain. This arrangement provided a radially
uniform flow of water over the samples containers, which we re
placed in two rows around tne perimeter on the bottom cf the
65
-------
tank. The outer row accommodated 27 sample containers, and the
inner row 18 sample containers. This experiment was started on
April 8, 1981, using the system shown in Figure 4 and the
sample arrangement shown in Figure 5.
The field-based study was conducted at the University of
Massachusetts Marine Station at Hodgkins Cove, on Cape Ann,
Massachusetts. The site has a mixed mud and sand bottom in
approximately 7 m of water and is subject to tidal currents of
medium strength. Fifteen tightly covered sample containers
we re placed in a weighted wooden box measuring 0.9 n x 0.6 m x
0.3 m (Figure 6). The box was covered with a sheet of 1-cm
mesh plastic grid to prevent intrusions by large predators and
straooed to a weighted plastic platform measuring 1.2 m x 1.0 m
x 0.2 m. Three such units were lifted by a winch over the side
of the 60-ft research vessel "Walter Phipps", sun* and
positioned adjacent to each other by SCU3A-equippea divers.
Approximately 1.5 hr after emplacement, the covers were removed
from the sample containers. This test system (rigure 7} was
deployed on April 10, 1981.
Upon collection, samples were sieved through a 0.25 mm
screen and preserved in 10% formalin. Eacn sample was
subsequently divided into a 0.5 mm and a 0.25 mm fraction, and
stained with Rose Bengal.
The 0.5 mm fraction of eacn sample was sorted under a
stereo dissecting microscope for the two- and four-week
recruitment periods of both experiments. Six-week samoles were
sorted only for the field-based experiment. All animals were
66
-------
SIDE VIEW:
Sea water source
Overflow trough
Drain from
overflow trough
TOP VIEW;
Drain
Overflow trough
nf low
Sample container:
15.5
190.5
FIGURE 4. Laboratory-based Experimental System
67
-------
Drain
Sample lable
Inflow
FIGURE 5. Arrangement of Test Containers for Laboratory-based
Experiment
68
-------
Rubber straps
1-cm-mesh
plastic grating
0.9 m
Sample container
Support (or 1cm-mesh
plastic grating
Support for
straps
v0.6m
Wooden box
with extra
weights
0.3m
platform
1.0
40-lb.
weight
1.2 m
FIGURE 6. Platform Used for One Recruitment Period in
Field-based Experiment
69
-------
A-10
B-10
C-10
A-ll
3-IJ
B-12
3-14
A-14
A-15
I
3-15
Wooden Box
Platform
Sample
Containers
A.
B.
C.
Key to Sample Labels
Control: Reference Sediment
Homogeneous Test: Drilling
Mud Mixed with Reference
Sediment
Surface Test: Drilling Mud
Deposited on the Surface of
Reference Sediment
1-5: Removed after 2 weeks
6-10: Removed after 4 weeks
11-15: Removed after 6 weeks
FIGURE 7. Arrangement of Test Containers for Field-based Experiment
70
-------
identified to the lowest possible taxon and counted. Meiofauna
(consisting of Nematoda, Arachnida, Ostracoda, and CopeDOda)
and the planktonic cyprid stage of cirriped larvae were
identified and counted but were excluded from data analysis
since most members of these taxa were not retained in the 0.5
mm fraction. Colonial species were also excluded.
The 0.25 mm fraction was sorted and animals identified
only for the four-week control and surface test samples of the
lab-based experiment, in order to determine whether sorting the
smaller fraction and including meiofauna had an effect on the
results. Data from both fractions, including meiofauna, were
combined and compared statistically to t^ose from the 0.5 mm
fraction alone, excluding meiofauna.
Three parameters were used for statistical analysis of
samples from each recruitment period of eacn experiment: (1)
number of individuals; (2) number of species; and (3) ratio of
numbers cf species and individuals. The number of individuals
indicates the overall abundance in a unit area. The number of
SDecies is a measure of variety or species richness. Both of
these determine the third parameter, number of species/number
of individuals (S/N), which is a simple estimator of diversity,
uncorrected for sample size or evenness of soecies
distribution. Analysis of variance and the Student-Newman-
Keuls multiple range test were performed to compare the
treatments for the above parameters. Student's t-test was used
for groups of data in which only two treatments were being
compared. In all statistical tests, a 95% confidence level
71
-------
was used (P < 0.05). Recolonizing populations were also
qualitatively characterized by distribution of individuals by
phylum and by species predominance. Species were considered
"predominant" on the following basis: each predominant species
occurred in at least 60% of the replicates and contributed at
least of the total animals in a treatment. Less abundant
species were also included until 15% of the total animals in a
sample were accounted for.
Within each experiment, patterns of community development
over time in the three treatments are compared, using percentage
change in abundance of all animals and of important tax a
between recruitment periods. Results are discussed with
reference to similar studies that have been performed. In
aodition, the lab- and field-based experiments are compared in
terms of methodology.
4.2 Resu It s
Appearance of samples at the time of collection was
similar in both experiments: each sample was covered with a
layer of detritus which increased over time. Detrital deposits
were greater in the field-based experiment. In the surface
test samples of both experiments, the layer of drilling fluid
did not wash out of samples and remained distinct under the
detritus.
Mean numbers (+_ standard deviation) of animals recovered
For all samDies analyzed in both experiments are displayed in
Figure 8. Numbers of animals increased over time in both
72
-------
Field-based experiment
Laboratory-based experiment
Control
Homogeneous test
Surface test
* ^ 1 oo
INOIV luu
/•
A
6
2
4
RECRUITMENT PERIOD (WKS)
FIGURE 8. Mean number of individuals collected after two-, four-,
and six-week recruitment periods for control, homogeneous
and surface test treatments in laboratory- and field-
based experiments. Data are mean of individuals for n = 5
replicates (except for lab-based two-weeK homogeneous test,
where n = 4). Vertical bars indicate standard deviations.
73
-------
lab- and field-based experiments, and the latter had a higher
number of animals than the former after both two- and four-week
recruitment periods.
4.2.1 Laboratory-Based Experiment
4.2.1.1. Two-Week Samples
After two weeks, 231 animals were collected from the
lab-based experiment, representing 30 species in five phyla
(Table XII). For all treatments combined, the most abundant
phylum was Annelida (50% of all animals), followed by Chordata
(26%), Arthropoda (16%) and Mollusca (8%). The three
predominant species were the polychaete Fabricia sabella, the
tunicate Molqula sp., and the tubificid oligochaete PeIosco lex
beneden i.
The mean number of individuals found in control samples
was 19.4 +_ 9.4, that for the homogeneous test treatment was
20.8 +_ 6.8, and that for surface test was 10.2 _+ 2.6 (Table
XIII).
The mean number of species for the three treatments were
6.8 + 2.2 fcr control, 9.6 +_ 2.5 for homogeneous, and 7.0 +_ 1.0
for surface test samples. Control, homogeneous, and surface
samples had mean S/N ratios of 0.43 _+ 0.23, 0.47 +_ 0.09, and
0.72 + 0.18, respectively. Although analysis of variance
showed no significant difference between treatments at P < 0.05
for any of the three parameters, differences for number of
individuals and S/N ratio were significant at P < 0.10.
A similarity between treatments was exhibited when fauna 1
distribution by phylum was considered (Table XIV). In all
74
-------
Table XII: TOTAL NUMBER OF INDIVIDUALS (N), SPECIES (S) AND PHYLA (P) PER TREATMENT
AND FOR ALL TREATMENTS COMBINED (5 REPLICATES/TREATMENT)
la. Laboratory-based Experiments
merit
Period
2
WEEKS
4 WEEKS
Control
Homog.
Test
Surface
Test
Treatments
Combined
Control
Homog.
Test
Surface
Test
Treatments
Combined
N
S
P
97
17
4
104 +
19
4
51
16
5
231
30
5
219
23
4
298
22
5
214
2 3
3
731
29
6
Adjusted
for 5 replicates
lb. Field-based Experiments
ment
-- 2 WEEKS --
- 4 WEEKS
6 WEEKS
Period
Control
Homog.
Surface
Treatments
Control
Homog.
Surface
Treatments
Control
Surface
Treatments
Test
Test
Combined
Test
Test
Combined
Test
Combined
N
292
260
274
826
495
555
299
1349
645
708
135 3
S
34
36
29
49
35
43
32
52
26
31
35
P
3
5
3
5
5
5
4
6
3
4
4
-------
Table XIII:
Number of individuals (N), number of species (S), and diversity index (S/N) for
control, homogeneous and surface test treatments in the laboratory- and field-based
experiments. Data are mean + standard deviation (n = 5 replicates/treatment except
for two week homogeneous test where n = 4). NA = samples not analyzed.
Experiment
Recruitment
Parameter
TREATMENT
Period
Control
Homogeneous Test
Surface
Test
2
N
19.4
+
9.4
20.8
+
6.8
10.2
+
2.6
T3
1
U
0
4
N
43.8
+
11.6
59.6
+
18.1
42.8
+
12.3
03
WKS
S
11.6
+
3.1
10.4
+
1.9
11
+
1.9
o
XI
S/N
0.277
+
0.092
0.180
+
0.033
0.268
+
0.057
flj
.J
2
N
50.4
+
22.0
52
+
18
54.8
+
20.3
WKS
S
14.2
+
3.03
15.6
+
3.8
14 .2
+
2.9
Leld-based
S/N
0.265
+
0.079
0.330
+
0.112
0.271
+
-0.055
4
N
99
+
22.5
111
+
34.4
59.8
+
16.4
WKS
S
18.8
+
2.2
19.8
+
3.6
17.4
+
2.7
S/N
0.20C
+
0.065
0.191
+
0.55
0.303
+
0.073
U)
6
N
129
+
48.6
NA
141.6
+
55.7
WKS
S
13.6
+
0.89
NA
15
+
3.5
S/N
0.114
+
0.02 5
NA
0.118
+
0.045
-------
Table XIV:
Laboratory-based Experiment: Faunal distribution by phylum for control, homogeneous
and surface test treatments over two recruitment periods. Data are % contribution
by phylum and total number of individuals in 5 replicates (in parentheses)
Recruitment
Period
Treatment
Control
Homog.
Test
Surface
Test
Moliusca
6.2 (6)
7.2 (6+)
11.8 (6)
Annelida
54.6 (53)
50.6 (42+)
41.2 (21)
PHYLUM
Arthropoda
10.3 (10)
20.5 (17+)
15.7 (8)
Chordata
28.9 (28)
21.7 (18+)
29.4 (15)
Other
1.9 (1)
Control
Homog.
Test
Surface
Test
5.4 (12)
6.7 (20)
7.0 (15)
65.8 (144)
81.2 (242)
68.2 (146)
28.3 (62)
11.4 (34)
24.8 (53)
0.5 (1)
0.7 (2)
+ numbers of individuals in 4 replicates
-------
Table XV:
Laboratory-based Experiment: List of predominant species for control, homogeneous and surface test treatments
over two recruitment periods. Occ. = Occurrence per 5 replicates, except in two week homogeneous test, where n = 4
CONTROL
HOMOGENEOUS TEST
SURFACE
TEST
Recruit-
ment
# of
* of
Cumul.
# of
% of
Cumul.
* of
» of
Cumul.
Period
Species
Indiv.
Occ.
Total
%
Species
Indiv.
Occ.
Total
%
Species
Indiv.
Occ.
Total
%
1
Fabr icia
1
Fabricia
1.
Molgula sp.
15
5
29.4
29.4
sabella
32
4
33.0
33.0
sabella
23
4
27.7
27.7
2
Molgula sp
28
5
28.9
61.7
2
Molgula sp.
18
4
21.7
49.4
2.
Fabricia sabella 12
4
23.5
52.9
3
Pcloscolex
3
Peloscolex
3.
Peloscolex
z
benedeni
14
3
14.4
76.3
benedeni
11
3
13.3
62.7
benedeni
5
3
9.8
62.7
WKS
4
Jassa
4.
Corophium sp.
3
2
5.8
68.5
fa lcata
4
3
4.8
67.5
5
Pleusymtes
5.
Dexamine thea
2
2
2.4
70.9
glabcr
4
1
4.8
72.3
6
Mytilidae
3
2
3.6
75.9
6.
CapitellidaeC
2
2
2.4
73.3
7.
Mytil idae
2
2
2.4
75.7
8.
Naticidae
2
2
2.4
78.1
1.
Fabr icia
1
Fabricia
1.
Fabricia
sabella
98
5
44 .7
44.7
sabclla
208
5
69.8
69.8
sabella
93
5
43.5
43.5
4
2
Pcloscolex
2
Peloscolex
2.
Peloscolex
WKS
benedeni
33
5
15.1
59.8
benedeni
22
5
7.4
77.2
benedeni
38
5
17.8
61.2
3
Corophium
3.
Corophium
sp.
23
5
10. 5
70.3
sp.b
16
4
7.5
68. 7
4
Marinogam-
4.
Aor idae
10
3
4.7
73.4
marus sp.
12
4
5.5
75.8
5.
Mytilidae
9
5
4.2
77.6
a -
b -
c -
probably Marinogammarus stoerensis
including Corophium bonelli and C. crassicomo
probably Capitella capitata (see text)
-------
treatments, Annelida was the most abundantly represented
phylum, followed in decreasing order by Chordata, Arthropoda,
and Mollusca.
In each of the three treatments, the same three species
were predominant (Table XV). Although surface test samples
showed a slightly different order of predominance, overall
percentages of abundance were very similar for the three
treatments.
4.2.1.2 Four-Week Samples
A total of 731 animals was collected after four weeks,
representing 29 species in six phyla (Table XII). Considering
all treatments together, annelids were predominant (73% of
fauna), followed by arthropods and molluscs (20* ana 6%,
respectively). The most aoundant species were the annelids
Fabricia sabella and Peloscolex benedeni, the ampniood
Co rophium sp. (including c. bone 11i and C. crassicorne) , and
juveniles of a Mytilid mussel.
The mean number of individuals found in control samples
was 43.8 + 11.6. The mean for homogeneous test samples was
59.6 + 13.1, and the mean for surface samples was 42.8 ^ 12.3
(Table XIII). Mean numbers of species were 11.6 +_ 3.1, 10.4 _+
1.9, and 11.0 _+ 1.9, respectively, for control, homogeneous and
surface test samples, and the three treatments had mean S/N
ratios of 0.28 + 0.09, 0.18 + 0.03, and 0.27 + 0.06
respectively. Treatments were not significantly different for
any of the three parameters (AN0VA, P >0.05).
79
-------
When data from the 0.5 mm and 0.25 mm fractions were
combined for each control and surface test sample and meiofauna
were included, mean numbers of individuals were 186.5 + 46.9
and 186.5 26.8, respectively, for the two treatments.
Corresponding mean numbers of species were 18.5 _+ 3.1 and
17.0 0.8; mean S/N ratios were 0.10 +_ 0.02 and 0.09 _+ 0.01.
Performance of Student's t-test showed no significant
difference between tne two means for any parameter (P > 0.05).
These results agreed with results obtained from the 0.5 mm
fraction alone, with meiofauna excluded.
Considering distribution of animals by phylum, annelias
were most abundant in each treatment, followed by arthropods
and molluscs (Table XIV). The percentage distribution was
somewhat different for homogeneous test samoles: annelids
accounted for a greater percentage of fauna found in this
treatment, and the otner two pnyla comprised correspondingly
lower percentages.
In each of the treatments, Fabricia sabella was the
predominant species although by a higher percentage in the
homogeneous test samples (70%, as compared with 45% and 44%,
respectively, for control and surface samples). (See Table
XV.) Second in abundance in each treatment was Peloscolex
beneden i.
4.2.1.3 Comparison of Recruitment Periods
Surface test samples exhibited the highest percentage of
growth, increasing 320% from 51 to 214 individuals (Figure 9a).
The number of animals recovered in homogeneous samples increased
80
-------
2 4
RECRUITMENT period (wks)
ino.v3004.
200f
100--
FIG- 9a. Aii Taxa
2 4
RECRUITMENT PERIOD (WKS)
FIG. 9b. Annelida
2 4
RECRUITMENT PERIOD (wks)
FIG. 9c. Fabricia sabella
Control
Homogeneous test
Surface test
FIGURE 9. Laboratory-based Experiment: Total Number of Individuals
for important Taxa in Control, Homogeneous and Surface
Test Treatments Over Two Recruitment Periods. (Numbers
are pooled data from 5 replicates except for two week
homogeneous test, where data from 4 replicates are
adjusted for comparison).
31
-------
187% from 104 to 298 animals. Control samples displayed the
smallest increase: 126%, from 97 animals after two weeks to 219
individuals after four weeks.
The phylum Annelida remained predominant and contributed
more than any other phylum to the increase in number of animals
(Figure 9b). Surface samples contained 595% more annelids
after four weeks than after two weeks. Corresponding
percentages for homogeneous and control samples were 357% and
172%, respectively.
The annelid species which accounted for this increase was
Fabricia sabe11a (Figure 9c). There were over six times more
F. sabe1 la in the four-week sampling of both homogeneous and
surface test samples. Control samples tripled in number of
this species. The oligochaete PeIosco lex benedeni also
increased in all treatments.
The tunicate Molqula sp., whicn occurred in all two-week
samoles and represented 26% of the fauna, was not present in
the four-week samples. This disaopearanee accounted for
corresoonding increases in predominance of other species and
phyla between the two recruitment periods. No other changes
occurred in the order of species predominance or relative
distribution by phylum from the two-week to the four-week
samples.
4.2.2 Field-Based Experiment
4.2.2.1 Two-Week Samples
After two weeks, 826 animals belonging to 48 species in
five phyla, were recovered from the field-based experiment
82
-------
(Table XII). Combining all treatments, Artnropoda was Dy far
the predominant phylum, constituting 72% of total individuals,
followed by Annelida (21%) and Mollusca (7%). Recently
metamorpnosed adults of a cirriped barnacle were the most
abundant species. Other predominant species were the amphipod
Harinogammarus sp. (probably Marinoqammarus stoerensis), the
isopod Edotea montosa and the polychaete Harmathoe sp.
Control contained samples had a mean number of
individuals of 58.4 _+ 22.0, which was similar to those for test
samples (52.0 * 18.0 and 54.8 _+ 20.3 for homogeneous and
surface samples; see Table XIII). Mean numbers of species for
control, homogeneous and sjrface test samples were 14.2 + 3.0,
15.6 +_ 3.8, and 14.2 _+ 2.9, respectively. Mean S/N ratios were
0.27 + 0.08, 0.33 + 0.11, and 0.27 + 0.06 for the three
treatments, respectively. Analysis of variance showed no
significant difference between treatments for any of these
parameters.
Distribution of animals by ohylum was the same in each
treatment: artnropods were considerably more abundant than
annelids and molluscs (Table XVI). The same four species
predominated in all three treatments (Table XVII). The order
of predominance was identical for control and homogeneous test
samples, and quite similar for surface samples, although
oercentage contribution by the predominant cirriped barnacle in
the latter was lower than in the other two treatments.
83
-------
Table XVI:
Field-based Experiment: Faunal distribution by phylum for control, homogeneous and
surface test treatments over three recruitment periods. Data are % contribution by
phylum and total number of individuals in 5 replicates (in parentheses).
Treatment
PHYLUM
Mollusca
Annelida
Arthropoda
Other
Control
"'.9 (23)
22.2 (65)
69.9 (204)
__
2
WKS
Homog.
Test
5.4 (14)
20.0 (52)
73.1 (190)
1.5 (4)
Surface
Test
7.7 (21)
20.1 (55)
72.2 (198)
—
Control
24.6 (122)
30.3 (150)
44.4 (220)
0.6 (3)
4
WKS
Homog.
Test
18.7 (104)
36.9 (205)
43.8 (243)
0.5 (3)
Surface
Test
27.1 (81)
28.1 (84)
44.5 (133)
0.3 (1)
Control
22.5 (145)
62.8 (405)
14.7 (95)
6
WKS*
Surface
Test
20.3 (144)
62.1 (440)
17.1 (121)
1
1 o
1 £*
1 u>
~Homogeneous test samples not analyzed
-------
o~jo
T3
<§-
e?.S
E8-
o 3
X)
•<
Table XVII:
Field-based Experiment: List of predominant species for control, homogeneous and surface teBt treatments over three
recruitment periods. Occ. - Occurrence per 5 replicates.
CONTROL
HOMOGENEOUS
TEST
SURFACE TEST
Recruit-
ment
• of
% of
Cunul
1 of
* of
Cisnul.
• of
* of
Cunul.
Period
Species
Indiv.
Occ.
Total
«
SpecieB
Indiv.
Occ.
Total
«
Species
Indiv.
Occ.
Total
«
1. Cirripedia
ee
5
30.1
30.1
1.
Cirripedia
81
5
31.2
31.2
1.
Cirripedia
64
5
23.7
23.7
2. Marinoqam-
2.
Marinogam-
2.
Edotea montosa
50
5
18.2
41.9
2
marua sp?
39
5
13.4
43.5
marus sp?
43
5
16.5
47.7
3.
Marinogaro-
W1CS
3. Edotea montoaa
29
5
9.9
53.4
3.
Edotea montosa
25
5
9.6
57.3
marus a p.®
46
5
16.8
58.7
4. Harmatho* sp.
26
5
8.9
62.3
4.
HarmathoV Bp.
20
5
7.7
65.0
4.
Harmathoe spi
25
4
9.1
67.8
5. CalllopiuB
5.
Capitellldae"
11
4
4.2
69.2
5.
Capitellldae*
15
5
5.5
73.3
laeviuaculua
13
3
5.5
67.8
6.
Calliopius
6.
Tellinidae
14
4
5.1
78.4
6. Capitellldae"
14
4
4.8
72.6
laevlunculua
10
4
3.8
73.0
7.
Corophium Bp.'5
11
4
4.0
82.4
7. Tellinidae
14
3
4.8
77.4
7.
Tellinidae
8
5
2.4
75.4
8.
Corophium sp?
8
5
2.4
77.8
1. Tellinidae
100
5
21.8
21.8
1.
Cirripedia
122
5
22.0
22.0
1.
Tellinidae
72
5
24.1
24.1
2. Capitellldae"
B7
5
17.6
39.4
2.
Tellinidae
96
5
17.3
39.3
2.
Ci rripedia
49
5
16.4
40.5
4
3. Edotea montosa
60
5
12.1
51.5
3.
Capitellldae9
59
5
10.6
49.9
3.
Capitellldae"
38
5
12.7
53.2
WKS
4. Cirripedia
58
5
11.7
63.2
4.
Edotea montosa
49
5
8.8
58.7
4.
Edotea montosa
22
5
7.4
60.6
S. Harmathoe Bp.
35
5
7.1
70.3
5.
Harmathoe sp.
48
5
8.6
67.3
5.
Harmathoe sp.
20
5
6.7
67.3
6. Marinogam-
6.
Polydora sp,c
48
5
8.6
75.9
6.
Polydora sp.c
11
5
3.7
71.0
marus sp?
29 '
5
5.9
76.2
7.
Corophium sp?5
10
4
3.3
74.3
8.
Mar inogam-
marus sp.e
10
4
3.3
77.6
1. Capitellldae"
344
5
53.3
53.3
1.
Capitellldae"
327
5
46.2
46.2
b
2. Tellinidae
131
5
20.3
73.6
2.
Tellinidae
139
5
19.6
65.8
WKS
3. Edotea montosa
63
5
9.8
83.4
3.
Edotea montosa
61
5
8.6
74.4
4.
Polydora spf
. 54
4
7.6
82.0
a - probably Capltella capltata (see text) b - Includes Corophium bonelli and C. craasicorne c - includes Polydora ligni
d - probably Tellina agilia e - prohably Marinogammarus stoerensis
-------
4.2.2.2 Four-Week Samples
Total number of animals recovered after four weeks was
1349, representing 52 species in six ohyla (Taole XII).
Arthropoda was the most abundant phylum in all treatments
combined (44% of total recovery); next were Annelida (33%) and
Mollusca (23%). The predominant species was a member of the
pelecypod family Tellinidae, which was probaoly Te1lina aqi1 is
but could not be positively identified. Cirripeds were next in
abundance, followed by a capitellid polychaete. The latter
taxon includes animals definitely identified as one of the
sibling species of Capite1 la capitata, as well as younger
animals which could be assigned only tentatively to this
genus. The isopod Edotea montosa and tne polychaete Harmathoe
sp. were also among the most abundant soecies.
Mean numbers of individuals were similar fcr centre! and
homogeneous test samoles: 99 _+ 22.5 and 111.0 + 34.4,
respectively (Table XIII). In surface samoles, 59.8 _+ 16.4
individuals were recovered. Mean number of species, similar
for all treatments, were 18.8 _+ 2.2, 19.8 _+ 3.6, and 17.4 _+ 2.7
species, respectively. Mean S/N values were 0.20 + 0.07 for
control, 0.19 + 0.06 for homogeneous, and 0.30 _+ 0.07 for
surface test samples. A significant difference between
treatments for the parameters number of individuals and S/N was
revealed by analysis of variance (P< 0.03). Student-Newman-
Keuls multiple range test showed tnis to ce due to a difference
between nomogeneous and surface test treatments.
86
-------
Arthropods were the oredominant phylum, accounting for
445S of the fauna in each treatment (Table XVI). Annelids and
molluscs were next in aoundance in each treatment. The
percentage contribution by Annelida was higher in homogeneous
test samples than in eitner of the other treatments.
In each treatment, the same four species were most
aoundant, but their order of predominance varied (Table XVII).
In control samples, tellinids were most abundant, followed in
decreasing order by capitellids, Edotea montosa, and
cirripeds. In both test treatments, cirripeds were relatively
more abundant than in the control treatment: in homogeneous
samples they were the predominant soecies, and in surface
samples they were second in abundance. The other three species
mentioned above remain in the same relative order of abundance
as in control samples.
4.2.2.3 Six-Week Samples
Since results of four-week samples indicated a depressed
recovery in surface test samples, it was decided to analyze
six-week samples for this treatment and compare them to those
of the control treatment. Homogeneous test samples were not
analyzed. A total of 1353 individuals were recovered in the
two treatments, representing 35 species in four phyla (Table
XII) .
The mean number of individuals for control samples was
129.0 +_ 48.6, while that for surface test samples was 141.6 +
55.7 (Table XIII). Mean numbers of species were 13.6 + 0.9 and
87
-------
15.0 + 3.5, and S/N values were 0.11 * 0.03 and 0.12 _+ 0.05,
for control and surface samples, respectively. Student's
t-test showed no significant (P > 0.05) difference between the
treatments for any parameter.
Tne two treatments resembled each other when considering
distribution of animals by phylum: annelids predominated,
followed by molluscs and arthropods. The treatments also
showed species predominance by the same three species, each
contributing similar percentages of the total number of
individuals.
4.2.2.4 Comparison of Recruitment Periods
Homogeneous test samples showed the highest rate of
growth between two-week and four-week samples, increasing 114%
from 260 to 555 individuals (Figure 10a). Control samples
increased 70% from 292 to 495 animals. In surface samples, 274
individuals were recovered after two weeks, and only 9% more
after four weeks (299 animals). The much slower rate of
increase in surface samples was distributed across almost all
species and phyla.
Arthrcpcda was the predominant phylum after both two and
four weeks, although other phyla exhibited more substantial
increases in number between the two periods. Control and
homogeneous test samples each contained a higher number of
arthropods after four weeks than two weeks (8% and 2 8%
increases, respectively; see Figure 10b). On the contrary,
surface samples showed a 32% decrease in arthropod recovery.
88
-------
700-
I NO 1 V
300-
200' •
100-
2 4 6
recruitment period (wks)
2 4 6
RECRUITMENT PERIOD (WKS)
FIG.lOaAll Taxa
FIG. 10b Arthropoda
INDIV
300"
200"
100--
2 4
RECRUITMENT period
FIG.iOcTellinidae
(wks)
-Control
¦ Homogeneous test
¦ Surface test
INDIV
100-¦
2 4
RECRUITMENT PERIOD
FIG-lCd Capitellidae
{WK s)
FIGURE 10. Field-based Experiment: Total Number of Individuals for
Important Taxa in Control, Homogeneous and Surface Test
Treatments Over Three Recruitment Periods. (Numbers are
pooled data from 5 replicates).
39
-------
Compared with the other two treatments, surface test
samples also displayed the smallest percentage increase in
number of annelids and molluscs. Annelid recovery increased
131% in control and 294% in homogeneous samples, but only 53%
in the surface treatment. Corresponding percentages of
increase for the phylum Mollusca were 430%, 643%, and 190% for
control, homogeneous, and surface samples, respectively.
Shifts in the predominant species between the two
recruitment periods reveal five species whose trends of
abundance are of interest (Table XVII). The cirriped barnacle,
which was most abundant in ail three treatments after two
weeks, decreased by about 30% in control and surface test
samples, but increased by 50% in homogeneous test samples after
four weeks. The amphipod Marinogammarus sp., second in
aDundance in all treatments after two weeks, decreased by at
least 25% in all treatments and was no longer predominant after
four weeks. The isopod Edotea mpntosa decreased by 56% in
surface samples but doubled in the other two treatments.
Two species exhibited significant increases in abundance
in all three treatments. Tellinids and capitellids each
accounted for around 5% of the total fauna in each treatment
after two weeks (Table XVII). 3y the time of the four-week
sampling, the numbers of tellinids recovered had jumped by 11
times in homogeneous test, over 6 times in control and 4 times
in surface samples (Figure 10c). In the four-week samples,
tellinids were the most abundant species in control and surface
test samples (22% and 24% of all animals, respectively) and
90
-------
second in abundance in homogeneous samples (17% of faunal
recovery). The rise in number of capitellids over the same
time period was most dramatic in control samoles (521%), almost
as high in homogeneous samoles (436%), and smallest in surface
samples (153%; see Figure lOd). After four weeks, caoitellids
were second in predominance in control samples (representing
19% of faunal recovery), and the third most abundant species in
homogeneous and surface samoles (11% and 13% of all animals in
each treatment, respectively).
Control and surface test samples from the six week
recruitment period revealed a continuation of patterns of
species distribution, but a reversal in numerical trends.
Control samples increased 30% in faunal recovery while surface
samples increased by 137% oetween four- and six-week samples
(Figure 10a). This escalation compensated for the depressed
recovery in surface samoles after four weeks: after six weeks,
the numbers of animals recovered in the two treatments were
very close (645 animals in control and 708 animals in the
surface samples).
Predominance in the two treatments after six weeks was
very similar. A shift occurred in relative predominance by
phylum between the four- and six-week periods (Table XVI).
Annelida was oy far the predominant phylum after six weeks,
comprising 62% of total recovery in each treatment. Mollusca
was next in abundance (around 21% for each treatment), followed
by Arthropoda (around 16% for each treatment). Capitellids and
tellinids continued tc increase and constituted, respectively,
about 50% and 20% of all animals in each treatment (Table XVI).
91
-------
4.3 Discussion
The data used for statistical analysis often exhibited a
large variability between replicates, making interpretation of
the results difficult. Consequently, although a 95% confidence
level was used to signify statistical significance, the finding
of a difference between treatments at the P < 0.10 level for
lab-based two-week samples deserves further investigation.
The mean number of individuals found after two weeks in
lab-based surface test samples was approximately half that of
control and homogeneous samples. The depressed recovery in
surface samples was more obvious in the phylum Annelida than in
other phyla, and was reflected in the smaller number of the
predominant species Fabricia sabe1la and Peloscolex benedeni.
Both depend in some manner on the substrate.
Fabricia sabe1 la is a tube-dwelling sabellid polychaete
that feeds by beating cilia on its branchial crown and
straining the resulting current of water. Small oarticles are
ingested and the organic material used as food; medium-sized
particles are used for tube-building. Depressed numbers of £.
sabe11a in surface test samples might have resulted from three
factors: a shortage of particles large enough for tube-building;
a reduced supply of organic material for nutrition; and a
clogging of the branchiae by very fine oarticles of drilling
mud, limiting food ingestion.
Oligochaetes similarly deoend on small particles of organic
material for food. Host ingest these along with sediment in the
course of burrowing; some graze off larger particles in the
92
-------
substrate such as sand or rock. In either case, the
oligochaete Psloscolex benedeni might have suffered from a
shortage of food in surface test samples.
Any of these deleterious effects would be expected to
disappear over time due to the accumulation of detrital
material settling from the incoming water. By the time of the
four-week sampling in the lab-based experiment, numbers of
these soecies in surface samples resembled those in control
sample s.
After four weeks, the mean number of individuals in homo-
geneous test samoles was nearly 5Q% higher than those of
control and surface samples. This was reflected in the number
of Fabric la sabe1 la in homogeneous samples, which was more than
two times that of either of the other treatments. It is
unclear which property of the homogeneous treatment accounted
for its ability to support sucn a higher number of this soecies.
In the field-based experiment, the populations that had
recoIonized each treatment were indistinguishable in size and
composition after two weeks. After four weeks, statistically
significant differences existed between the high number of
individuals in homogeneous test samples and the low number in
surface samples. The control samples supported a 65% larger
population than the surface test samples (not statistically
significant at P < C.35), and an 11* smaller population than
the homogeneous treatment.
The difference in number of individuals between control
and homogeneous test samples after four weeks can be accounted
93
-------
for by the recovery of over twice as many cirriped barnacles in
homogeneous samples. An increase of cirripeds between the two-
and four-week periods occurred only in this treatment. If the
more heterogeneous grain size' in homogeneous test samples is in
some manner responsible for this enhancement, it is unclear why
it did not develop after the two-week recruitment oeriod.
The higher percentage contribution by Annelida in
homogeneous samples might be explained by the physical nature
of the substrate. The mixture of reference mud and drilling
mud had less of a tendency to pack together than reference mud
alone and this might have facilitated burrowing by annelids.
A depressed number of animals found in surface test
samples was observed in all phyla. A combination of factors
may explain the finding of a lower recovery in this treatment
after four weeks but not after two weeks: increased
predominance by species that depend more directly on the
substrate, a reduced rate of increase of some species, and
mortality in other species.
Recolonization of the defaunated sediment in this
experiment presumably occurred by two mechanisms: the settling
of planktonic larval stages and the immigration of adults from
the surrounding substrate via crawling or suspension oy
currents. A count of the pooulation after a Deriod of time
reflects the number of larval stages that have settled and
survived, and the number of adults that nave immigrated and
survived. Larvae have been shown to be capaDle of
discriminating between potential substrates and delaying
metamorphosis until a suitable substrate is found (Thorson,
94
-------
1966). Survival of both metamorpnosed larvae and immigrated
adults depends on factors such as the availability of food, the
nature of tne substrate, and the general quality of the
environment. Suppressed numbers in surface test samples could
be due to reduced settlement by larvae or greater mortality of
all animals in this treatment.
The predominant soecies after two weeks was a cirriped
barnacle, a filter-feeder which lives on the surface of the
substrate. Like the polychaete Fabricia sabella, cirripeds
depend on straining a current of water for procurement of food
and prooaDly for gas exchange. Suppression of the population
of cirripeds in surface samples might have been expected to
occur due to clogging of branchiae by very fine particles of
drilling fluid, as observed with F. sabe11a in the lab-based
experiment. However, surface samples supported nearly as many
cirripeds as each of the other two treatments, providing
evidence that potential suffocation by drilling fluid did not
cause significant mortality in this species. It might have
been prevented oy the layer of detritus deposited by the water
column. The layer accumulated at a much *aster rate in the
field-based experiment than in the laboratory system.
In the period between collection of two- and four-week
samples the population composition changed: numbers of a
capitellid polychaete and a tellinid pelecypod grew rapidly.
These are both deposit feeders, which ingest and rework the
sediment. The rate of increase of both species was higher in
control and homogeneous samples than in surface samples, which
95
-------
could reflect the interaction of these species with the
subsurface layer of drilling mud in the latter treatment.
The isopod Edotea montosa and the polychaete Ha rma thee
sp. , two species which are predominant in the four-week
samples, increased between two- and four-week periods in
control and homogeneous samples, but declined in surface
samples. Since no ether species were observed to "bloom" over
the same period, these decreases suggest that the surface test
treatment was unable to support the original recolonizing
papulations of these two species, resulting in some mortality.
It is believed that the lower recovery of individuals in
surface test samples after four weeks was caused by a
combination of the factors discussed above. Any effects by the
surface layer of drilling fluid on the recruited population
disappeared after six weeks, as shown by the strong resemolance
oetween the two treatments in population size and composition.
Two explanations could account for the relative increase
of animals between four and six weeks in surface samoles and
control samples: either animals moved into the layer of
drilling fluid in surface samples, or the detrital deposition
had finally accumulated a layer thick enough to act as a new
substrate. Since the drilling fluid had such a distinct effect
after four weeks, the second case is believed to be more
probable. Tne layer of deoosited material incluaed detritus,
sand, and smaller inorganic particles swept up from the
surrounding bottom, and appeared to be suitable as a substrate.
96
-------
The effect of suppressing numbers of individuals found in
surface samples could have been caused by physical or chemical
aspects of the drilling fluid. Three pieces of evidence
suggest a physical mechanism. First, if the effect was
chemical, adverse effects would be expected to occur to a
lesser extent in the homogeneous test samples. This was not
the case. In fact, when homogeneous samples differed from the
other two treatments, they contained slightly higr.er numbers of
animals. Secondly, the effect ceased when animals were no
longer in direct contact with the layer of drilling fluid, yet
chemical effects would probably have persisted since toxicants
could continue to leach out of the drilling fluid. Finally,
organic and trace metal analyses of this particular PESA
drilling fluid and liquid phase toxicity testing showed this
mud to have a relatively low toxicity.
Barite, a non-toxic weighting agent, is a major component
of drilling fluids. Cantelmo e_t a_l (1979) found that barite
mixed with a sand substrate enhanced the population density of
meiofauna, presumably because of increased sediment
heterogeneity, but that a cover of barite over sand
significantly decreased meiofaunal population density. Tagatz
and Tobia (1978) found adverse effects on macrofauna in
developing communities after ten weeks when barite either
covered a sand substrate, or was mixed in a ratio cf 1 part
barite to 3 parts sand. Both of these ajtnors suggested that
since barite is non-toxic to many marine organisms, the effect
of barite is due to its changing of the sediment granulometry.
97
-------
Tagatz st_ al ( 1978) tested the effects of a used
lignosulfonate drilling mud on recolonization over a period of
eight weeks. Their results showed considerably more pronounced
adverse effects of drilling fluid than the data presented
here. There are at least two possible explanations for this
apparent discrepancy. Sand was used as a reference substrate
by Tagatz et a_l., while our study used a natural, defaunated
sediment. The water supply was pumped from a sandy-bottom
environment and probably contained larvae "searching" for a
sand substrate. The change in grain size caused by the
addition of drilling fluid to sand was presumably much more
extreme than that caused by its addition to a fine-grained
reference mud; the adverse effects might have been more
pronounced as a consequence. Another possibility is that the
useo drilling fluid tested by Tagatz et al contained more toxic
components than the PESA mud used in the present study.
In general, these data show that a used DESA drilling
fluid affected recolonization when layered on top of defaunated
sediment, but not when mixed with it. In both experiments,
deposition of a new layer of material on too of the drilling
fluid seemed to reduce or reverse the effects, and by four to
six weeks after the beginning of the experiment, effects were
no longer obvious.
98
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4.4 Evaluation of Methodology for Solid Phase Recolonization
Tests
The laboratory-based experiment required five months of
prelininary work (described in Progress Report #• 2; New England
Aquarium (1981)) to ensure that the system provided a uniform
flow of water over all sample containers. Once this condition
had been satisfied, the system offered easy access for
deployment and retrieval of samples. Minor maintenance was
required every other day during the course of the experiment.
Tne field-based system required three days of preliminary
work in the laboratory to prepare the experimental equipment.
Deployment of the samples was fairly easy when using a research
vessel equipped with a winch. Two divers were required to
assist during deployment and to retrieve samples every two
weeks; tnese operations were therefore limited by the weatner.
In this exDeriment, only extremely stormy conditions would have
prevented access, since the site was very close to the dock
where the divers entered the water.
More animals were collected in the field-based than the
lab-based experiment after both two and four weeks of
recruitment time. This may have been dje to the different
locations used for this study. Since a difference between
control and test treatments would presumably oe more obvious
with a larger number of animals in each treatment, tne
field-based system appeared preferable to the lab-based study.
From January through March, low water temperatures would
prohibit use of the field-based system in northern latitudes,
while experiments could still be conducted under laboratory
99
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conditions. However, planktonic larvae occur in very low
numbers in the water column during these months, so there is
little advantage to running a recoIonization experiment during
the winter in temperate climates. In general, it is felt that
the amount of time and/or maintenance required to ensure
unbiased water flow in a laboratory-based system makes this a
less attractive alternative than a field-based system.
Recolonization studies are an improvement on solid phase
toxicity tests as a method of assessing the impact of
contaminated sediment on the benthic environment. The study
described above was a more sensitive measure of the effects of
releasing drilling mud, because it considered development of
benthic communities rather than the ability of a contaminant to
kill adult animals over a ten-day test period (see discussion
of solid Dhase toxicity tests in NEA, 1980). Although the
method is a valid approach, we have concluded, based on the
present study, tnat it requires too much time for efficient
evaluation of whole drilling muds.
100
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