PD89-134118
Effects of Atrazine on
'Zostera marina' in
Chesapeake Say, Virginia
Virginia Inst. of Marine Science
Gloucester Point
Prepared for
Environmental Protection Agency, Annapolis, MD
Aug 81
\
J
EPA Report Collection
Regional Center for Environmental Information '
U.S. EPA Region HI
Philadelphia, PA 19103
s*
-------
TECHNICAL REPORT DATA
(Please read Imlrucnoin on the mrrtr btlurt compli-ting/
1 REPORT NO.
EPA/6QO/3-88/Q5Q
RECIPIENT'S ACCESSION NO
PB80 134118/AS
4 TITLE AND SUBTITLE
EFFECTS OF ATPAZINE ON Zostera marina IN CHESAPEAKE
BAY, VIRGINIA
REPORT DATE
August, 1982
6. PERFORMING ORGANIZATION CODE
7. AOTHOR(S)
Hershner, Ward, Illowsky, Deslistraty, Martorona
a PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, VA 23062
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R805953 and X003245
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Chesapeake Bay Program
2083 West Street
Annapolis, MD 21401
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING ACIENCY CODE
EPA/600/05
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This project was designed to assess the effects of agricultural herbicides
on submerged aquatic vegetation in the Lower Chesapeake Bay. Atrazine was
selected for testing because it is the most widely utilized herbicide in the
Bay region. Zostera marina was the submerged vegetati.n studied.
This project began with two surveys. The first survey, conducted in 1979,
covered forty eight stations throughout the Virginia portion of the Chesapeake
Bay. The survey was designed to indicate typical atrazine loading for the lower
Bay. A second survey in 1980 was limited to the Severn River, and was intended
to indicate the duration of peak atrazine loading. Information generated in the
two surveys was utilized to design dosing experiments.
Field dosing experiments, utilizing large plexiglass enclosures, measured
effects of short-term atrazine exposure on the net production of the Zostera
community. Greenhouse experiments, utilizing a flow-through dosing system, measured
effects of long-term (21 day) atrazine exposure on the morphology of mature
Zostera plants.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS c. COS AT I Field/Group
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS iTha Report/
UNCLASSIFIED
21. NO. OF PAGES
20 SECURITY CLASS (This page I
UNCLASSIFIED
23. PRICE
EPA Fwm 2220-1 (R«*. 4-77) PREVIOUS EDITION is OBSOLETE
-------
TABLE A3.1. (continued)
coordinates: latitude 37ol9'54", longitude 76°28'15"
location: north west branch of Severn River, east shore, small inlet
1100 meters upstream of Brays Landing, 10 meters offshore
(USGS Achilles Quadrangle)
access: boat
depth: 0.5 meters
sediment: silt and clay
SAV: none
SR-3A, Severn River northwest channel
coordinates: latitude 37°19'37", longitude 76°28'24"
location: northwest branch of Severn River, main axis, 800 meters
upstream of Brays landing, IL meters offshore (USGS
Achilles Quadrangle)
access: boat
depth: 0.7 meters
sediment: silt and clay
SAV: none
SR-4, Cod Point
coordinates: latitude 37°19I23", longitude 76o27'18"
location: northwest branch of Severn River, north shore, westend of
Bryant Bay, end of Cod Point, 15 meters offshore (USGS
Achilles Quadrangle)
access: boat
depth: 1 meter
sediment: silt, sand and clay
SAV: none
SR-5, School Neck Point
coordinates: latitude 37O19'21", longitude 76°26'29"
location: northwest branch of Severn River, north shore, eastend
of Bryant Bay, 25 meters offshore of School Neck Point
(USGS Achilles Quadrangle)
access: boat
depth: 1 meter
sediment: sand and silt
SAV: none
SR-6, Turtle Neck Point
coordinates: latitude 37°19'18", longitude 76°25'15"
location: northshore of Severn River, mouth of river, 200 meters
offshore southwest of Turtle Neck Point (USGS Achilles
Quadrangle)
access: boat
depth: 1 meter
sediment: sand and clay
SAV: Ruppia maritiina
Ware River
WR-1, Goshen
(continued) 22
-------
TABLE A3.1. (continued)
SR-1, Warner Hall north drainage
coordinates: latitude 37°20'39", longitude 76°29'6"
location: northwest branch of Severn River, head of northern most
tributary, paralleling Rt. 629, 3100 meters upstream of
Bray's landing (USGS Achilles Quadrangle)
access: boat
depth: 0.5 meter
sediment: silt and clay
SAV: none
SR-lA, Warner Hall north drainage
coordinates: latitude 37020'28", longitude 76°29'0"
location: northwest branch of Severn River, mid-axis of northern-
most tributary paralleling Rt. 629, 250 meters downstream
from SR-1 (USGS Achilles Quadrangle)
access: boat
depth: 0.5 meters /
sediment: silt and clay .' -
SAV: none
SR-lB, Warner Hall, Severn River headwater
coordinates: latitude 37°20'20", longitude 76°28'56"
location: northwert branch of Severn River, mouth of northernmost
tributary, 500 meters downstream of SR-1 (USGS Achilles i
Quadrangle) /
access: boat /
depth: 0.5 meters
sediment: silt and clay
SAV: none
SR-2, Warner Hall cemetery ^
coordinates: latitude 37°20'14", longitude 76°28'37"
location: northwest branch of Severn River, east shore, small inlet
2000 meters upstream of Brays Landing, due south of
Warner Hall cemetery, 5 meters offshore (USGS Achilles
Quadrangle)
access: boat or Rt. 629 and wading
depth: 0.5 meters
sediment: silt and clay
SAV: none
SR-2A, Warner Hall cemetery (2)
coordinates: latitude 37°20'5", longitude 76°28'40"
location: northwest branch of Severn River, 2000 meters upstream
of Brays Landing, west of SR2, 15 meters offshore (USGS
Achilles Quadrangle)
access: boat
depth: 0.5 meters
sediment: silt and clay
SAV: none
SR-3, Eagle Point west drainage '
9 1
(continued)
-, A
-------
TABLK M.I. (c.-ni i n.ir.l )
SAV: none
YR-1, Carter Creek
coordinates: latitude 37°19'22", longitude 76°34'24"
location: nortli shore of Carter Creek, 1000 meters upstream of
mouth (Blundering, Point), 20 meters offshore (USGS Clay
Bank Quadrangle)
access: boat
depth: 1 meter
sediment: clay and silt
SAV: none
YR-2, Mumfort Island
coordinates: latitude 37°16'6", longitude 76°31'0"
location: north shore of York River, south west of southern Mumfort
Island, 1800 metors north of Gloucester Point, 50 meters
offshore of island (USGS Clay Bank Quadrangle)
access : boa t
'lepth: 1 ,Tieter
sediment; sant!, clay and silt
SAV: none
YR-3, Allen's Island
coordinates: latitude 37°15'25", longitude 76°25'20"
location: north shore of York River, 50 meters off south shore of
island (I'SGS Achilles Quadrangle)
access: boat
depth: 1 meter
sediment: sand and silt
SAV: Zostera mar in"
YR-4, Guinea Marsh
coordinates: latitude 37°16'24", longitude 76°20'44"
location: north side of York River mouth, 800 meters east-south east
of last Guinea Marsh island (USGS New Point Comfort
Quadrangle)
access: boat
depth: 1.3 meter
sediment: sand and silt
SAV: Zostera marina
YR-5, Browns' Bay
coordinates: latitude 37°18'2", longitude 76°23'39"
location: east of Blevins Creek mouth, 10 meters offshore (USGS
Achilles Quadrangle)
access: boat
depth: 1.3 meter
sediment: sand and silt
SAV: Zostera marina
Severn River
(cont inued) 20
-------
TABLE A3.1. (continued;
depth: 1 meter
sediment: sand and silt
SAV: none
JR-2, Chickahominy
coordinates: latitude 37°14'22", longitude 76°51'55"
location: north shore, 500 meters downstream of Ch' .kahominy River
mouth, 50 meters offshore (USGS Surry Qu .'rangle)
access: boat
depth: 1 meter
sediment: sand clay and silt
SAV: none
JR-3, James River Bridge
coordinates: latitude 36°57'40", longitude 76°30'5l"
location: south shore, 100 meters downstream of James River Bridge,
20 meters offshore (USGS Bena's Church Quadrangle)
access: Rt. 17 and wading
depth: 1 rnete*
sediment: sand and silt
SAV: none
York River
YR-1A, Sweet Hall Marsh
coordinates: latitude 37O34'10", longitude 76°54'28"
location: Pamunky River, northshore, 50 meters upstream of impound-
ment outfall at Sweet Hall Landing, 10 meters offshore
(USGS New Kent Quadrangle)
access: Rt. 634 and wading
depth: 1 meter
sediment: sand,gravel and silt
SAV: none
YR-lB, Water Fence Landing
coordinates: latitude 37°35'30", longitude 76°47'57"
location: Mattaponi River, northshore, public boat ramp at Water
Fence Landing, 5 meters offshore (USGS West Point
Quadrangle)
access: Rt. 611 and wading
depth: 1 meter
sediment: silt
SAV: none
YR-1C, Gressitt
coordinates: latitude 37°28'0", longitude 76°43'35"
location: north shore of York River, 3100 meters upstream of
Propotank River, 100 meters offshore (USGS Gressitt
Quadrangle)
access: Rt. 667 and wading
depth: 1 meter
sediment: sand and silt
(continued) 19
-------
TABLE A3.1. 1979 VIRGINIA SURVEY SITES
Chesapeake Bay
CB-1, Dameron Marsh
coordinates: latitude 37°47'14", longitude 76°18'16"
location: southside of Ingram Bay, opposite Fleeton, north west
corner of Dameron Marsh, 200 meters offshore (USGS
Reedville Quadrangle)
access: Rt. 606, private lane and wading
depth: 1 meter
sediment: sand
SAV: none
CB-2, Fleets Bay
coordinates: latitude 37°39'36", longitude 76°20'10"
location: Fleets Bay, end of Poplar Neck, midday between Dvmer Creek
and Tabbs Creek, 5 meters offshore just south of unnamed
impoundment (USGS Fleets Bay Quadrangle)
access: Rt. 646, Rt. 647, private lane and wading
depth: 1 meter
sediment: sand
SAV: none
CB-3, Cricket Hill/Gwynn's Island
coordinates: latitude 37°29'12", longitude 76°18'5"
location: Mil ford Haven, northwest shore at mouth of Lanes Creek,
southside of land at terminus of Rt. 669 in Cricket Hill,
10 meters offshore (USGS Mathews Quadrangle)
access: Rt. 223, Rt. 669 and wading
depth: 1 meter
sediment: sand and silt
SAV: none
CB-4, Plum Tree Island
coordinates: latitude 37°10'35", longitude 76°25'24"
location: south of Poquoson River, 50 meters offshore of north east
terminus of Plum Tree Island bombing range (identified as
Marsh Point) (USGS Poquoson East Quadrangle)
access: boat
depth: 1.3 meter
sediment: sand
SAV: none
James River
JR-1, Hopewell
coordinates: latitude 37018'55", longitude 77°13'7"
location: southshore, 200 meters upstream of Benjamin Harris Bridge,
15 meters offshore (USCS Westover Quadrangle)
access: Rt. 156 and wading
1H
(cont inued)
-------
REFERENCES
Orth, R. J., K. A. Moore, and H. H. Gordon. 1979. Distribution and abundance
of submerged aquatic vegetation in the lower Chesapeake Bay, Virginia.
U.S. EPA Final Report. 600/8-79-029/SAV1. 199 pp.
17
-------
occurrence is available indicates that vegetation has persisted in the
presence of atrazine at four stations and disappeared at six stations. At
station1; where atrazine was not detected during the survey, vegetation has
persisted at eleven stations and disappeared at one station. This analysis is
suggestive of a correlation between the presence of atrazine and the
disappearance of submerged aquatic vegetation. Unfortunately, there are
insufficient numbers of samples in each treatment response category to allow a
test of the significance of this correlation (x* test requires a minimum of 5
sampl-s per category).
The analysis of the survey data we have employed for this report is
"observational" and based on the assumption that the sampling was in fact
representative of conditions in the lower Chesapeake Bay. With this
assumption the survey results indicate several things.
First, atrazine concentrations in the lower Chesapeake Bay waters are
generally below 1 ppb. Second, concentrations of atrazine above 1 ppb in
water seem related to runoff events following spring application of
herbicides. Third, in every case in the survey program, concentrations above
1 ppb were only found in upriver stations well removed from present or former
Zostera beds. Fourth, concentrations of atrazine in waters over existing or
former Zostera beds was generally 0.2 ppb or less.
16
-------
well below 1 ppb. For the rest of the year atrazine concentrations were near
or below the detection "imit.
All three Mobjack Bay stations currently support extensive beds of
submerged vegetation. These beds have apparently been relatively stable
through the recent past. Atrazine concentrations in water samples were always
below the detection limit.
None of the Rappahannock River stations currently support submerged
vegetation. There were extensive beds of vegetation reported in the lower
river in the early 1970's. The three down-river stations in this system are
all apparently at sites which once supported submerged vegetation. Water
sample analysis found atrazine concentrations below the detection limit prior
to field applications in spring. Following application, atrazine was found in
excess of 1 ppb at Port Royal, with concentrations generally decreasing to the
detection limit at the river mouth. Atrazine vas detected throughout the
river system in late summer, with concentrations well below 1 ppb at the three
down-river stations. Concentrations at those stations were below the
detection limit during the final survey round.
In the Potomac River system none of the stations support Zostera. The
upriver station at the Potomac River Bridge supports an extensive bed of
Potomageton perfoliatus and Vallisneria americana. Water sample analyses
found atrazine concentrations below the detection limit prior to spring field
applications. Atrazine concentrations were highest at the Potomac River
Bricge station in survey rounds two and three. Concentrations were relatively
uniform throughout the river in survey round four. All concentrations,
however, were well below 1 ppb.
Most of the Eastern Shore stations are in areas which currently support,
or recently supported, submerged vegetation. Despite the intense agricultural
land use on the Eastern Shore, water samples generally contained very little
atrazine. No concentrations above 1 ppb were detected.
The principal objective of the 1979 survey program was to identify
concentrations of atrazine potentially impacting Zostera marina i.n the lower
Chesapeake Bay. The sampling program was designed to include those periods we
believed, a priori, would include the maximum concentrations in Bay waters,
i.e. immediately after field applications and shortly after harvesting. These.
two times should correspond with maximum runoff of sediments and chemicals
from the fields.
One type of analysis of the data collected in the 1979 survey has been
suggested by Dr. D. Leav (personal communication). Dr. Leav correctly
observes that there is no assurance that the atrazine concentrations detected
in this survey program are "worst case" concentrations given the frequency of
the sampling. A conservative analysis of the data (i.e. one which ignores
much of the information content of the sampling design) would compare the
presence or absence of atrazine at each station with the loss or retention of
submerged aquatic vegetation at that station. This approach reduces the
information to a binominal data set with atrazine as a treatment. Analysis of
those stations 'or which a good record of submerged aquatic vegetation
15
-------
TABLE 3.2. CONCENTRATIONS OF ATRAZINE IN SELECTED SEDIMENT SAMPLES FROM LOWER
CHESAPEAKE BAY DURING 1979
(all concentrations in parts per billion; based on dry
weight)
Circuit #1 Circuit #2 Circuit #3 Circuit #4
Station Date
SRI
SR2
SR3
SR4
SR5
SR6
WRL
WR2
WR3
MB1
MB 2
MB3
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-10-79
4-10-79
4-10-79
4-10-79
4-10- .'9
4-10-79
Atrazine
33 83
N.Q.*
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
Date
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
3-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
Atrazine
<5.0
N.Q.*
N.Q.*
Lost
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
Date
6-25-79
6-25-79
6-25-79
6-25-79
6-25-79
6-25-79
8-8-79
8-8-79
8-8-79
8-8-79
8-8-79
8-8-79
Atrazine
25.13
35.08
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
"5.0
<5.0
<5.0
Date Atrazine
11-15-79
11-15-79
11-15-79
11-15-79
11-15-79
11-15-79
11-16-79
11-16-79
11-16-79
11-16-79
11-16-79
11-16-79
21.33
13.01
13.71
<5.0**
5.38
O.O**
<5.0
<5.0
<5.0
<5.0
<5.0
^5.0
* not quantitatable due unresoluable interferences
** trace
14
-------
TARI.K J.I. (continued)
Station
PR1
PR2
PR3
PR4
ESI
ES2
ES3
ES4
ESS
ES6
ES7
ESS
Circuit #1
Circuit #2
Circuit #3
Circuit #4
Date Atrazine
4-9-79
4-9-79
4-9-79
4-9-79
4-16-79
4-26-79
4-26-79
4-26-79
4-25-79
4-25-79
4-25-79
4-25-79
<0
<0
<0
10
<0
<0
<0
lo
lo
lo
lo
lo
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
Date Atrazine Date Atrazine
6-8-79
6-8-79
6-8-79
6-8-79
6-12-79
6-12-79
6-12-79
6-12-79
6-12-79
6-12-79
6-12-79
6-12-79
0
<0
0
10
0
0
lo
0
0
0
lo
lo
.20
.10
.14
.10
.71
.62
.10
.12
.12
.12
.10
.10
8-10-79
8-10-79
8-10-79
8-10-79
8-16-79
8-16-79
8-16-79
8-16-79
8-16-79
8-16-79
8-16-79
8-16-79
0
0
0
10
<0
<0
<0
<0
<0
lo
<0
<0
.53
.21
.12
.10
.10
.10
.10
.10
.10
.10
.10
.10
Date Atrazine
12-4-79
12-4-79
12-4-79
12-4-79
10-25-79
10-25-79
10-25-79
10-25-79
10-25-79
10-25-79
10-25-79
10-25-79
0
0
0
0
<0
<0
<0
lo
0
lo
0
lo
.28
.27
.28
.31
.10
.10
.10
.10
.13
.10
.17
.10
(a) station not initially occupied
(b) station occupied by bee swarm
13
-------
TABLE 3.1. CONCENTRATIONS OF ATRAZINE IN WATER SAMPLES FROM THE LOWER
CHESAPEAKE BAY DURING 1979
(all concentrations in part per billion)
Circuit #1
Circuit #2
Circuit #3
Circuit #4
Station
CBl
CB2
CB3
CB4
JRl
JR2
JR3
YROA
YROB
YROC
YRl
YR2
YR3
YR4
YR5
SRl
SRlB
SR2
SR3
SR3A
SR4
SR5
SR6
WRl
WR2
WR3
WR4
WR5
MBl
MB2
MB3
RRl
RR2
RR3
RR4
RR5
(continued)
Date Atrazine
4-14-79
4-14-79
4-14-79
4-14-79
4-25-79
4-25-79
4-25-79
3-22-79
3-22-79
3-22-79
3-22-79
3-22-79
3-22-79
3-22-79
3-22-79
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-3-79
4-10-79
4-10-79
4-10-79
4-10-79
4-10-79
4-10-79
4-10-79
4-10-79
4-19-79
4-19-79
4-19-79
4-19-79
4-19-79
'0
<0
<0
1°
<0
~0
1°
1°
<0
<0
~0
<0
<0
<0
1°
0
<0
1°
0
<0
10
10
^0
<0
<0
IQ
<0
<0
10
<0
<0
lo
o
0
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.33
(1)
.10
.10
(a)
.32
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
.10
Date Atrazine
6-8-79
6-8-79
6-8-79
6-8-79
6-5-79
6-5-79
6-5-79
5-29-79
5-29-79
5-29-79
5-29-79
5-29-79
5-29-79
5-29-79
5-29-79
3-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
5-14-79
b-14-79
5-30-79
5-30-79
5-30-79
6-8-79
6-8-79
6-8-79
6-8-79
6-8-79
<0.
<0.
<0.
1°'
0.
0.
1°-
<0.
~0.
-------
system maps included in the appendix, Figure A3.1 through Figure A3.15. The
concentrations of atrazine in water samples are reported in Table 3.1. The
concentration of atrazine in selected sediments samples are reported in Table
3.2.
DISCUSSION
All discussion of Zostera marina distributions are based on Orth et al.,
1979.
The four Chesapeake Bay stations are allocated in areas which have or had
Zosteri beds. Zostera was not found in any of the samples collected for this
survey, but it exists in siriificant quantities in all the areas except the
Cricket Hill/Gwynn's Island dite. In that area, Zostera is still found in
small beds at the entrances to Milford Haven. Water samples collected at the
Chesapeake Bay stations never contained detectable amounts of atrazine.
None of the James River sites have any Zostera, nor have they had any in
the recent past. Atrazine was detected in water samples collected after
spring field applications. Concentrations were generally highest at the
upriver sites, but no sample ever exceeded a 1 ppb level.
In the York River system, sites at the head of the river do not now have,
nor previously had, submerged aquatic vegetation. The sites at Carter Creek
and Mumfort Island, in the middle of the system, formerly had extensive grass
beds, but neither site supports Zostera presently. The sites at the mouth of
the river all have extensive grass beds currently. Water samples from the
York system contained detectable amounts of atrazine only in the
post-application survey round and the later summer survey round. At those
times the concentrations were detectable only at sites above Carter Creek.
None of the detectable concentrations exceeded 1 ppb.
The Severn River system was the most intensively sampled system in this
survey. As in the York River, Zostera is only found near the river mouth at
the Turtle Neck Point station. Zostera may have previously occurred at the
next two stations upriver, School Neck Point and Cod Point, but there are no
records of any further upriver extensions. Analysis of water samples always
detected atrazine at the station in the headwaters. The result is not
unexpected since the station is located in the principal drainage channel for
much of the agricultural land in the drainage basin. Atrazine concentrations
in water samples exceeded 1 ppb only in the second survey round, completed
immediately after spring field applications. Concentrations were regularly
near or below the detection limits at the three downstream stations.
Concentrations never exceeded the detection limits at the river mouth where
the grass beds are currently found.
The Ware River system has extensive grass beds at the three down-river
stations. These beds have apparently been relatively stable through the
recent past. The upstream stations do not now have, and may never have had,
any grass beds. Atrazine was detected in water samples at all stations
following the spring field applications. At that time concentrations were
relatively uniform along the length of the river, with a maximum concentration
11
-------
Figure 3.1 - Locations of Lower Chesapeake Bay Survey Stations
10
-------
SECTION 3
1979 LOWER CHESAPEAKE BAY SURVEY PROGRAM
INTRODUCTION
The 1979 survey of the lower Chesapeake Bay was designed to identify the
levels of atrazine in water and sediments during one growing season. A
preliminary assumption was that atrazine levels would fluctuate seasonally,
reaching maxima immediately after field applications ir. the spring and,
perhaps again, following harvesting in the fall. The survey was therefore
designed to sample a large number of sites fjur times during the year, with
timing selected to correspond to spring and fall farming activities. The
results of the survey were intended to establish the actual range of atrazine
concentrations to which Zostera marina might be exposed.
METHODS
Forty-eight sampling stations in the lower Chesapeake Bay were identified
and occupied four times during 1979. Stations were selected primarily to
provide a wide coverage of the Bay shoreline and major tributaries. Specific
site selection was governed principally by available access. Wherever
possible, however, sites which either have or had Zostera beds were selected.
Sample collection was scheduled so that the first sampling round occurred
prior to any farming activities in the spring. The second round occurred
immediately after the first major rainstorm following spring application of
herbicides to the fields. The third round was generally late summer and prior
to the fall harvesting of crops. The fourth round was conducted after most
field" were harvested.
Samples were collected either from a small boat or by wading t'i the
nearshore site. Sub-surface water was collected in solvent rinsed, amber
glass bottles with teflon lined lids. Sediments were collected with an 18 cm*
coring tube. Several cores were taken at each station and the top 5 cm of
each core was collected and stored in either glass jars with aluminum foil
lined caps or equivalent containers. Water samples were refrigerated and
sediments were frozen until analysis.
See the analytical methods section (Section 2) of this report for sample
analysis techniques.
RESULTS
The survey stations are listed in the appendix to this section, Table
A3.1. The sites are located on a general area map, Figure 3.1 and on river
-------
REFERENCES
Gaskill, A., Jr. and R. K. M. Jayanty. 1981. Second performance audit of
the VIMS herbicide monitoring program. Report submitted to Environmental
Protection Agency, Research Triangle Park, North Carolina. 25 pp.
Mattson, A. M., R. A. Kahrs, and R. T. Murphy. 1970. Quantitative
determination of triazine herbicides in soils by chemical analysis.
Residue Reviews 32:371-390.
-------
2. A linearity plot of standards, covering the range and attenuation at
which the extracts were analyzed, was developed at the beginning of each
GC run. A standard within 10% of the sample peak height was injected
immediately after each positive sample.
3. Sample blanks and samples fortified over a range of atrazine
concentrations were carried through the analyses periodically to assure
consistency in recovery and reproducibility.
4. A limited access laboratory was maintained. The lab, lab instruments,
and glassware were used only for atrazine determinations. Only personnel
involved directly with the atrazine determinations were permitted access.
5. All glassware used for these analyses was detergent-washed, rinsed with
tap water, disti1led-deionized water, acetone, toluene, and hexane.
6. All reagents and supplies to come in contact with the samples, such as
glass wool, sodium sulfate, XAD-2 resin, cellulose extraction thimbles,
teflon boiling chips, etc. were exhaustively extracted by Soxhlet in
acetone, toluene, and hexane, or, in some cases, methylene chloride.
7. Solvents were checked for purity periodically by concentration of 500 ml
to 1 -nl for subsequent analysis by GC. (Burdick and Jackson glass
distilled solvents were used). Disti1led-deionized water was checked for
contamination by extraction of one liter and GC determinations.
Ultra-high purity hydrogen and helium, and zero grade air were used as GC
gases; high purity nitrogen was employed for concentration of small
volumes of extracts (Linde/Union Carbide specialty gases).
8. Water samples were collected in amber glass bottles with teflon lined
caps and refrigerated in the dark until analysis. Sediment and
vegetation samples were collected in glass jars with aluminum foil-lined
caps or equivalent containers and frozen until analysis.
9. All chromatograms were labeled, dated, and stored for raw data retrieval.
Standard lab sheets were maintained for documentation of sample number,
substrate, station, dates of collection, extraction, and analysis,
volumes of sample extracted and injected, as well as peak heights of
samples and standards.
10. Samples were extracted as soon as possible after collection, however, in
some cases several r.ionths elapsed before lab workup took place.
External quality assurance consisted of analyses of "blind" or unknown QA
samples submitted by outside agencies such as EPA Annapolis Field Office and
Research Triangle Institute, Research Triangle Park, N.C. Each of these
agencies also conducted on-site evaluations of the VIMS herbicide laboratory.
Those performance evaluations involved fortification of estuarine water at
levels of atrazine between 0.21 ppb and 65.5 ppb. The VIMS recoveries were
usually well within 10% of the true values (Gaskill and Jayanty, 1981).
-------
X I
reduce emulsions. t'he extract was then partitioned against methylene chloride
(3 x. 50 ml) which was then passed through anhydrous granular sodium sulfate
and rotary evaporated just to dryness. The residue was quantitativelv
tranferred to an alumina column (25 pn Grade V; Kontes K-420 280, 22 n. o.d.)
in carbon tetrachloride (10 ml). The column was rinsed with an additional 20
ml carbon tetrachloride which was discarded. The column was eluted with
carbon tetrachloride (80 ml) and then ethyl ether/carbon tetrachloride (1:20,
100 ml). The eluate was rotary evaporated just to dryness and quantitatively
transferred to graduated centrifuge tub.'s with methylene chloride. The
extract was concentrated to dryness under nitrogen and volui..-1 adjusted with
toluene. The MDL for atrazine in sediment was set at 5.0 ppb.
GAS CHP1MATOGRAPH1C PARAMETERS
Analysis of water and sediment extracts were performed using a Tracor 560
gas chromatograph equipped with a model 702 nitrogen-phosphorus detector under
the following parameters:
Column: 3Z Carbowax 20 M 80/100 Chromasorb WHP
(well conditioned) 4' x 2 mm i.d. glass
Temps: Oven, 210°C; injection port, 230°C;
detector , 275°
Flows: (carrier) Hf 40 ml/min, ultra high purity
(plasma gases) H2 3.0 ml/min, ultra high purity
Air set at 40 psi at regulator; zero grade
NP source power: 810, background set at 75% FSD at 1 x 4
attenuation with zero off
Chart speed: 0.2V/min
Linearity plots wore made with each GC run. Standards within 10% of the
atrazine value in environmental extracts were injected after all positive
samples. Calculations were based on the analytical standard immediately after
each positive sample.
QUALITY ASSURANCE PROGRAM
A rigorous internal and external laboratory and analytical quality
assurance program was maintained throughout the course of the project.
Internal laboratory and analytical quality assurance included the
following.
1. Stock atrazine standards wi-re usu.il Iy prepared every three months;
working standards were prepared each month. Stock standards were stored
in a freezer. Working standards and sample extracts were refrigerated
between gas chromatographic analyses.
-------
SECTION 2
ANALYTICAL METHODS
INTRODUCTION
The methods utilized for analysis of atrazine in all of the succeeding
work in this report are based on standard ctiromatographic procedures.
Procedures were modified as detailed below aft»>r consultation with personnel
..•orking in the Ciba Geigy Corporation laboratories in Greensboro, North
Carolina.
All of the analytical work was conducted in a small laboratory set up
exclusively for this project. A rigorous quality assurance program was
undertaken to ensure the accuracy of the ntrazine concentrations reported.
WATER
Estuavine water was collected in amber glass bottles with teflon lined
caps and stored under refrigeration until analysis. Subsamples were filtered
through Reeve-Angel 802 and Wh.itman 2V filter papers. Powdered sodium sulfate
(poproximaiely 3-5 gm) was dissolved in the water in an effort to reduce
possible emulsions. All water samples were extracted with methylene chloride
(3 x 50 ml) which was then passed through anhydrous granular sodium sulfate
and reduced in volume by rotary evaporation to approximately 1 ml. Extracts
were quantitatively transferred to graduated centrifuge tubes with methylene
chloride, evaporated just to dryness under nitrogen, and volumes adjusted with
toluene. Most water extracts were sufficiently clean for direct GC analysis.
The minimum detection limit (MDL) for atrazine in water was set at 0.10 ppb .
SEDIMENT
The procedure used is modified from Mattson et al., 1970 on the basis of
discussions with Ciba Geigy Corporation personnel in Greensboro, North
Carolina.
Homogenized sediment samples (100 gm wet) were refluxed one hour in
water/acetonitrite (':10, 300 ml). The resulting extract was filtered through
Reeve-Angel 802 and Whatman 2V filter papers. Using the water content
determination from a dried subsample and the volume of recovered extract, the
dry-weight equivalent of sediment was calculated. The extract was placed on a
steam bath under nitrogen, reduced in volume to approximately 100 ml,
transferred to a separatory funnel, and diluted to one liter with water.
Powdered sodium sulfat (.ipproximatel y 5 gm) was dissolved in the water to
-------
interesting question. It is apparent from this work that detection of
herbicide impacts on Zostera requires fairly sensitive analytical
methods. The sublethal effects potentially caused by typical herbicide
loadings are of a magnitude which is not especially amenable to analysis
by the morphometric or production measurements used in some of our
studies. If those types of analyses are attempted, our experience
indicates efforts must be made to obtain large numbers of replicates and
special attention must be given to collection of ancillary data which
can be used to factor out response variations aue to the composition of
the natural community.
-------
(twenty one days) sublethal stress caused by exposure to atrazine
concentrations of 0.1 ppb, 1.0 ppb and 10 ppb was indicated by a change
in the ratio of adenylate concentrations (termed "energy charge").
Summarization of the adenylate experiments suggested that mature Zostera
is able to withstand exposure to low levels of atrazine (10 ppb and
less) for periods in excess of 21 days. Exposure to higher levels of
atrazine (100 ppb and 1000 ppb were tested) apparently elicits
physiological changes which can support the plant for only shorter
periods of time.
From all of these investigations we are led to believe that the
effects of atrazine on mature Zostera marina are probably not a major
causative factor for the recent declines in distribution within the
Virginia portion of the Chesapeake Bay. Our work indicates that while
atrazine can produce lethal and sublethal effects on Zostera, the
herbicide is not found in areas presently or formerly inhabited by
Zostera at concentrations high enough or persistent enough to exceed the
plant's ability to resist the imposed stress. This conclusion must be
considered in light of several limitations of these investigations.
virst, we have only addressed effects on mature plants. No work is
reported here on reproduction, germination or seedling growth. Second,
we have only addressed the effects of atrazine as a sole stressor. No
work was undertaken to evaluate additive or synergistic effects with
other chemicals. The investigation of light and atrazine interaction
was inconclusive and light was the only environmental parameter
addressed.
Third, all of the work conducted here was undertaken with Zostera
plants and their naturally occurring epiphyte community. For purposes
of this study, which was designed to address potential management
questions, analysis of effects on the natural assemblage was
appropriate. However, interpretation of results of the dosing studies
must be cognizant of the lack of any data partitioning effects among the
assemblage's components. A fourth consideration is that the work
reported here is focused on dissolved atrazine which we believed to b3
the principal mode of exposure for Zostera. We have not analyzed the
impacts of atrazine sorbed to suspended sediments which may also be a
significant mode of exposure. A final consideration is the lack of any
quantitative data assessing the condition of Zostera returned to control
conditions after exposure to atrazine.
With all of the limitations of this investigation in mind, we
believe our data suggest management or regulation of agricultural
herbicide usage will not prove a panacea for the decline of Zostera
marina in the Chesapeake Bay.
RECOMMENDATIONS
The limitations 'f this investigation suggest several avenues for
additional work. The effects of atrazine on Zostera germination and
seedling growth remain a significant question. Synergistic effects of
atrazine with other chemical and physical stressors also remains an
-------
seasonal loadings of atrazine in Bay waters. In 1980 a second survey
was conducted in the Severn River, This survey was designed to gather
information about the duration of the peak spring loadings identified by
the first survey program. Together these survey programs provided
information on the in situ levels of atrazine experienced by Zostera
marina.
The second line of research was a variety of experiments designed
to identify effects of atrazine on Zostera. Using the information
collected in the survey programs, we selected a range of atrazine
concentrations and two general exposure periods for testing. The
selections were made to ensure that we tested both typical and extreme
conditions.
We selected three test parameters in the investigation for effects.
Oxygen production was monitored during short term in situ exposures of
the entire Zostera community. Aboveground morphometrics were monitored
during long term laboratory exposure of individual Zostera plants.
Finally adenylate energy charge was monitored during both short and long
term laboratory exposures.
CONCLUSIONS
The survey of Virginia waters of the Chesapeake Bay indicated that
Zostera marina in either its current or recent distributions has
generally not been exposed to levels of atrazine in excess of 1 ppb.
Using the Severn River as a model system, a survey program suggested
that even in "worst case" situations, the exposure of Zostera to
elevated levels of atrazine (in excess of 1 ppb) was short term (one
week or less). With this information in mind field and laboratory
dosing experiments were undertaken to test the effects of naturally
occurring atrazine concentrations on Zostera. Field studies indicated
that Zostera productivity, as measured by oxygen production, is
consistently depressed by atrazine concentrations of 1000 ppb.
Concentrations of 100 ppb frequently caused depression of productivity
but results at this and lesser concentrations were so variable as to
prevent statistically significant conclusions. Field experiments
designed to test effects of simultaneous exposure to atrazine and
reduced light produced no evidence of either additive or synergistic
' .fects.
Long-term exposure (21 days) of Zostera to atrazine in greenhouse
experiments demonstrated that atrazine could produce significant effects
on Zostera morphology at concentrations greater than 60 ppb. The
morphometric test parameters proved so variable and the range of
concentrations tested was so wide that no statistically significant
conclusions could be drawn.
Analysis of adenine nucleotide concentrations in Zostera tissues
proved to be a potentially sensitive indicator of stress. In short terra
exposures (six hour), adenylate concentrations were altered by atrazine
concentrations of both 10 ppb and 100 ppb. In long-term exposures
-------
SECTION 1
PROJECT OVERVIEW
INTRODUCTION
The decline of submerged aquatic vegetation in the Chesapeake Bay
during the 1970's led to much speculation about potential causes. Among
the factors considered were agricultural herbicides. The initial
hypothesis was that increased levels of herbicides were being carried
into the Bay by storm runoff producing concentrations sufficient to kill
the submerged vegetation. Preliminary literature reviews and land use
studies indicated that herbicide use was increasing in the Bay
watershed, thus providing at least circumstantial evidence for the
hypothesis. The project reported here was designed to specifically
investigate the hypothesis and produce evidence of the degree to which
agricultural herbicides were affecting submerged aquatic vegetation.
RESEARCH APPROACH
Atrazine has been used throughout this investigation as our model
herbicide. It was selected because it is the herbicide utilized in the
largest quantity within the lower Chesapeake Bay watershed. Atrazine is
a triazine herbicide whose principal mode of action is disruption of the
Hill reaction in photosynthesis. Its principal application is for
control of weeds in cornfields. The herbicide is typically applied as a
preemergent spray to fields in the spring of the year. It has found
increasing use with the spread of no-till planting methods and is
sometimes applied in combination with other agricultural chemicals.
Zostera £ja_r_in_£ is the species of submerged aquatic vegetation which • •
has been studied. It is the predominate subtidal vegetation of the
lower Chesapeake Bay and because of the recent declines in distribution
has been the focus of other studies in Virginia undertaken as part of
the EPA's Chesapeake Bay Program.
The research approach utilized in this study was to first determine
the level of Zostera marina's exposure to atrazine, and then test for
effects caused by that level of exposure. To that end, this project has
been divided into two general lines of investigation. The first effort ,1
was a survey program to monitor levels of atrazine in water and ,
sediments in the lower i,ay. Forty eight sites were sampled four times '
during 1979. The samplings were generally timed to occur before and '
immediately after spring application of herbicides and before and after
fall harvesting. This schedule was intended to allow detection of peak
-------
CONTENTS (continued)
Page
Section 6. (continued)
Methods 121
Results U',3
Discussion 126
Appendix D 131
Section 7. Adenylate Energy Charge Studies 159
General Introduction 159
References 161
Method Development 164
Introduction 164
Methods 164
Results 177
Discussion 225
References 240
Atrazine Experiments 245
Introduction ?45
Methods ?.46
Results 254
Discussion 267
References 276
vri
-------
CONTENTS
Page
Abstract ill
Section 1. Project Overview 1
Introduction 1
Research Approach 1
Conclusions 2
Recommendations 3
Section 2. Analytical Methods 5
Introduction 5
Quality Assurance Program 6
References 8
Section 3. 1979 Lower Chesapeake Bay Survey Program 9
Introduction 9
Methods 9
Results 9
Discussion 11
References 17
Appendix A 18
Section 4. 1980 Severn River Survey Program 43
Introduction 43
Methods 43
Results 44
Discussion 44
References 51
Appendix B 52
Section 5. Field Dosing Studies 53
Introduction 53
Methods 53
Results 54
Discussion 54
References 66
Appendix C 67
Section 6. Greenhouse Studies . 121
Introduction 121
-------
ABSTRACT
This project was designed to assess the effects of agricultural
herbicides on submerged aquatic vegetation in the lower Chesapeake Bay.
Atrazine was selected for testing because it is the most widely utilized
herbicide in the Bay region. Zostera marina was the submerged vegetation
studied.
The project began with two surveys. The first survey, conducted in 1979,
covered forty eight stations throughout the Virginia portion of the Chesapeake
Bay. The survey was designed to indicate typical atrazine loading for the
lower Bay. A second survey in 1980 was limited to the Severn River, and was
intended to indicate the duration of peak atrazine loading. Information
generated in the two surveys was utilized to design dosing experiments.
Field dosing experiments, utilizing large plexiglass enclosures, measured
effects of short-term atrazine exposure on the net production of the Zostera
community. Greenhouse experiments, utilizing a flow-through dosing system,
measured effects of long-term (21 day) atrazine exposure on the morphology of
mature Zostera plants.
Adenine nucleotide concentrations and a ratio of those concentrations
(termed adenylate energy charge) was assessed in Zostera exposed to various
concentrations of atrazine in laboratory dosing studies. Adenylate
determinations proved a more sensitive indication of stress than either the
oxygen production measurements or the morphometric determinations utilized in
the field and greenhouse studies. In combination with those studies, the
adenylate studies provide evidence for a resistance in Zostera to low (less
than 10 ppb) levels of atrazine, and short term adaptation to atrazine
concentration around 100 ppb.
The entire series of investigations is concluded to indicate that
atrazine effects on mature Zostera marina plants are probably not the
principal cause for the recent decline in distribution of eelgrass in the
lower Chesapeake Bay. Several limitations of the study and suggestions for
future work jre included.
This report was submitted in fulfillment of Contracts R805953 and X003245
by the Virginia Institute of Marine Science under the sponsorship of the U. S.
Environmental Protection Agency and the Ciba-Geigy Corporation. This report
covers the period September 1, 1978 to August 31, 1982.
ill
-------
NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse*
ment or recommendation for use.
ii /
-------
EFFECTS OF ATRAZINE ON ZOSTERA MARINA
IN CHESAPEAKE BAY, VIRGINIA
DRAFT FINAL REPORT
by
Carl Hershner
Keith Ward
Jerome tllowsky
Damon Delistraty
Jeffrey Martorana
Virginia Institute of Marine Science
of the College of William and Mary
Gloucester Point, VA 23062
Contract Nos. R805953 and X003245
Project Officer
David Flemer
U.S. Environmental Protection Agency
2083 West Street
Annapolis, MD 21401
-------
Id
EPA/600/3-88/050
August 1982
7 7*100
:~T^V-^ILJL_- ~-. i_i:i;j:
BALTIMORE -^j7£*? ,-£
~
Hershner
Ward
e Illowsky
i s t r a t y
effitafcs Martorana
Gloucest?.er
fal Protection Agency
Street
is, MD 21401
REPRODUCED BY
U.S. DEPARTMENT OF COMMERCE
NATIONAL TECHNICAL INFORMATION SERVICE
SPRINGFIELD, VA. 22161
-------
TABLE a '.. 1 . (oMitinu.nl)
coordinates: latitude 37°23I54", longitude 76O29'15"
location: south shore of Ware River, 600 meters downstream of public
landing at end of Deacon's Neck, 25 meters offshore
(USGS Ware Neck Quadrangle)
access: boat
depth: 1 meter
sediment: silt and clay • "-
SAV: none
I
WR-2, Bailey's Wharf J
coordinates: latitude 37°23'15", longitude 76°27'48"
location: south shore of Ware River, 25 meters offshore of northside
of Bailey's Wharf (USGS Ware Neck Quadrangle)
access: boat
depth: 1 meter
sediment: silt and clay
SAV: none X
WR-3, Wilson Creek ,
coordinates: latitude 37O21'57", longitude 76°28'8" '
location: south shore of Ware River, mouth of Wilson Creek, 50 mecars
offshore of west side of Roanes Wharf (USGS Achilles
Quadrangle)
access: boat
depth: 1 meter /
sediment: sand and silt
SAV: Ruppia maritima
WR-4, Windmill Point
coordinates: latitude 37°21'57", longitude 76°26'51"
location: south shore of Ware River, north of Oldhouse Creek mouth,
50 meters offshore of west side of Windmill Point (USGS
Achilles Quadrangle)
access: boat -
depth: 1 meter
sediment: sand
SAV: Zostera marina and Ruppia maritima
WR-5, Four Point Marsh
coordinates: latitude 37o20'30", longitude 76°24'34"
location: south shore of Ware River, mouth of river, between Ware
River Point and Tow Stake Point on Four Point Marsh,
300 meters south of Ware River Point (USGS Achilles
Quadrangle)
access: boat-
depth: 1 meter ;
sediment: sand ' "*";
SAV: Zostera marina and Ruppia maritima
Mob j ack ..Bay
MB-1, Whites Neck
.... 23
• (continued;
-------
TABLE Ail.l. (continued)
coordinates: latitude 37°22'5", longitude 76°21'15" x
location: northeast shore of Mobjack Bay, between North River and
East River, 100 meters offshore between Minter Point and
Pond Point at southern end of Whites Neck (USGS New Point
Comfort Quadrangle)
access: boat
depth: 1 meter
sediment: sand -,:
SAV: Zostera marina and Ruppia maritima ;,
MB-2, Bay Shore Point
coordinates: latitude 37°21'42", longitude 76°20'20" \
location: northeast shore of Mobjack Bay, south of East River : it'i, \
200 meters offshore, 500 meters south of Bay Shore PC < -^
(USGS New Point Comfort Quadrangle)
access: boat
depth: 1 meter
sediment: sand
SAV: Zostera marina and Ruppia maritima <
x_^ •
MB-3, Pepper Creek
coordinates: latitude 37°20'26", longitude 76°19'53"
location: northeast s;icre of Mobjack Bay south shore of Pepper /
Creek at mouth, 150 meters offshore (USGS New Point /
Comfort Quadrangle) j. •
access: boat
depth: 1 meter
sediment: sand
SAV: Zostera marina and Ruppia maritima -~
Rappahannock River .-,
— g>,
RR-1, Port Royal
coordinates: latitude 38°10'34", longitude 77°11'12"
location: north shore of river, east side of Rt. 301 bridge, 5
meters offshore (USGS Port Royal Quadrangle)
access: Rt. 301 and wading
depth: 1 meter ^
sediment: silt and clay /
SAV; none ,
i
RR-2, Tappahannock ,'
coordinates: latitude 37°56'22", longitude 7C°50'33"
location: north shore of river, east side of Rt. 250 bridge, 5 meters
offshore (USGS Tappahannock Quadrangle) /
access: Rt. 360 and wading I
depth: 1 meter
sediment: sand
SAV: none „•.
RR-3 Butylo
coordinates: latitude 37°46'6", longitude 76°40'57"
(continued)
-------
TABLE A3.1. (continued)
location: south shore of river, McKans Bay, north side of cause-
way to marmade island, 200 meters offshore (USGS Morattico
Quandrangle)
access: Rt. 600 and wading
depth: 1 meter
sediment: silt
SAV: none
RR-4, Rosegill Farm
coordinates: latitude 37038'2", longitude 76°33'33"
location: south shore of river, 1000 meters downstream from
Bailey Point at mouth of Urbanna Creek, 25 meters
offshore from dam forming Rosegill Lake (USGS Urbanna
Quadrangle)
access: Rt. 227, private lane and wading
depth: 1 meter
sediment: sand and clay
SAV: none
RR-5, Stingray Point
coordinates: latitude 37°33'21", longitude 76°17'59"
location: mouth of river, southern shore, 500 meters south of
Stingray Point, 5 meters offshore (USGS Deltaville
Quadrangle)
access: Rt. 33 and vading
depth: 1 meter
sediment: sand
SAV: none
Potomac River
PR-1, Potomac River Bridge
coordinates: latitude 38°21'38", longitude 77°0'52"
location: south shore of river, 300 meters upstream from Rt. 301
bridge, 25 meters offshore (USGS Dahlgren Quadrangle)
access: Rt. 301 and wading
depth: 1 meter
sediment: sand and clay
SAV: Potamogeton perfoliatus and Vallisneria americana
PR--Z, Ragged Point
coordinates: latitude 38°8'32", longitude 76°36'50"
location: south shore of river, 800 meters south of Ragged Point,
just north of Long Pond, former Pond-a-River Campground,
10 meters offshore (USGS Piney Point Quadrangle)
access: Rt. 728 and wading
depth: 1 meter
sediment: sand
SAV: none
PR-3, Coan River
coordinates: latitude 37°50'10", longitude 76°27'0"
(continued) 25
-------
TABLE A3.1. (continued)
location: south shore of river, south of Coan River mouth, 1200
meters east of Walnut Point, 250 meters west of Balls
Creek mouth, 50 meters offshore (USGS Heathsville
Quadrangle)
access: Rt. 630 and wading
depth: 1 meter
sediment: sand and clay
SAV: none
PR-4, Smith Point, Ginny Beach
coordinates: latitude 37°54'5", longitude 76°15'13"
location: south shore of river, 1850 meters upstream of Little
Wicomico River mouth, 5 meters offshore (USGS Burgess
Quadrangle)
access: Rt. 649 and wading
depth: 1 meter
sediment: sand
SAV: none
Eastern Shore
ES-1, Pocomoke River
coordinates: latitude 37°58'30", longitude 75°37'52"
location: south shore of river, between Pitts Creek and Bullbegger
Creek, north side of Pitts Neck, public dock at end of
Rt. 709 (USGS Saxis Quadrangle)
access: Rt. 709
depth: 1 meter
sediment: silt and clay
SAV: none
ES-2, Saxis
coordinates: latitude 37056'10", longitude 75°43'5"
location: south shore of Pocomoke Sound, north of Saxis, south
of North End Point, 20 meters offshore (USGS Saxis
Quadrangle)
access: Rt. 695 and wading
depth: 1 meter
sediment: sand
SAV: none
ES-3 Chesconessex Creek
coordinates: latitude 37°45'1", longitude 75°O'36"
location: south of Chesconessex Creek, just north of unnamed
inlet midway between Chesconessex Creek and Back Creek,
50 meters offshore (USGS Chesconessex Quadrangle)
access: Rt. 782 and wading
depth: 1 meter
sediment: sand
SAV: none
ES-4, Davis Wharf
(cont innc'l)
26
-------
TABLE A3.1. (continued)
coordinates: latitude 37°33'3", longitude 75°52'44"
location: north shore of Occohannock Creek, due south of Davis
Wharf, 25 meters offshore (USGS Jamesville Quadrangle)
access: Rt. 615 and wading
depth: 1 meter
sediment: sand and silt
SAV: none
ES-5, Occohannock Creek
coordinates: latitude 37°33'28", longitude 75°56'3"
location: north shore of Occohannock Creek near mouth, between
Powells Bluff and Johns Point, 50 meters offshore of
unnamed impoundment (USGS Jamesville Quadrangle)
access: Rt. 612, private lane and wading ^
depth: 1 meter
sediment: sand
SAV: none '
ES-6, Vaucluse Shores
coordinates: latitude 37°24'18", longitude 75°59'6"
location: north of Hungars Creek mouth, 500 meters offshore from
Great Neck (USGS Franktown Quadrangle)
access: boat
depth: 1.3 meters
sediment: sand and clay
SAV: Zostera marina
ES-7, Hungars Creek
coordinates: latitude 37O25'5", longitude 75°57'41"
location: mid-axis Hungars Creek, between Sparrow Point on north
shore and Masden Gulf on south shore (USGS Franktown
Quadrangle)
access: boat
depth: 1.3 meters
sediment: sand and clay
SAV: Ruppia maritima
ES-8, Picketts Harbor
coordinates: latitude 37°11'19" longitude 75°59'59"
location: north of Butlers Bluff on Chesapeake Bay shore, 10 meters
offshore of old range tower at Picketts Harbor (USGS
Townsend Quadrangle)
access: Rt. 646 and wading
depth: 1 meter
sediment: sand
SAV: none
27
-------
INDEX MAPS
Figure A3.1. Index maps for lower Chesapeake Bay survey stations.
-------
-------
GWYNN I
HOLE IN THE »ALL
WINTER HARBOR
HORN HARBOR
- -o.
NEW POINT COMFORT
T
A.
<£>
Ui
Ul
0.
4
Ul
•x.
CB-2
Figure A3.3. Locations of survey stations RR-5 (Stingray Point) and
CB-3 (Crickett Hill/Gwynn's Island).
JJ
-------
-------
-------
-------
-------
'-\\
CM
60
eg
(U
u
0)
10
3
to
1-4
an
0)
<0
z
01
0)
(0
o
-------
? ., i % v • A :£
'-——*f E ^Vv* 4Vi
<~ \ V VVK -r? >JyJ.
~"~ A 'YR-4
Figure A3.9. Locations of survey stations WR-1 (Goshen), WR-2
(Bailey's Wharf), WR-3 (Wilson Creek), WR-4 (Windmill
Point), WR-5 (Four Point Marsh), MB-1 (Whites Neck
Point), MB-2 (Bay Shore Point), MB-3 (Pepper Creek),
3R-1 (Warner Hall Plantation north drainage), SR-2
(Warner Hall Cemetery), SR-3 (Eagle Point Plantation
west drainage), SR-4 (Cod Point), SR-5 (School Neck
Point), SR-6 (Turtle Neck Point), YR-2 (Mumfort
Islands), YR-3 (Allen's Island), YR-4 (Guinea Marshes),
and YR-5 (Brown's Bay).
36
-------
/
-------
CO
I
OC
-------
-------
//
-------
/
Figure A3.14. Locations of survey stations ES-4 (Davis Wharf), ES-5
(Occohannock Creek), ES-6 (Vaucluse Shores), ES-7
(Hungar's Creek) and ES-8 (Picketts Harbor).
41
-------
-------
SECTION 4
1980 SEVERN RIVER SURVEY PROGRAM
INTRODUCTION
While the 1979 survey of atrazine concentrations in the lower Chesapeake
Bay provided evidence of the general level of concentrations, it did not
provide evidence of the duration of exposure Zostera beds experienced. To
address this question, a survey program was established in the Severn River
during 1980. The program involved repeated sampling of the Severn River
stations following the first major rainfall after field application of
atrazine.
Stations in the Ware River (WR-1 and WR-3/ and in the York River (YR-2
and YR-4) were also occupied three times during 1980 to help relate the 1980
data to 1979 survey results.
METHODS
Six stations were occupied in the Severn River during the 1980 survey.
They were the same stations occupied during the 1979 survey. Water samples
were collected and analyzed as indicated in the analytical methods section of
this report (Section 2). Samples were collected at approximately high tide on
each sampling date. Sampling was undertaken on April 17 just prior to
application of atrazine to fields at the head of the northwest branch of the
Severn River. The fields were treated on April 22 and the first rainfall
after application occurred two days later, April 24. Sampling began on April
25 and included six collections over an 8 day period. The next major
rainstorm occurred on May 18-20. A second set of collections was therefore
conducted on May 20, 21, 22 and 23.
Rainfall records were collected from two rain gauges. One is installed
at the Virginia Institute of Marine Science at Gloucester Point. It is
approximately 10 km south of the fields at the head of the northwest branch of
the Severn River. The second rain gauge was situated at Goshen on the Ware
River. That gauge is approximately 8 km north of the northwest branch fields.
Estimates of water volume in the northwest branch of the Severn River and
estimates of land use acreages in the drainage basin were developed by
planimetering areas of USGS topographic maps (Achilles, VA and Clay Bank, VA
quadrangles).
43
-------
RESULTS
The concentrations of atrazine in water samples from the 1980 survey
stations are reported in Table 4.1.
The 1980 rainfall records for the gauges at VIMS and on the Ware River
are reported in Table 4.2.
The water volumes in the northwest branch of the Severn River and the
land use areas in its drainage basin are reported in Table 4.3.
DISCUSSION
The initial sampling on April 17, 1980 found values in the Severn River
near or below the detection limits, as did the initial 1979 sampling. The
York River station samples were also below detection limits as they were in
1979. The presence of atracine in the Ware River samples, even at relatively
low levels, was unexpected. The values are in excess of any found d.iring the
1979 survey.
The samplings conducted after field application of atrazine and
substantial rainfall catalogued the transport of atrazine into the estuary.
The rainstorm on the 24th of April delivered approximately one inch of rain to
the fields in the northwest branch drainage basin. The day after the
rainstorm atrazine was found in detectable amounts at only the two headwater
stations. During the following two days concentrations of atrazine decreased
in the headwaters and rose to detectable amounts throughout the remainder of
the river. Three days after the rainstorm, concentrations in the river were
relatively uniform at levels very near the detection limit and well below 1
ppb.
Late on April 27 a second rainstorm moved through the area depositing
aproximately one more inch of rain on the fields. This rain event, falling on
fields already well saturated, produced rapid and large increases in atrazine
concentrations throughout the river. Within 24 hours the station at the river
mouth, an area supporting extensive Zostera beds attained a 1 ppb level of
atrazine in the water. The concentration decreased within tvo days, despite
continued rainfall, to less than 0.3 ppb. Atrazine concentrations in water at
upstream stations remained well above 1 ppb for at least four days following
the second rainstorm. During that time, however, the concentrations declined
to approximately one-fourth those attained immediately after the second
rainstorm.
The next major rainfall event occurred in May on the 18th, 19th and 20th.
This time atrazine concentrations again rose above the detection limit
throughout the river, but they exceeded 1 ppb only at the station in the
headwaters.
If the 1980 survey results are taken as representative of long term
experience in the Severn River, several observations are important. First,
Zostera marina beds at the mouth of the river are exposed to levels of
atrazine approaching 1 ppb infrequently and only for short periods of time
44
-------
o
OO
•*
o
z
5
^2
a
z
o
»>H
O
s
2
JgJ
O
— ,
aa
O
Z
i
te.
i/)
CiJ
0. C
Z o
5 *•"'
CO — •
a: .-i
W .0
H
< u
3 V
D.
Z
H^ 0)
w w
z
z — •
80!
^
Hj
eo
H
eg
Z
en
CM
m
X
fj
CM
X
«
Z
_
CM
X
eg
J£
O
CM
X
eg
z
CM
^
a.
0
en
u
a
CM
00
.
ON
_*
— '
^
t_t
CM
O
-*
,
o
0
0-
V |
(0
O
^J
7
CO
m
m
O
0
en
o
00
en
d
CM
CM
d
o
CM
rt
m
r-
•
»H
00
d
-«
d
-^
.
0
o
o,
^1
o
5,
^J
f
OS
CO
ON
en
O
CM
O
CM
~->
O
0
en
O
*
_J
O
o
-<
,
o
o
0-
V 1
o
5,
1
as
vO
en
O
0
0
0
d
\j 1
NO
CM
d
CM
CM
d
00
CM
d
^
o
^
CM
d
o
.
0
0
o
o
d
i
os
CO
CM CM O -<
*4 «i^ ^M f*4
o o o d
vl
f
— o o o
o 0,0 d.
v| v| v|
NO en O O
CM en ^^ ^H
d d o. d,
v| v|
-H m CM
-------
V)
O —i —< O CM OO OO OO
OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO O
CS
00
00
OOOOOO— 'OOOOOOOOOOOOOOOOOOOOOOO
M
oS
W
u.
o
Q
Wc"> or in CM O r^ 00
*** ^5 co >j M? co c^ CM o? ^s
ooooooooooooooooooo'oooooo oooooo CM
M3 co r- M3
—' O CM .»
OOOOOOOOOOOOOOOOOO X X X X X X XOOOOOO O
(d
CMCMCMCsCMCMCMCMCMCMncO
U
at
00 in OOO CMW>-«CMO* CM O CM
OOOOOO X X X X X X X X X X X X XOOOOOOOOOO
O ^ ^ MO O ^ CM
CM O *^ M3 f*^ CM CM
oooooo o'odoooooooooooooo' ooo ooo
u.
z
PO
u
u.
CMCMCMCMCMCMCMCMCMCM
o
00
CQ
CM — -- -1 o f-- f>
-------
vO
O
O -4- O vj
inOO —
OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO -*
05
O »f O CO O —« O O -* CM
ooooooooodoooooo'ooooooooooooooo -•
u
w
Q
CMCMCMCMCMCMCMCMCSCMCOCO
I
QO
CM
-^ 10 in i— i
OOO-H
o o -< o
oooooooooooooooo-
V)
— O P» O 00 -H
oooo'oooooooooooooooooooooooooo
vO
o»
CNCMCMCMCMCMCMCMCNCMCO
co co o Or* -H cM-
CM -- -»
CO
o
OOO X X X X X X XOOOOOOOOOOOOOOOOOOOOO ->
00
vD
CT>
O m
3 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO co
<—* CM O O *C^ CO >O O 00
ooooo xoooo'oooooo — oooooooooooooo CM
-. I- -4 d
CMCMCMCMCMCMCMCMCMCMCOrO H
47
-------
TABLE 4.3. NORTHWEST BRANCH OF SEVERN RIVER
total drainage basin « 35.54 kin2
open water » 4.36
intertidal marsh » 0.68
pasture and residential area " 6.33 km2
cropland area « 5.57 km2
forested area » 18.60 km2
branch mean low water volume * 7.01 x 10' liters
tidal volume » 1.96 x 10^ liters
branch mean high water volume * 8.97 x 10' liters
48
A
-------
(less than two days). If grass beds once grew throughout the lower one-half
of the river system, the upstream beds may have occasionally been exposed to
atrazine concentrations exceeding I ppb, but much less than 10 ppb. The
exposures, however, were probably limited to spring and probably did not last
for more than a week or two.
In general, the data collected in this survey program suggest that
Zostera marina, in its present distribution, is unlikely to experience levels
of atrazine in water in excess of 1 ppb for more than one to two days during a
growing season. This conclusion requires several major assumptions. The
first assumption is that the Severn River is a good model for the lower
Chesapeake Bay system. The second assumption is chat the spring runoff event
monitored in 1980 is an unusual event and fairly represents a "worst case"
situation.
The latter assumption is probably a reasonable one. The combination of
two major rainstorms dropping approximately two inches of rain within several
days of field application of atrazine is unusual. A review of daily rainfall
from a number of recording stations in the region (U.S. Environmental Data
Service) indicate that during the eleven year period 1971 through 1981, a
greater amount of rain has fallen in late April on only two occasions. An
average and median amount over the twelve year period was approximately 1.2
inches of rainfall. Data from the Williamsburg, Virginia, station is
summarized in the appendix to this section as an example (Table 64.1).
The first assumption that the Severn River is a good model for lower
Chesapeake Bay systems is more tenuous. The 1979 survey program suggests the
Severn River stations, despite a limited geographic range, experience atrazine
loadings which cover Nie entire range of exposures in the lower Bay.
Furthermore, the general trend of concentations from headwaters to river mouth
seems typical of the other river systems sampled in 1979. The general pattern
of land use in the Severn River is not unusual for the larger rivers,
particularly in respect to the proportion of croplands. The topography of the
drainage basin is atypically flat, but this factor is somewhat compensated by
the proportionately reduced scale of the entire system. The major difficulty
with using the Severn River as a model for other Bay systems is the lack of
specific information about circulation within the river. In the absence of
information about water parcel residence times in the river, particularly
during runoff events, extrapolation of herbicide exposures to other systems
must remain intuitive.
The principal objective of the 1980 survey program was to evaluate the
duration of Zostera marina exposures to atrazine during a growing season.
Building on data collected during the 1979 survey, a program was instituted to
monitor the spring runoff events in the Severn River. Fortunately for this
effort, there was an unusual amount of rain immediately after field
applications of atrazine in the Severn drainage basin. The monitoring program
determined that existing grass beds within the Severn River were exposed to
atrazine concentrations as high as 1 ppb for a period cf less than two days.
Reaches of the river which may have conta'ned Zostera beds in the past, were
exposed to water concentrations of atrazine in excess of 1 ppb, but less than
10 ppb, for a period of approximately one week.
49
A'-
-------
In general, the 1980 survey program is presumed to indicate that Zostera
marina, in its present distribution, rarely is exposed to atrazine
concentrations in excess of 1 ppb.
50
-------
REFERENCES
U.S. Environmental Data Service. Climatological Data-Virginia. Volumes
81-91. Published by U.S. Department of Commerce.
51
-------
TABLE B4.1. SUMMARY OF ELEVEN YEAR RAINFALL DATA FOR WILLIAMSBURG, VIRGINIA,
GAUGING STATION
Total April Amount During Last
Year Rainfall (in.) 15 Days in April
1971 1.71 0.52
1972 3.80 2.29
1973 3.42 1.61
1974 1.48 0.34
1975 3.19 0.99
1976 0.77 0.00
1977 3.87 1.29
1978 4.20 4.06
1979 3.88 1.16
1980 3.05 1.62
1981 2.62 1.14
Average ' 2.91 1.37
Median 3.19 1.16
52
-------
SECTION 5
FIELD DOSING STUDIES
INTRODUCTION
Short term effects of atrazine on the Zostera marina community were
investigated by in situ dosing experiments. Since the mode of action of
atrazine is to block the Hill reaction in photosynthesis, the short term
studies were designed to measure changes in oxygen production. Oxygen
production was also selected as a test parameter to provide correlations with
the data sets being generated by Wetzel e£ al_. (1979) as part of their studies
of production in the Zostera community.
The concentrations of atrazine selected for study ranged up to and
including 1000 ppb . The survey program results suggested the 1 ppb and 10 ppb
concentrations should be of greatest interest since they represent the range
of values actually found in Bay waters. Higher concentrations were included
to ensure detection of an effect on oxygen production.
Some of the field dosing experiments were designed to test the hypothesis
that atrazine acts in an additive or synergistic fashion with reduced light
levels to produce significant impacts on the Zostera community. The
hypothesis was suggested by the high probability of co-occurrence of maximum
atrazine concentrations and high turbidity during post planting spring runoff
events. The hypothesis was tested by adding greenhouse shading material to
some of the dosing enclosures. Effective insolation was thus reduced to 802,
702 or :>OZ of natural conditions.
METHODS
_In situ dosing of the Zostera marina community was accomplished with
hemispherical plexiglass enclosures. The enclosures are identical to those
used by Wetzel _e£ a_l (1982). Each dome enclosure has a volume of
approximately 260 liters. Six domes were generally used simultaneously to
provide control and atrazine treatments.
Before "setting" the domes, the working platform was positioned and
anchored. The grass bed in the vicinity of the platform stern was surveyed
for uniformity by divers. Each of the six domes was carefully positioned on
the bottom and the four inch vertical flange on the perimeter of the dome was
driven into the sediment. This provided a "seal" effectively isolating the
dome contents from the surrounding system. Ambient water was then pumped
through the dome to flush it for approximately one hour. An experiment was
initiated by closing all portals in the dome, so that a closed loop between
53
-------
the dome and an onboard pumping station was created. Atrazine was introduced
to the closed system through septa in the apex of the dome. Atrazine
standards, prepared in the laboratory, were injected by 50 ml glass syringes.
The standards consisted of technical grade atrazine dissolved in 100 ml
methanol (for 1 ppb through 100 ppb atrazine treatments) or 200 ml methanol
(for J000 ppb atrazine treatment).
The amount of atrazine was selected to give the appropriate nominal
concentration of atrazine in water. Methanol controls were run in the first
several experiments to identify effects due to the atrazine carrier.
Domes were usually "set" at or near sunrise. Experiments typically
lasted until near sunset. Dissolved oxygen in the domes was monitored hourly
by inserting an oxygen meter probe (YSI or Orbisphere) into a port on the
pumping station. Near termination of the experiment water samples were taken
for atrazine analysis (500 ml). Samples were also collected for dissolved
oxygen determination by Wir.kler titration as a check on the oxygen meters.
Shading experiments were conducted by making individual shades for domes.
The shades were constructed of greenhouse shade cloth (a coarse woven nylon
material/. Insolation is controlled by coarseness of the weave in the
material. For these experiments the material used blocked 20%, 30% or 502 of
incident light without altering the spectrum of the transmitted light.
RESULTS
Data from the experiments are presented in the appendix to this section
(Appendix C). Tables C5.1 through C5.18 present the hourly dissolved oxygen
concentrations (in ppm). Figures C5.1 through C5.18 are graphs of the oxygen
concentrations versus time. Tables C5.19 through C5.36 contain the calculated
oxygen production rates (in mg 02 m~* hr~^) for each experiment.
For analytical purposes each experiment was divided into five time
periods based on the sun's declination (morning 0700-1000, noon 1100-1400,
afternoon 1500-1800, evening 1900-2300, night 0000-0600). Within each time
period the oxygen production values for each treatment were averaged and the
mean values compared using the F Test. Table 5.1 presents the results of the
analysr-s for each period of each experiment.
Experiments with significant differences between the mean rates of oxygen
production were further analyzed by the multiple range test in order to
indicate probable associations among the treatments. Table 5.2 presents the
results of these analyses.
Table 5.3 presents analyses of water samples collected from the domes at
the conclusion of dosing. The samples were taken as a check on the nominal
concentrations assumed for each treatment.
DISCUSSION
Review of the field dosing experiments indicates consistent and
significant negative effects of atrazine dosing were only detected at the
54
-------
TABLE 5.1. STATISTICAL EVALUATION OF GUINEA MARSH DOME STUDY DATA USING 5*
LEVEL F TESTS TO TEST DIFFERENCES BETWEEN MEAN OXYGEN PRODUCTION
RATES FOR EACH EXPERIMENTAL PERIOD
Exp.
5.1
5.1
5.1
5.1
5.1
5.1
5.2
5.2
5.2
5.2
5.2
5.3
5.3
5.3
5.3
5.3
5.5
5.5
5.5
5.4
5.4
5.4
5.4
5.4
5.4
5.4
5.6
5.6
5.6
5.6
5.6
5.7
5.7
5.7
5.7
5.8
5.8
5.8
Period
afternoon
evening
night
morning
noon
afternoon
morning
noon
afternoon
evening
night
morning
noon
afternoon
evening
night
noon
noon
afternoon
morning
noon
afternoon
evening
night
morning
noon
morning
noon
afternoon
evening
night
morning
noon
afternoon
evening
morning
noon
afternoon
Date
5-29-80
5-29-80
5-29-80
5-30-80
5-30-80
5-30-80
6-23-80
6-23-80
6-23-80
6-23-80
5-23-80
6-25-80
6-25-80
6-25-80
6-25-80
6-25-80
7-14-80
7-14-80
7-14-80
7-15-80
7-15-80
7-15-80
7-15-80
7-15-80
7-16-80
7-16-80
7-18-80
7-18-80
7-18-80
7-18-80
7-13-80
7-29-80
7-29-80
7-29-80
7-29-80
7-30-80
7-30-80
7-30-80
F Ratio
1.004
1.279
0.399
2.184
1.235
0.221
1.721
2.906
0.003
2.723
1.130
5.047
2.414
1.239
3.421
0.467
17.272
8.703
2.061
1.672
15.876
0.324
40.525
2.937
38.735
84.010
12.826
12.380
0.754
6.318
1.598
0.506
3.583
1.187
3.305
5.519
2.429
0.244
F Probability
0.4777
0.3154
0.7553
0.1428
0.3399
0.8776
0.2156
0.0784
0.9997
0.0909
0.3609
0.0101
0.0766
0.3318
0.0272
0.7993
0.0000
0.0002
0.1415
0.1924
0.0000
0.8915
0.0000
0.0252
0.0000
0.0000
0.0000
0.0000
0.5942
0.0007
0.1909
0.7683
0.0201
0.3715
0.0416
0.0072
0.0753
0.9375
Significant at
.05 Level
-
-
-
-
-
-
_
_
-
-
*
-
-
*
-
*
*
_
-
*
-
*
*
*
*
*
*
-
*
-
-
*
-
*
*
-
-
(continued)
55
-------
TABLE 5.1. (continued)
Exp.
5.9
5.9
5.9
5.10
5.10
5.11
5.11
5.11
5.12
5.12
5.12
5.13
5.13
5.13
5.13
5.14
5.14
5.14
5.15
5.15
5.15
5.16
5.16
5.16
5.17
5.17
5.17
5.18
5.18
5.18
*Morning
Noon
Afternoon
Evening
Night
Period
morning
noon
afternoon
morning
noon
morning
noon
afternoon
morning
noon
afternoon
morning
noon
afternoon
evening
morning
noon
afternoon
morning
noon
afternoon
morning
noon
afternoon
morning
noon
afternoon
morning
noon
afternoon
0700-1000
1100-1400
1500-1800
1900-2300
0000-0600
Date
7-31-80
7-31-80
7-31-80
8-1-80
8-1-80
8-12-80
8-12-80
8-12-80
8-13-80
8-13-80
8-13-80
8-14-80
8-14-80
8-14-80
8-14-80
8-15-80
8-15-80
8-15-80
9-8-80
9-8-80
9-8-80
9-9-80
9-9-80
9-9-80
9-10-80
9-10-80
9-10-80
9-11-80
9-11-80
9-11-80
F Ratio
8.821
0.094
0.109
6.669
34.920
0.664
7.782
0.093
0.945
3.289
0.124
1.934
0.259
0.763
2.035
1.935
6.336
i.049
1.345
3.775
0.203
0.434
4.010
0.488
0.309
0.760
0.657
1.171
2.479
0.143
F Probability
0.0010
0.9921
0.9884
0.0144
0.0000
0.6652
0.0005
0.9925
0.4871
0.0277
0.9852
0.1620
0.9294
0.5906
0.1355
0.1619
0.0017
0.4683
0.3601
0.0163
0.9570
0.8168
0.0128
0.7761
0.8984
0.590
0.6605
0.3782
0.0709
0.9784
Significant at
.05 Level
*
_
_
*
*
-
*
-
-
*
-
-
-
-
-
-
*
-
-
*
-
-
*
-
-
-
-
-
-
56
-------
TABLE 5.2. STATISTICAL EVALUATION OF GUINEA MARSH DOME STUDY DATA
USING 52 LEVEL STUDENT-NEWMAN-KEULS MULTIPLE RANGE TEST
Experiment/
Perion
Atrazine
Concentration
Shading
Level
Results of S-N-K
52 Level
5.3 morning
5.3 evening
5.4 noon
(7/15/80)
5.4 evening
(7/15/80)
5.4 night
(7/15/80)
1000 ppb
100 ppb
1 ppb
10 ppb
MEOH control
control
MEOH control
control
10 ppb
1 ppb
100 ppb
1000 ppb
1000 ppb
100 ppb
1 ppb
10 ppb
MEOH control
control
control
10 ppb
1 ppb
MEOH control
100 ppb
1000 ppb
MEOH control
1 ppb
10 ppb
control
100 ppb
1000 ppb
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
control
c""itrol
corrrol
control
control
control
control
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
(continued)
57
-------
TABLE 5.2. (continued)
Experiment/
Period
5.4 morning
(7/16/80)
5.4 noon
(7/16/80)
5.5 morning
5.5 noon
5.6 morning
5 . 6 noon
Atrazine
Concentration
1000 ppb
100 ppb
10 ppb
1 ppb
MEOH control
control
100 ppb
1000 ppb
10 ppb
1 ppb
MEOH control
control
1000 ppb
100 ppb
control
MEOH control
1 ppb
10 ppb
1000 ppb
MEOH control
100 ppb
control
1 ppb
10 ppb
1000 ppb
10 ppb
100 ppb
control
1 ppb
MEOH control
1000 ppb
100 ppb
10 ppb
1 ppb
MEOH control
control
Shading Results of S-N-K
Level 51 Level
control *
control *
control *
control *
control * *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control *
control * *
control *
control *
control *
(continued)
58
-------
TABLE 5.2. (continued)
Experiment/
Period
5.6 evening
5 . 7 noon
5. 7 evening
5.8 morning
5.9 morning
5. 10 morning
5. 10 noon
Atrazine
Concentration*
1 ppb
MEOH control
control
10 ppb
100 ppb
1000 ppb
10 ppb
MEOH control
control
10 ppb
MEOH control
control
control
MEOH control
10 ppb
control
10 ppb
MEOH control
MEOH control
control
10 ppb
10 ppb
control
MEOH control
MEOH control
10 ppb
10 ppb
control
MEOH control
control
10 ppb
control
control
10 ppb
10 ppb
control
10 ppb
control
Shading
Level
contr jl
control
control
control
control
control
51%
51%
51%
control
control
control
control
control
control
51%
51%
51%
51%
51%
51%
control
control
control
51%
51%
control
control
control
51%
30%
30%
control
control
30%
30%
control
control
Results of S-N-K
5% Level
*
*
*
* *
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* *
* *
* *
*
* *
* *
* *
*
*
*
* *
*
*
*
*
*
*
(continued)
59
-------
TABLE 5.2. (continued)
Experiment/
Period
Atrazine
Concentration
Shading
Level
Results of S-N-K
5% Level
5.11 noon
5.12 noon
5. 14 noon
5.15 noon
5. Ib noon
control
10 ppb
I ppb
control
10 ppb
1 ppb
10 opb
control
1 ppb
control
10 ppb
1 ppb
1 ppb
10 ppb
10 ppb
1 ppb
control
control
1 ppb
control
10 ppb
1 ppb
control
10 ppb
10 ppb
I ppb
control
10 ppb
control
1 ppb
30%
30%
30%
control
control
control
30%
30%
30%
control
control
control
control
30%
control
30%
30%
control
20%
20%
20%
control
control
control
20%
20%
20%
control
control
control
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* *
* *
* *
*
*
*
*
*
*
L.
* Atrazine concentrations are ranked in order of lowest mean
productivity rates (IDR 02 m~2 hr~l ) to highest mean productivity
rates.
60
-------
TABLE 5.3. ATRAZINE CONCENTRATION IN WATER, NOMINAL VS MEASURED
CONCENTRATIONS
(samples generally taken at termination of experiment)
Experiment
Date
Treatment Norn.
Cone. & % Shade
Measured
Concentration
5.1
5.2
5.3
5-29-80
6-23-80
6-25-80
5.4
7-15-80
5.5
7-1A-80
5.6
7-18-80
5.7
7-29-80
(continued)
control
MeOH control
100 ppb
100 ppb - 100%
control
MeOH control
10 ppb
100 ppb
control
MeOH control
1 ppb
10 ppb
100 ppb
1000 ppb
control
MeOH crntrol
1 ppb
10 ppb
100 ppb
1000 ppb
control
MeOH control
1 ppb
10 ppb
100 ppb
1000 ppb
control
MeOH control
1 ppb
10 ppb
100 ppb
1000 ppb
control
control-51%
MeOH control
MeOH-51%
10 ppb
10 ppb 51%
61
0.16 ppb
0.11 ppb
2.54 ppb
65.15 ppb
0.28 ppb
0.10 ppb
1.48 ppb
6.14 ppb
72.49 ppb
515.15 ppb
0.16 ppb
0.17 ppb
1.47 ppb
6.38 ppb
71.85 ppb
761.90 ppb
0.21 ppb
£0.10 ppb
0.81 ppb
4.85 ppb
61.44 ppb
709.29 ppb
0.24 ppb
0.24 ppb
0.58 ppb
0.58 ppb
6.51 ppb
9.87 ppb
A
-------
TABLE 5.3. (continued)
V
Experiment
Date
Treatment Norn.
Cone. & % Shade
Measured
Concentration
5.8
7-30-80
5.9
7-31-80
5.10
5.11
8-1-80
8-12-80
5.12
8-13-80
5.13
8-14-80
5.14
8-15-80
control
control 51%
MeOH Control
MeOH - 51%
10 ppb
10 ppb - 51%
control
control -51%
MeOH control
MeOH - 51%
10 ppb
10 ppb - 51%
control
control - 30%
10 ppb
10 ppb - 30%
control
control - 30%
1 ppb
1 ppb - 30%
10 ppb
10 ppb - 30%
control
control - 30%
1 ppb
1 ppb - 30%
10 ppb
10 ppb - 30%
control
control - 30%
1 ppb
1 ppb - 30%
10 ppb
10 ppb - 30%
control
control - 30%
1 ppb
1 ppb - 30%
10 ppb
10 npb - 30%
0.18 ppb
no sample
no sample
no sample
6.30 ppb
7.86 ppb
6.51 ppb
8.81 ppb
0.63 ppb
0.36 ppb
7.41 ppb
6.93 ppb
0.72 ppb
0.69 ppb
6.58 ppb
6.35 ppb
1.02 ppb
0.85 ppb
7.18 ppb
6.54 ppb
(continued)
b2
-------
TABLE 5.3. (continued)
Experiment
Date
Treatment Norn.
Cone. & % Shade
Measured
Concentration
5.15
5.16
5.17
5.18
9-8-80 control
control
1 ppb
1 ppb -
10 ppb
10 ppb
9-9-80 control
control
1 ppb
10 ppb
10 ppb
9-10-80 control
control
1 ppb
1 ppb -
10 ppb
10 ppb
9-11-80 control
control
1 ppb
- 20%
20%
- 20%
- 20%
- 20%
- 20%
20%
- 20%
- 20%
1 ppb - 20%
10 ppb
10 ppb
- 20%
0.13 ppb
0.60 ppb
6.59 ppb
£0.10 ppb
0.26 ppb
6.79 ppb
£0.10 ppb
0.59 ppb
7.77 ppb
63
-------
highest concentration, 1000 ppb. Productivity, as measured by oxygen
production, was frequently reduced by 100 ppb atrazine concentrations but the
difference from controls was not always statistically significant as
determined by multiple range testing. The data for lower concentrations of
atrazine was even more variable, preventing significant conclusions about
effects. A priori expectations were for a graded response of oxygen
production reduction positively correlated with atrazine concentration. A
number of the experiments produced results which fit these expectations (see
Figure C5.6 for example) however, we have found no basis in any of the
information we collected for conclusions based only on selected experiments.
We felt constrained therefore to analysis of the entire data set and caution
against any selective interpretations.
The in situ enclosure techniques proved unable to distinguish moderate
effects of atrazine from control responses. The principal reason for this
appears to be the natural variability of the Zostera community. Despite our
efforts to cb:ain a homogeneous set of enclosed communities for each
experiment we were obviously unable to achieve a reduction in variation
sufficient to permit statistically significant detection of anything other
than major effects. Detailed sampling of the enclosed communities in each
experiment may have permitted better resolution of the data, but unfortunately
suitable data was not collected during these studies.
The shading experiments generally produced the expected reduction in
production, but no statistically significant evidence of either additive or
synergistic effects with atrazine dosing was developed.
The results of analysis of the water samples collected from the domes
revealed a persistent sub-part-per billion level of atrazine within the
control domes. The results are not due to analytical errors. Great care was
exercised in the field to minimize any chances for cross-contamination.
Specific sets of experimental gear were routinely used for the control and
dosed treatments and each enclosure was run as a closed system throughout- the
experiments. Ambient atrazine concentrations were always below our detection
limits at the experimental site. Nevertheless, low-level contamination of the
controls remained a persistent problem.
The water samples also indicate a fairly consistent recovery of 60-702 of
the injected spike at. the conclusion of each experiment. Attempts were made
to investigate partitioning of the atrazine spike among water, sediments,
plants and epiphytes within the domes during the course of the experiments.
Satisfactory sampling methods proved to be an intractable problem. Despite
several attempt? to collect usable samples of each substrate we had not solved
the methodology problem by the conclusion of this project.
In summary, the results of the field dosing experiments appear to be
limited by tUa methodology. The finding that atrazine concentrations of 100
ppb and greater generally produced a significant effect on short-'tenn net
productivity of the Zostera community is in general agreement with the results
of the greenhouse dosing studies reported in the following section.
Conclusions about effects of lower concentrations of atrazine on Zostera
64
A
-------
communities, either the presence or absence of effects, are generally not
supported by the data generated in this investigation.
A more intensive use of the in situ enclosure methodology may permit
better definition of effects in the future. Specifically, greater replication
of both control and low-level doses will be required. Much of the current
data may have been more useful if information about the enclosed community
(e.g. macrophyte and epiphyte biomasses) has been available. This information
would permit efforts to normalize the observed oxygen production effects,
factoring out nonhomogeneity of the enclosed communities. From our
experience, development of this information requires a major commitment of
resources (see also Orth et al. 1982). As indicated by the analytical
problems we have had, however, the commitment is essential.
Questions raised by this study which remain unanswered include
description of the partitioning of atrazine among components of the enclosed
Zostera community, and analysis of the response of Zostera to other forms of
atrazine exposure (e.g. atrazine sorbed to suspended sediments). Both of
these questions are important to efforts to extrapolate this type of
experimental data to natural communities.
65
-------
REFERENCES
Orth, R. J., K. A. Moore, M. H. Roberts, and G. M. Silberhorn. 1982.
The biology and propagation of eelgrass, Zostera marina, in the
Chesapeake Bay, Virginia. Final Report. U.S. EPA Grant No. R805953.
Wetzel, R. L., P. A. Penhale, R. F. van Tine, L. Murray, A. Evans, and K. L.
Webb. 1982. Primary productivity, community metabolism, and nutrient
cycling. In: Functional Ecology of Submerged Aquatic Vegetation in the
Lower Chesapeake Bay. R. L. Wetzel, ed. Final Report U.S. EPA,
Chesapeake Bay Program, Annapolis, MD.
66
-------
TABLE C5.1. DOME STUDY, 29-30 MAY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. ID Dome 7 Dome 8 Dome •> Dome 10
1800
1900
2000
7100
2200
2300
2400
0100
0200
0300
0400
0500
0600
0700
0800
0900
1000
1100
1200**
1300
1400
1500
1600
1700
1800
25°C
25°
23°
22.5°
23°
23°
23°
24°
24°
23°
*
23°
22.5°
22°
22°
22°
22°
23°
24°
24.5°
25°
25o
26°
25.5°
25.5°
7.9
7.7
7.2
7.6
6.6
5.5
6.3
5.3
5.6
4.8
4.6
5.0
5.4
5.6
5.4
5.5
4.8
5.6
5.9
9.0
9.0
8.8
11.1
11.5
8.4
7.P
6.7
6.0
4.8
5.3
4.4
4.0
3.4
2.6
1.8
2.6
2.4
3.0
3.5
4.0
5.6
6.2
6.4
10.9
11.5
12.0
12.6
12.6
8.5
7.9
6.9
5.9
4.2
3.9
2.2
1.2
1.4
0.6
0.1
0.6
0.2
0.3
0.2
0.8
2.0
3.0
3.2
6.5
7.4
8.4
9.0
9.0
8.2
7.8
7.1
6.5
5.3
4.8
4.2
3.0
3.0
1.8
1.3
1.6
1.0
0.6
0.2
0.7
1.1
1.6
1.4
3.0
3.3
3.6
3.8
3.9
7.9
6.8
6.2
5.4
4.3
3.8
2.9
2.6
2.0
0.8
0.6
0.9
0.3
0.0
0.0
0.0
0.2
0.4
0.0
0.2
0.2
0.2
0.2
0.4
dome 7 - control
dome 8 - 100 ml MeOH
dome 9-100 ppb atrazine
dome 10- 100 ppb atrazine
*no readings taken
**Probe malfunction-membrane replaced after 1300 reading
67
-------
TABLE C5.2. DOME STUDY, 23-24 JUNE 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
Cdissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 7 Dome 8 Dome 10
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
0100
0200
0300
0400
0500
0600
0700
0800
0900
1000
22°C
22°
22.5C
23°
23.5C
24°
25°
25°
25.5C
25.5C
25°
2?S
24°
24°
24°
24°
2V
23°
23°
23°
23°
23°
23°
23°
23.5C
6.1
6.4
6.9
7.7
8.0
8.6
9.4
9.4
8.4
8.0
7.4
7.0
6.6
6.4
6.2
5.8
5.9
6.3
7.1
9.4
10.0
10.8
11.4
11.5
11.7
11.5
10.6
9.9
8.2
7.2
5.7
4.3
5.0
5.2
5.5
5.5
5.6
5.4
5.1
5.2
5.6
1.2
0.65
0.5
0.15
0.1
0.2
0.2
0.25
0.6
5.7
6.3
7.4
10.0
10.8
12.0
12.8
13.1
13.2
12.8
11.9
10.8
8.8
7.4
5.6
4.1
0.7
0.25
0.05
0.15
0.1
0.25
0.15
0.4
1.1
6.4
7.0
7.6
9.3
10,
11,
12.0
12.3
12.4
11.6
10.4
8.8
7.2
5.7
4.4
2.9
0.75
0.4
0.3
0.15
0.15
0.2
0.3
1.0
2.10
5.4
5.3
6.1
6.2
6.2
6.2
6.0
6.1
5.8
4.6
3.7
2.4
1.5
0.6
0.15
0.1
0.1
0.05
0.05
0.05
0.05
0.1
0.1
0.1
0.1
dome 5 - control (measured concentration »0.16 ppb)
dome 7 - 100 mIMeOKmeasured concentration = 0.11 ppb)
dome 8-10 ppb atrazine (measured concentration » 2.54 ppb)
dome 10 - 100 ppb atrazine (measured concentration » 65.15 ppb)
*no readings taken
68
I.
>
-------
TABLE C5.3. DOME STUDY, 25-26 JUNE 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 7 Dome 6 Dome 8 Dome 10 Dome: _9_
0900
1000
1100
1200
1300
•400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
0100
0200
0300
0400
0500
0600
0700
0800
0900
- — _r
23°C
23°
2V°
24°
24°
25°
25°
25°
24.5°
24°
24°
23.5°
23.5°
23°
*
23°
23°
23°
23°
23°
*
23°
23°
23°
23°
5.5
6.1
6.5
7.1
7.6
8.2
8.2
7.4
6.9
6.6
6.5
6.1
5.7
5.3
4.8
5.2
4.3
4.7
4.5
4.5
4.8
5.0
5.1
5.4
6.2
6.7
7.6
8.7
9.5
9.9
9.6
8.8
8.0
6.7
5.2
3.8
2.5
0.7
0.2
0.2
0.2
0.2
0.35
0.1
0.10
0.3
5.4
6.0
6.5
7.3
8.1
9.2
9.5
10.0
9.0
8.6
7.4
6.1
5.0
3.6
2.55
0.75
0.55
0.55
0.45
0.15
0.1
0.75
0.35
4.9
5.6
6.0
6.7
7.8
8.7
9.0
9.0
8.2
7.4
6.0
4.4
2.9
2.65
0.35
0.2
0.2
0.1
0.1
0.1
0.1
0.25
5.2
5.8
6.3
7.0
8.1
9.0
9.6
9.3
8.4
7.6
6.3
4.9
3.5
2.4
0.85
0.25
0.15
0.05
0.1
0.15
0.05
2.2
0.1
5.4
5.8
5.5
5.8
6.1
6.3
6.1
5.6
4.6
3.3
2.2
1.2
1.05
0.25
0.15
0.1
0.1
0.05
0.05
0.8
0.05
1.3
0.05
5.3
5.6
5.0
4.8
4.6
4.6
4.1
3.7
2.8
2.0
1.5
1.0
0.35
0.35
0.20
0.15
0.2
0.2
0.2
0.2
0.1
0.15
0.35
dome 5 - control (measured concentration * 0.28ppb)
dome 7 - MeOH (measured concentration • 0.10 ppb)
dome 6-1 ppb atrazine (measured concentration » 1.48 ppb)
dome 8-10 ppb atrazine (measured concentration "6.14 ppb)
dome 10- 100 ppb atrazine (measuied concentration « 72.49 ppb)
dome 9 - 1000 ppb atrazine (measured concentration * 515.15 ppb)
*no readings taken
69
-------
TABLE C5.4. DOME STUDY, 15-16 JUV* 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
'Ambient
Dome 7 Dome 8 Dome 9 Dome 10
Time
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2COO
2100
2200
2300
2400
0100
0200
0300
0400
0500
0600
0700
0800
0900
1000
1100
1200
1300
1400
1500
dome
dome
dome
dome
dome
Temp.
25°C
25°
25.5°
26°
26°
26.5°
27°
27.5°
28°
2V
2b°
2J°
28°o
27.5°
27°
27°
26.5°
26°
26°
26°
26°
26°
25.5°
25.5°
25°
25.5°
26°
26.5°
27°
27.5°
28°
28.5°
LJU
5.0
6.1
6.9
7.2
7.3
7.9
8.6
8.4
8.7
8.6
8.7
8.7
8.0
6.8
6.0
5.8
6.1
6.2
6.0
5.9
5.6
5.0
5.1
4.3
4.7
5.6
6.9
7.1
7.4
8.0
9.2
9.8
uome j
5.9
8.1
6.8
9.5
9.9
12.0
13.4
14.5
15.4
16.0
15.9
15.8
14.2
12.2
10.3
8.3
6.5
4.7
3.0
1.7
0.95
0.45
0.15
0.20
0.25
0.95
2.2
3.3
4.4
5.7
7.3
8.7
1 LHJUIC
-------
TABLE C5.5. DOME STUDY, 14 JULY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen.concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8
Dome 9 Dome 10
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
26.5°C
27°
27°
28°
28°
29°
29°
29.5°
30°
30°
30.5°
4.2
5.5
6.9
7.1
7.4
7.8
8.4
8.6
8.6
8.6
8.5
4.5
5.2
6.4
7.7
8.9
10.1
11.3
11.9
12.4
12.8
12.2
4.8
5.3
6.7
8.0
8.9
9.3
9.5
9.8
9.2
8.7
7.6
4.8
5.7
7.0
8.4
9.6
10.8
12.0
12.6
13.0
13.0
12.2
4.9
6.1
7.9
9.7
11.9
12.4
13.5
14.0
13.9
13.4
12.2
4.9
5.6
6.5
7.1
7.5
7.9
8.5
9.3
8.3
7.8
6.8
5.0
4.4
3.4
2.5
1.85
1.35
0.9
0.65
0.35
0.2
0.2
Dome 5 - control (measured concentration 0.16 ppb)
Dome 6 - MeOH (measured concentration 0.17 ppb)
Dome 7-1 ppb atrazine (measured concentration 1.47 ppb)
Dome 8-10 ppb atrazine (measured concentration 6.38 ppb)
Dome 9 - 100 ppb atrazine (measured concentration 71.85 ppb)
Dome 10- 1000 ppb atrazine (measured concentration 761.90 ppb)
71
-------
TABLE C5.6. DOME STUDY, 18-19 JULY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved .oxyijen concentrations in parts per million)
Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
(dissolved .ox
Ambient
Time
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
0100
0200
0300
0400
0500
0600
0700
Temp.
25.5°C
25.1°
25.2°
26.0°
26.8°
26.9°
27.4°
27.8°
28.2°
28.2°
28.4°
28.5°
28.5°
28.5°
28.0°
28.0°
28.0°
28.0°
28.0°
27.5°
28.0°
28.0°
27.5°
27.5°
DO
3.2
4.1
5.0
6.1
6.9
7.1
7.5
8.2
9.3
10.6
10.8
11.0
10.4
9.9
8.5
7.4
7.8
7.0
6.5
6.0
5.6
5.1
4.3
3.6
Dome
3.2
3.5
4.2
5.3
5.9
6.9
8.2
9.7
10.6
11.2
11.0
10.2
9.2
7.3
5.6
3.25
2.30
2.00
0.70
0.30
0.25
C.20
0.15
0.15
3.3
3.6
4.7
6.0
6.7
7.3
9.2
10.4
11.4
12.0
12.0
11.3
10.2
8.3
6.5
4.05
2.70
1.10
0.35
0.15
0.20
0.10
0.10
0.15
3.4
3.9
4.8
5.8
6.4
7.3
8.6
9.8
10.5
11.1
11.0
9.9
8.3
6.4
4.0
1.70
0.50
0.35
0.10
0.10
0.10
0.10
0.10
0.10
3.4
3.5
4.2
5.0
5.2
5.8
6.7
7.4
7.9
7.9
7.5
6.2
4.4
2.45
0.65
0.10
0.10
0.05
0.05
0.05
0.05
0.05
0.05
0.10
3.3
3.8
4.5
5.2
5.4
5.7
6.4
6.9
7.1
6.9
6.5
5.4
4.3
2.95
1.60
0.50
0.10
0.05
0.05
0.05
0.05
0.05
0.05
0.05
3.5
2.9
2.2
1.6
1.1
0.7
0.4
0.3
0.3
0.2
0.2
0.2
0.20
0.15
0.10
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Dome 5 - control (measured concentration = 0.21 ppb)
Dome 6 - MeOH (measured concentration - £0.10 ppb)
Dome 7-1 ppb atrazine (measured concentration » 0.81 ppb)
Dome 8 - 1C ppb atrazine (measured concentration • 4.85-ppb)
Dome 9 - 100 ppb atrazine (measured concentration » 61.44 ppb)
Dome 10- 1000 ppb atrazine (measured concentration - 709,29 ppb)
72
-------
TABLE C5.7. DOME STUDY, 29 JULY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
27.2
26.9
26.9
27.4
27.7
28.0
28.5
28.8
29.2
29.5
29.6
29.4
29.1
28.7
28.3
28.1
3.06
5.03
5.10
4.70
5.35
6.24
7.00
7.05
7.50
7.95
8.10
8.14
8.62
7.76
7.40
6.62
2.08
1.70
1.55
2.10
3.25
3.75
4.93
5.56
6.48
7.00
7.06
6.39
4.86
3.28
2.30
1.01
1.71
0.91
0.57
0.62
1.02
0.95
1.22
1.22
1.26
1.02
0.67
0.16
0.055
0.05
0.13
0.20
2.24
1.55
1.38
1.90
3.14
3.59
4.73
5.33
6.22
6.65
6.39
5.50
3.55
1.87
1.01
0.20
1.17
1.03
0.65
0.72
1.12
1.02
1.34
1.33
1.33
0.98
0.44
0.04
0.042
0.04
0.1C5
0.140
1.96
1.26
1.08
1.69
2.95
3.30
4.33
4.80
5.58
5.64
5.08
3.83
1.83
0.67
0.20
0.14
1.99
1 .12
0.73
0.73
1.04
0.93
1.56
1.13
1.18
0.83
0.43
0.03
0.03
0.06
0.09
0.07
Dome 5 - control (measured concentration =• 0.24 ppb)
Dome 6-51% Shade (measured concentration - 0.24 ppb)
Dome 7 - MeOH (measured concentration - 0.58 ppb)
Dome 8 - MeOH 51% (measured concentration » 0.58 ppb)
Dome 9-10 ppb atrazine (measured concentration « 6.51
Dome 10-10 ppb 51% (measured concentration - 9.87 ppb)
ppb)
73
-------
TABLE C5.8. DOME STUDY, 30 JULY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
27.0
27.1
27.6
27.6
28.0
28.4
28.7
29.0
29.2
29.4
29.3
29.1
28.8
3.38
4.70
6.20
6.72
6.80
7.75
8.16
8.55
8.60
8.50
8.68
7.82
6.86
3.14
3.23
4.06
4.92
5.96
7.07
8.40
8.27
9.18
9.65
9.07
8.20
6.23
2.82
2.13
1.83
1.77
1.94
2.22
2.48
2.38
2.56
2.42
1.58
1.16
0.45
3.33
3.77
4. GO
5.54
6.55
7.57
8.65
8.49
9.08
9.46
8.69
7.29
5.07
2.69
1.89
1.48
1.30
1.42
1.61
i.73
1.70
1.88
1.55
1.09
0.90
0.30
3.10
3.09
3.47
4.05
4.65
5.42
6.22
6.58
6.95
7.26
6.45
4.49
2.75
2.74
2.29
1.90
1.81
1.86
2.09
2.85
2 ,,40
2.34
2, .07
1.50
0.85
0..78
Dome 5 - control (measured concentration = 0.18 ppb)
Dome 6 - control 51%
Dome 7 - Me OH
Dome 8 - MeOH -512
Dome 9-10 ppb atrazine (measured concentration » 6.30 ppb)
Dome 10- 10 ppb - 51% (measured conrentration = 7.86 ppb)
-------
TABLE C5.9. DOME STUDY, 31 JULY 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0830
0930
1030
1130
1230
1330
1430
1530
1630
1700
1800
1900
2000
2100
,..-, . *
27.6
27.5
27.6
28.0
28.2
28.6
29.0
29.3
29.7
_
30.2
30.1
29.8
29.6
2.88
5.37
6.82
7.60
8.00
8.73
9.34
9.32
9.75
10.2
10.0
9.7
8.7
8.3
2.71
2.93
3.79
4.67
5.51
6.17
6.74
6.98
7.80
6.6
6.3
5.0
3.1
1.65
2.94
4.34
6.83
8.24
8.85
9.05
9.49
9.78
11.95
9.4
*
*
*
*
2.94
3.09
4.00
5.38
8.20
5.69
6.12
5.88
-
4.9
4.0
1.7
0.2
0.15
2.56
2.20
1.21
1.15
1.22
1.02
0.96
1.98
-
0.7
0.35
0.25
0.2
0.15
2.54
2.55
3.02
3.67
4.16
4.39
4.62
5.95
4.42
3.1
2.4
4.20
0.15
0.10
1.42
0.71
0.58
0.46
0.47
0.34
0.22
1.18
1.70
0.15
0.15
0.20
0.10
0.05
Pome 5 - control
Dome 6 - control 517= shade
Dome 7 - MeOH
Dome 8 - MeOH 51% shade
Dome 9-10 ppb atrazine (measured concentration 6.51 ppb)
Dome 10 - 10 ppb - 51% shade (measured concentration 8.81 ppb)
* discontinued due to pump failure
75
-------
TABLE C5.10. DOME STUDY, 1 AUGUST 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Some 5 Dome 6 Dome 7 Dome 10
0900 28.0 4.02 4.37 4.03 4.46 4.10
1000 27.9 6.38 4.67 3.47 4.81 3.14
1100 28.2 6.73 5.05 3.28 5.41 2.97
1200 28.6 7.08 5.71 3.38 5.80 2.80
1300 29.0 7.15 6.17 3.28 5.98 2.41
1400 29.3 7.80 6.66 3.15 6.03 1.91
IbOO 29.8 7.64 7.03 3.09 6.00 1.59
1600 29.8 7.40 7.37 2.83 5.73 1.10
Dome 5 - control
Dome 6 - control 30%
Dome 7-10 ppb
Dome 10-10 ppb 30%
No water samples taken.
76
-------
TABLE C5.ll. DOME STUDY, 12 AUGUST 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Tetnp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
28.9
28.5
28.8
29.1
29.6
29.7
30.8
30.1
30.4
JO. 5
30.5
30.3
5.90
6.59
6.97
7.05
7.34
7.39
8.00
8.94
9.56
8.75
8.58
8.24
5.39
4.03
3.53
3.54
3.77
3.95
4.25
4.98
5.07
4.61
3.32
1.70
5.72
4.44
3.83
3.46
3.24
3.00
2.91
3.38
3.40
2.82
1.86
1.06
5.71
4.71
4.40
4.39
4.73
5.00
5.29
6.18
6.56
6.27
5.20
3.54
5.67
4.10
3.29
2.94
2.85
2.66
2.68
3.14
3.09
2.55
1.59
0.97
5.69
4.65
4.32
4.39
4.69
4.82
5.09
5.93
6.09
5.66
4.43
2.89
5.41
3.38
2.38
1.85
1.63
1.39
1.47
1.92
1.96
1.58
0.84
0.38
Dome 5 - control
Dome 6 - control 30%
Dome 7-1 ppb atrazine (measured concentration = 0.63 ppb)
Dome 8 - Ippb 30% (measured concentration = 0.36 ppb)
Dome 9-10 ppb (measured concentration = 7.41 ppb)
Dome 10-10 ppb 30% (measured concentration = 6.93 ppb)
77
-------
TABLE C5.12. DOME STUDY, 13 AUGUST 1980, GUINEA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
26.1
26.7
28
28
28
28
28
28.5
29
29
29
29
28.5
3.14
3.90
9.0
11.0
9.20
12.6
11.4
13.6
14.5
18.9
18.4
15.5
16.4
2.89
3.46
5.60
6.20
7.70
8.80
10.2
11.25
12.40
13.0
11.4
9.1
5.1
3
2
2
3
3
4
5
5
5
5
3
2
0
.12
.50
.80
.00
.35
.50
.10
.7
.9
.3
.55
.00
.75
3
2
5
6
7
9
10
12
13
13
12
10
7
.09
.60
.60
.20
.20
.80
.40
.9
.6
.8
.4
.6
.5
3.
2.
3.
3.
3.
4.
5.
6.
6.
5.
3.
2.
0.
16
64
00
00
70
90
40
0
0
5
73
00
75
3.18
3.63
5.80
6.30
7.60
9.80
10.80
12.65
13.20
13.4
12.0
10.1
7.00
3.19
3.22
4.30
4.45
4.85
5.80
6.30
6.90
6.80
6.50
5.10
3.25
1.35
Dome 5 - control
Dome 6 - control 30%
Dome 7-1 ppb atrazine (measured concentration = 0.72 ppb)
Dome 8 - 1 ppb 30% (measured concentration = 0.69 ppb)
Dome 9-10 ppb atrazine (measured concentration • 6.58 ppb)
Dome 10-10 ppb 30% (measured concentration =6.35 ppb)
A
-------
TABLE C5.13. DOME STUDY, 14 AUGUST 1980, GUINEA MARSH STATION, SET IN 20STERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0330
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
2130
2230
2330
•"-
27
27
27
27.5
28
28.5
29
29
29
29
29
29
29
28
28
28
4.20
6.20
8.20
8.20
8.50
10.00
9.8
10.20
10.10
9.20
8.20
7.90
7.70
10.00
9.00
8.40
3.40
3.40
3.30
3.60
4.35
5.15
6.00
5.50
5.60
4.80
3.80
2.60
1.40
0.25
0.25
0.20
3.20
2.50
1.90
1.80
2.30
2.80
3.00
2.55
2.20
1.90
1.05
-
0.55
0.30
0.25
0.20
3.65
3.50
3.40
3.80
4.30
4.95
5.15
4.40
3.60
2.75
1.30
-
0.15
0.15
0.15
0.10
3.40
2.70
2.20
2.20
2.90
3.50
3.75
3.00
2.30
1.55
-
-
0.15
0.10
0.10
0.10
3.50
3.00
2.80
3.10
3.70
4.25
4.45
3.35
2.50
1.40
-
-
0.10
0.10
0.10
0.05
3.30
2.40
1.80
1.65
2.05
2.45
2.50
1.70
1.70
0.55
-
0.20
0.05
0.10
0.10
0.05
Dome 5 - control
Dome 6 - control 30%
Dome 7-1 ppb atrazine (measured concentration = 1.02 ppb)
Dome 8-1 ppb 30% (measured concentration = 0.85 ppb)
Dome 9-10 ppb atraziue (measured concentration =• 7.18 ppb)
Dome 10- 10 ppb 30% (measured concentration * 6.54 ppb)
79
-------
TABLE C5.14. DOME STUDY, 15 AUGUST 1980, i! iNhA MARSH STATION, SET IN ZOSTERA
MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Ambient
Time Temp. DO Dome 5 Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
26.9
27
27.5
28
28
28.5
29.0
29.0
29
28.5
5.60
5.40
7.20
7.80
8.00
8.20
8.30
8.30
8.00
7.80
4.65
4.20
4.30
4.70
5.40
5.70
5.80
6.00
5.70
5.50
4.30
2.90
2.10
1.80
1.65
1.55
1.35
1.20
0.90
0.60
4.65
3.40
2.90
2.60
2.55
2.20
1.80
1.35
1.05
0.85
4.25
2.60
1.60
1.00
0.95
-
0.80
0.40
0.25
0.10
4.35
2.70
2.00
1.75
1.80
1.75
1.35
1.00
0.85
0.55
4.25
2.70
1.70
1.20
1.05
0.85
0.60
0.35
0.30
0.05
Dome 5 - control
Dome 6 - control 30%
Dome 7-1 ppb atrazine
Dome 8-1 ppb 30%
Dome 9-10 ppb atrazine
Dome 10-10 ppb 30%
No water samples taken.
80
-------
TABLE C5.15. DOME STUDY, 8 SEPTEMBER 1980, GUINEA MARSH STATION, SET IN
ZOSTERA MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
(dissolved ox;
Ambient
Time
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
Temp.
25
25
25
25.5
26.0
27.0
27.5
27.5
27.5
27.5
26.5
26
DO
3.2
3.5
4.2
5.4
6.8
8.2
9.5
9.7
9.8
8.0
7.5
7.3
Dome 5
3.4
3.5
4.3
5.0
6.4
7.6
8.6
8.5
8.5
7.7
6.3
4.8
3.2
2.9
3.0
3.3
3.8
4.6
5.2
4.9
4.3
3.6
2.8
1.4
3.8
3.7
4.4
5.1
6.6
7.8
8.7
8.6
8.3
7.1
5.7
3.9
3.4
3.2
3.0
3.8
4.2
4.7
5.0
4.3
3.7
2.6
1.7
1.0
3.6
4.0
4.6
5.5
6.7
8.6
9.4
9.6
7.0
5.5
3.2
3.4
4.4
4.8
5.8
6.9
7.9
7.7
7.3
6.2
4.8
3.0
Dome 5 - control
Dome 6 - control 20% (measured concentration » 0.13 ppb)
Dome 7-1 ppb atrazine
Dome 8-1 ppb 20% (measured concentration =0.60 ppb)
Dome 9-10 ppb atrazine
Dome 10 - 10 ppb 20% (measured concentration • 6.59 ppb)
61
-------
-X*
TABLE C5.16. DOME STUDY, 9 SEPTEMBER 1980, GUINEA MARSH STATION, SET IN
20STERA MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Time
Ambient
Temp. DO
Dome 5
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
25
25.5
25.5
26
26
?6.5
27
27
27
27
4.2
4.2
3.7
3.8
4.6
4.5
4.6
4.1
4.1
4.6
3.7
3.5
3.3
3.6
4.6
5.5
6.2
6.8
6.7
5.4
Dome 6
3.7
3.2
2.8
2.9
3.4
3.7
4.1
4.3
3.8
2.8
Dome 7
3.8
3.6
3.4
3.7
4.6
5.5
6.4
6.9
6.5
5.3
Dome 8
3.8
3.4
3.0
3.1
3.6
3.9
4.2
4.2
3.6
2.6
Dome 9 Dome 10
3.7
3.6
3.3
3.6
4.4
5.2
5.8
5.7
4.6
3.1
3.8
3.4
3.1
3.2
3.7
3.9
4.3
4.3
3.8
3.0
Dome 5 - control
Dome 6 - control 20%
Dome 7-1 ppb atrazim
Dome 8 - 1 ppb 20%
Dome 9-10 ppb atrazine
Dome 10- 10 ppb 20%
No water samples taken.
-------
TABLE C5.17. DOME STUDY, 10 SEPTEMBER 1980, GUINEA MARSH STATION, SET IN
ZOSTERA MARINA COMMUNITY
(dissolved oxygen concentrations in parts per million)
Dome 6 Dome 7 Dome 8 Dome 9 Dome 10
(dissolved ox
Ambient
Time
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
Temp.
24.5
25.0
25.0
25.0
24.5
24
24
24
24
23.5
23.5
23.5
DO
4.2
4.3
4.4
4.3
4.0
4.0
4.2
4.3
4.3
4.2
4.0
3.9
Dome 5
4.2
3.5
3.3
2.8
2.5
1.8
1.8
1.6
1.4
1.2
0.80
0.60
4.3
3.6
3.2
2.9
2.4
2.0
1.7
1.3
1.0
0.8
0.40
0.20
4.2
3.6
3.2
2.6
2.2
1.5
1.3
1.0
0.7
0.3
0.20
0.20
4.0
3.3
3.0
2.8
2.5
1.9
.8
0.60
0.50
4.1
3.4
3.1
2.6
2.3
1.9
1.7
1.5
1.2
1.0
0.70
0.40
4.1
3.3
2.9
2.3
1.8
1.3
1.0
0.8
0.20
0.20
Dome 5 - control
Dome 6 - control 20% (measured concentration = £0.10 ppb)
Dome 7-1 ppb atrazine
Dome 8-1 ppb 20% (measured concentration * 0.26 ppb)
Dome 9-10 ppb atrazine
Dome 10 - 10 ppb 20% (measured concentration • 6.79 ppb)
83
A
-------
TABLE C5.18. DOME STUDY, 11 SEPTEMBER 1980, GUINEA MARSH STATION, SET IN
ZOSTERA MARINA COMMUNITY
(dissolved oxy
Ambient
Time
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
Temp.
21.5
22
23
23.5
23.5
22.5
22.8
23.5
24.5
25
24
DO
3.9
4.2
4.5
5.0
5.5
5.1
5.1
6.0
6.7
7.1
6.3
Dome 5
4.0
3.9
3.9
4.2
4.6
5.0
5.6
6.2
7.3
7.6
7.6
4.0
3.8
3.4
3.4
3.3
3 3
3.1
3.6
4.1
4.0
3.6
4.1
3.8
3.6
3.7
3.9
4.2
4.4
4.9
5.6
5.8
4.8
4.0
3.6
3.2
3.2
3.2
3.3
3.2
3.6
4.0
4.0
3.4
4.0
3.9
3.7
4.2
4.4
5.0
5.4
6.1
6.9
7.2
6.6
3.2
3.3
3.5
3.2
3.8
4.2
4.0
3.4
Dome 5 - control
Dome 6 - control 20% (measured concentration » £0,10 ppb)
Dome 7-1 ppb atrazine
Dome 8 - 1 ppb 20% (measured concentration = 0.59 ppb)
Dome 9-10 ppb atrazine
Dome 10- LO ppb 20% (measured concentration - 7.77 ppb)
84
-------
O
00
2 S I
• H »H
N H
0
X ki •
4J »
O « >
tn
I *-•
9\ &.I
CM O. I-
. o
>.OCM
•§-'•
4-1 T>
(0 C (N
3 O
O — i O
£. S
4; O —
.M i-i in
u C
O en
(n O (D
3 -<
0) -^ J3
ki o a
i
en «->
x e •
x o «
o u u
TJ 00 O
4) C T3
o ^ •*
« O hi
10 > <8
•H e TS
Q -H ;
U
MI
N39XXO Q3ATOSSIO
35
-------
o
CO
41 •
C -a
3 V
I 41
«-l C
CM -H
14
- eg •
X u *-»
•O *J 41
3 <0 S
u ^
•o o
^^^H
£ I
<0 T3 »-
Wi C JC
nj iq
X (X CM
3 O in
O —.
x -e
•^ - c
~* «
41 O
.3 is';
« c E
o ••»
-------
-~/r.
N30AXO Q3ATOSSIO
60
87
-------
:\
I
in
01
§
•o
U)
I*
IB
x
nj
V
*r*
O
11
c
NCM
S'B
to
01
.O Ml
O. (Q
0-H
—• O
O
<0
o
Ifl
I
o
o
in
O
<\J
o
o
o
o
•<
o
CD
.-*'"" T
-^
O
^
o
(O
^- —
a
o
0 O
9 to
o
rg
O
-s
o
O 0
2 u
3
a
•-« k.
u a,
C CC
o
(J
§
X Tl
E C
- N
"o u -
4j fj ^
5 J3 —<
u &H
OC
con
— O >
_> O
o ~*T
> -o
C c I-
•fH {5 £
uidd N39AXO a3ATOSSia
U
01
k4
3
00
-------
o
o o
in (M
o o
- o
o
o
o
10
v
• H
. NfM
o *J
— i ..£»
.3- O
1.1
I
O
«M
^> 0>
•o c
g -H
W N
a CD
U 4-1 4)
i -.a
•O ^ H
O.
£ a •
a n
Wi O >
C8 —
CO 41 >—
41 C
C -H 1/1
• *4 N •
3 eg ui
O fc
u 10
a 41
• i—<
^ js rfa
a a ra
i- an
£ !~ 2
O 4)
iJ C DC
O
eg u
3
a o
JC T3
^« u
B 4) 4)
a 6 e
a -H
^ « N
-* , c e
X O ft •'•
O O O.H
a.
•O 60
« c o »
> -H o >
i > o
O 1-4 _4»*
« O I
CO > T3
•H C C kl
a -H m £
uidd N3QAXO Q3A10SSIO
4)
M
DO
-------
1
0
«
1
o
o
0
(M
o
o
o
o
00
1
o
K
1
o
1
o
1
0
*T—
o
o
1
o
-\e
in
O
N39A.XO 03MOSSIQ
u
60
90
-------
8-3
' X O
•-I O
(»N C
CM <"J
£
X 41
T> B
g-5
n O
v • .
(Q 4J '
co o ^
3 g1
41 •
Q ^^
• 41
«-» B
n
t-i «
3 ~
O »•«
10 ••-(
U H
4J
<0 •
-
—I £
- o
N30AXO 03A10SSICJ
to c j: CM
3 O « O
(0 O
4) TJ H e
> 01 "9 —'
B £ C CM
Q. 00 *f^ •
O. N in
%_/ . (8
e "3 u e
41 lJ 10 ...I
>-l > - 09
O — '•x >
n o *c
m > _ o
•H c in. Q
Q -H ^» ^x
in
O
at
60
• i-4
u<
91
-------
GO
O r- •
00 O m
ON I-
—I 4-1 (0
e w
X O -J
t-i o .a
3 CO
c o
a*
{- i
z o
o u
0 2
_^-— -^, — "J
<5^ v
"^>\ ^x
^ ^v^ \
~^\x
^
o o Q o ooqo
c\i_o<*>ot>Ku'10
uidd u'nnivfi n^Ainccin
ppbATRAZINE \
>l% SHADE \
2
XA
X"
vc
o
*
UJ
a
4
UJ Z
Z (A
3 #
(E m
i-
< ->
0
S «
a »-
1 §
- v
*?
« * 5
% 4 s?
> ^— s
' t 5
f f u
>^
i ''*
w
O
O
«
o
o
.i
Q
* K
eg
.8
«
.8
«
•?.
-I
g
•«
o
.0
o
-s
o
o o o o
(0 CM —
§J= (0
•
0) <0
^ -J> >
3 ^v 0.
OK B^<
£ -* 1
S 4) ^ '" *
3 B — -001
O -H O U 1
z iJ I- -o B
— 4J CO
at c ,-z CM
Ul 3 O 0) O
2 ca O
H 4) T3 C B
> 01 <0^
^ «J 1) ^
B X c CN
Ou « -H •
cx N in
>-_^ •« fQ
B O u B
0) ki co CO
go 'a'*
o 0. B
•e MO H
Hi C —i
> -H •
i > - 0)
0 -" <~- >
at O »*
« > -^ o
.* B in Q
O
V
u
92
-------
o *-• •
OO O U1
Ov hi
— " 4-1 »1
C HI
X O r-1
i-l O .Q
a a
— C
en «
0» 01
B1"
V
-o as
0)
•o
(0 •
.C at
09 01
o
hi x^
4-1 *t
C —i
O in
o >-»
— 0) •
O C ^
C •!-( 01
ONE
j; as .H
4-1 w. H
• « u
-s B i
O 0)
I- T5 B
4-1 CO
e .e CM
o w o
u
•O 60
•o e S
ai a —'
•o
CD ai r^
£ e cs
(0 -H •
N m
. (0
e
01
00
x
e ^ x^
o a oi
o a. e
• 1-4
BOO H
C —
— > -co
O ^ <-N >
(0 O K
01 > —i Q
N30XXO Q3ATOSSia
"
00
• •4
bu
93
-------
O 01 --»
co c u
O\ •<-! B
-^ N -H
3 A
< a—.
at
-« M
O JC
TJ T) I
3 W B
at ca
Ul
z
M
4
£
O <
c
1- *
I 1
V
v\
\
x>
i
i ~i i i i 1 i 1 —
OQO°OOOO
«j-O0>a>r'-«>in
Q
K»
UJ
0 Q
s i
*>
u» u
Q Z
x <
w or
, h-
o <
« a
5 S
8 2
« /
< /
* /
< X
« /
*f
*j
. x'
^ -4
"
«fl -C
«s u c
OJ 4J CO
.5 "^
0.--I
H
• o
X™^ ^H •
W 01
IJ • >
3 ^^
0 »« Q
£ 0 O
(U O
E — —
•H O •
.u ki in
4J
CO C (0
3 O V
w y — i
4) TJ W H
-ils
a a
a. w
c o ai
01 U, OS
00 4^
X C
S S «
o
B ro
O *H N«X
U
60
>•»!
tu
94
-------
O —i
00 —'
t»<
—> o> in
c
u -H in
n N 01
3 <8 —i
eo u .a
3 4J «9
< « H
M
ja o
-H D. "
o.
hi
X—i 01
T3 •*
3 -O Oi
u oi M
••g
JJ .C •
S ID a
o «
"S i
JZ
to
U O) ^"^
w e K
s: -H o
N ro
W <8 >^
01 ki
C 4J V •
•H to c ^->
9 -H 01
U X> N B
O. ro --H
O. U H
• 4J
»~. —i IB •
go CD
I* -.0 >
3 x-x a
O K G—I
£ O I
v^ fO O U
^-- —i j:
V
E i-l -OfM
••< O 4) I
*J h T3 S
u n>
w c jc D <8 ^
•O
'-v 10 0) O\
B J= C CM
D. 0) -H .
a. N in
^x *• IQ
^H Ij T3
B O <-> c
t-i «J cs
60 4J
x c .a ^
x o a. w
o a a B
N30AXO a3Aiossia
SCO H
C —i
•o
V
"-I > • OJ
O -. ^v >
CO O K
10 > o tD
•^ c _/
O
01
t»
• H
(h
95
-------
>r^
N
4-1 —H O
o
(M
o
o
o
00
o
(O
o
10
o
(VJ
Li
o
8
2 3
? £
s s
to
hi
3 -
O i
N39AXO (J3A10SSICI
e
a;
t«
x
X
o
•o
«J
f-4
o
01
01
-,4 r*.
N 4;
<8 g
»- —
<»1 u H
v-« (D
—i .O en
O a >
u a
U —I
C O I
O —« p
•> £
•o
T5 WCM
tl) TJ I
t3 «9 S
«> £
JS « tN
a> o
•o
• C 00
•-I
-------
•*.
V
o •—>
00 H
9> O
-
g
01 V
3 C to
60-fl -4
3 N •
-H CD
JS .fl
a. o
4J -H O
0) U
•o
tl 11 14
§T3 01
CD vu
•a £ at
09 Ofi
£
a -
i- at •
a C oo
X •-« tu
N P
IS <8 5
41 W T3
C «
• H CJ <^
ti)
B
3 »-v
O »<
£ O
0)
B <-"
•H o
a c
3 O
oo o
>
a
•o
01 01
N39AXO 03A10SSIO
c o
4) t*
60 u
x e
So
o
•a t>o
at c
So
•rf B
a •<•*
U
01
u
M
C 00
« e
01
c —
a E
to
97
-------
o -~>
00 »*
o
••^ fo ^5
>-' s
4J x^
(0 41
3 c . G."«
T3 O.H
4J —< O
CO 4J
•a
ft) OJ W
•O J5 tU
05 06
I «
««
fO fsJ ISJ
.8
o
.8
N
<.
K
o A
00
<3
4
o
o
o
o>
o
CD
O
<0
I
o
I
O
I
p
10
I
Q
5 C 09
S -
a.
fj-i t- a.
~ « -
— L: m c c^ i
3 5
co o
v -a
> tu
.8
e A
a. a
a
N39AXO Q3MOSSIQ
c p
0) hi
DO U
X C
X O
o u
TJ 60
oj e
So
a >
•H e
a •-"
•a
cues
•o l
ra B
CO CM
O
I M
e
C CM
• rl CO
N •
ca m
u
AJ -Q
to c
10
J3
D.'^
Ou 41
O -H
— H
in
U
53
to
-------
I4J
? 14J
is< Z
E 5
< £
a «
s i
O a
•• ..'C
^•".^-••-'r^
^^^r''
S9 . -^--^ ^^
f ^f /'"
< f« / •*
r i s
V 4^ <
^o^C-.^ tei
•^Si. ^^ ^
^^Co,
^S^&ik
Ot\
_i
0
IT
H*
z
o
o
^^t
•*
INE/20% SHADE
% SHADE
E/20% SHADE
N O ±
< CM M
(E \ <
t- -i
I §1
-•* 4 •
"" •' ' X
•« ,•"
« ,-•''
« x"
•X
V
\
^**
s*
•^^
X
i i i i i i
o Q o a o Q
O 0^ CD ^" ^ ^
"™
/»
1
o
V
>
^^
C
o
CJ
00
c
>
o
>
c
m
o
00
-*
NM'
01
c
N
CO
eg
r^
a.
a.
o
^H
•
Ol
co
^
-«
1
hi
TJtN
•o
eg
n
•O
eg
01
c
• f4
tt
4J
CO
^3
a.
a
o
—>
*~*.
^4
o
cs
^^
'g
CM
O
J
en
in
e
CO
V
• *4
H
0)
>
§
"— '
U
Ol
60
b.
99
-------
V
1) tC
e — i
•H •
H if,
CO
Q u 01
Ol <« — <
u A
a .o ra
4) CL S-H
en a
o
>> Ol 0>
•o -o <*-
£5,8
o
uj o
Q r«
4
%O UJ
0s Q
0 <
*~ (/) UJ
uj . , a
UJ z W v° 4
z 1 - « *
^ ^ M O
* a 3 \ >5
_i tr £ * _, «?•
0 t- 4 t (0 0
t£ < *
r^ •^***^ /^*
\. r" *'
cv*^ %i
^^ v
^"^to. ^^M
^^^fe. X^
~V^*
Ji ^^
^^•^
s^^
tr^
^f
&
o
-8
o
.8
0
.8
.1
CSI ^^,
•1 I
-S ^
t 2
—
-§
™"
o
.0
!2
.§
Q
o
o»
o
ooo oooooooo
o ^ u)«f^r K>ra —
-*
^
C, 1.
C 01 •
•i-i -< C *•*
5 .'S 1
*^v co '<^
»< W H
• O *->
/-v CN CO •
CO ^» OJ
U J3 >
3^0.
O O Q^«*
j: I- l
C — < JS
!U O
E 0 -JON
Ol CO
CO TJ £ «S
3 CO CO O
CO .£
U - CO -^
^ O 01 -*
B w> e n
a. u -^ •
a. c N u-i
x^ O CO
U U T3
e u c
01 00 CO CO
60 C
X -M ^ V-*.
x > a. 01
O r-* O. B
o •*•*
T3 > 0 H
0) C — i
— l "to
o -^> >
a O M
co co o Q
-M o 01 a
Q _ ^f *^
u
-------
t
6 I- «
01 «•• Ol
w W •-*
a jo
u ja CB
to a H
&
o o
UJ
a
S _8
en uj
O
o? <
o *
IM ^
•^ UJ 0
UJ 2 o^
Z ^i 0
N, < ~
1 *
-i 5 i- j
0 P < 0
a < cr
5-! 5
s_sis
\U V
>p> f «
/x>«*
/v* ^
/*« KT
/'* - v
r^ ^VX N£
fjf <] V\ ^ UJ
-^i' .- u < o
y"*« -^ ? *?
/S ' ^ a x
+Sj^ jr' < >f>
*Ss' *- ' 5 So-0
^-w' -= °S
^^&-* — o
•^ 0
ft
o
.8
*i
o
.8
o
-8
M
.8
cst
««
g 5
• * o
I
8 I
•^ a
o •-
-8
o
.g
0
- O
0
. 0
at
0
1 I 1 I
o o o o o
V »r» rv —
wdd N39XXO 03A10SSIO
>, Oi 01
U | «
4-J JC CC
n 09
01 • •
§0) «
c 01
T3 — B
N 0
JC «1 TJ
ID U
« w «7
r o
CO O. ^-*
01 O-
C 01 •
O -'N e
^ CO •«
M U f-i
• O u
~* tv n
(U -^ W
1. JO >
3 — a.
o o r i -
f. U 1
N-/ U O >•
01 O
E u *ocs
u T3 -0 B
a> re
3 <0 e 6
> - w ^_-
^\ *O 01 in
E W c <*>
D. " •- •
a e N m
^ o a 01
o — a B
o — •
•o > o (-
i— - •>
o * ^** ^
« o
-------
tl OO
41 f
•§ :
41 «-
0) A 10
CO O. H
O.
— O
UJ
o
O O
- o
UJ u
z a
£ <
o
03
-[-
O
O
(C
o
o
i
O
i >
0 O
I\J _
8
M ^»
O o
Z I
i:
•"00 N30AXO 03A10SSIO
- TJ U
X 41 41
T3 •« <*«
3 <0 4)
*J .C «
a a
41 * •
§ s«
•O —«
N _
^: « -o
to b
U *J ^~
n 10 M
X O
J3 PM
TO a^
4) a
C 41 •
— —. e **.
S •-« 41
U - N B
X-N OJ .-,
M V- H
• O «-i
>-• «s «J •
« •*-* 01
^ JO >
3—0.
o o a~*
£ b I
^* *J O U
C — £
41 O
6 O -OCM
•- 41 I
" TJ TJ B
II (0
IB -o ^: «N
3 m « o
« x:
W. « « -~s
^. o 41 vO
•B u c «»>
Q. u .^ .
O. C N U-l
^* O C
41 Ml W «
00 C •
>* ••* xt ***•
X > Q. 01
O —. O. B
O •-<
•a > o H
41 e -H
O -<-« >
*> O M
on 00 O Q
•^ O> CM O
in
U
3
60
102
-------
TABLE C5.19. GUINEA MARSH DOME SET, 29-30 MAY 1980
Date Time*
5/29/80 1730
1830
1930
2030
2130
2230
2330
5/30/80 0030
0130
0300
0430
0530
0630
0730
0830
0930
1030
1130
1230
1330
1430
1530
1630
Control
-200.00
-366.67
-233.33
-400.00
166.67
-300.00
-133.33
-200.00
-266.67
-133.33
266.67
- 66.67
200.00
166.67
166.67
533.33
200 . 00
66.67
1500.00
200.00
166.67
200.00
0.0
rag 0^ in
MEOH
-200.00
-333.33
-333.33
-566.67
-100.00
-566.67
-333.33
66.67
-266.67
- 83.33
166.67
-133.33
33.33
- 33.33
200 . 00
400.00
333.33
66.67
1100.00
300.00
333.33
200.00
0.0
2 hr-1
100 ppb**
Light
133.33
-233.33
-200.00
-400.00
-166.67
-200.00
-400.00
0.0
-400.00
- 83.33
100.00
-200.00
-133.3^
-133.33
166.67
133.33
160.67
- 66.67
533.33
100.00
100.00
66.67
33.33
100 ppb**
Dark
-366.67
-200.00
-266.67
-366.67
-166.67
-300.00
-100.00
-200.00
-400.00
- 33.33
100.00
-200.00
-100.00
0.0
0.0
66.67
66.67
-133.33
66.67
0.0
0.0
0.0
66.67
* Time - midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
103
-------
TABLE C5.20. GUINEA MARSH DOME SET, 23-24 JUNE 1980
Date
6/23/80
6/24/80
Time*
0730
0830
0*30
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
2130
2230
2330
0130
0230
0330
0430
0530
0630
0730
0830
Control
133.33
266.67
766.67
200.00
266 67
200.00
33.33
66.67
- 66.67
-300.00
-233.33
-566.67
-333.33
-500.00
-466.67
—
233.33
-1450.00
- 50.00
-116.67
- 16.67
33.33
0.0
16.67
116.67
mg 02
MEOH
200.00
366.67
866.67
266.67
400.00
266.67
100.00
33.33
-133.33
-300.00
-366.67
-666.67
-466.67
-600 . 00
-500.00
-
-377.78
-150.00
- 66.67
33.33
- 16.67
50.00
- 33.33
83.33
233.33
m-2 hr-1
10 ppb**
200.00
200.00
566.67
333.33
333.33
233.33
1*0.00
33.33
-266.67
4466.67
-S400.00
-533.33
-500.00
-433.33
-500.00
—
-238.89
-116.67
- 33.33
- 50.00
0.0
16.67
33.33
233.33
366.67
100 ppb**
- 33.33
266.67
33.33
0.0
0.0
- (.6.67
33.33
-100.00
-400.00
-300 . 00
-433.33
-300.00
-300.00
-150.00
- 16.67
—
0.0
- 16.67
0.0
0.0
0.0
16.67
0.0
0.0
0.0
* Time - midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
104
-------
TABLE C5.21. GUINEA MARSH DOME SET, 25-26 JUNE 1980
mg 05 m~2 hr'1
Date
6/25/80
6/26/80
Time*
0830
0930
1030
1130
1230
1330
1430
1530
1630
IV 30
1830
1930
2030
2200
2330
0030
0130
0230
0400
0530
0630
0730
Control
266.67
166.67
300.00
366.67
266.67
133.33
-100.00
-2S6.67
-266.67
-433.33
-500.00
-466.67
-433.33
-300.00
-166.67
0.0
0.0
0.0
25.00
- 83.33
0.0
16.67
MEOH
200.00
166.67
266.67
266.67
366.67
100.00
166.67
-333.33
-133.33
-400.00
-433.33
-366.67
—
-408.33
-600.00
- 66.67
0.0
- 33.33
- 50.00
- 16.67
216.67
-133.33
1 ppb**
233.33
133.33
233.33
366.67
300.00
100.00
0.0
-266.67
-266.67
-466.67
-533.33
-500.00
- 83.33
-383.33
- 50.00
0.0
- 33.33
0.0
0.0
0.0
50.00
0.0
10 ppb**
200.00
167.67
233.33
36C.67
300.00
200.00
-100.00
-300.00
-266.67
-433.33
-466.67
-466.67
-366.67
-258.33
-200.00
- 33.33
- 33.33
16.67
8.33
- 33.33
716.67
-700.00
100 ppb**
133.33
-100.00
100.00
100.00
66.67
- 66.67
-166.67
-333.33
-433.33
-366.67
-333.33
- 50.00
-266.67
- 16.67
- 16.67
0.0
- 16.67
0.0
125.00
-250.00
416.67
-416.67
1000 ppb**
100.00
-200.00
- 66.67
- 66.67
0.0
-166.67
-133.67
-300.00
-266.67
-166.67
-166.67
-216.67
—
- 25.00
- 16.67
16.67
0.0
0.0
0.0
- 33.33
16.67
66.67
* Time - midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
105
-------
TABLE C5.22. GUINEA MARSH DOME SET, 15-16 JULY 1980
mg 02 in hr~*
Date Time*
7/15/80 0730
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
1830
1930
2030
2130
2230
2330
7/16/80 0030
0130
0230
0330
0430
0530
0630
0730
0830
0930
1030
1130
1230
1330
Control
733.33
-433.33
900.00
133.33
700.00
466.67
366.67
300 . 00
200.00
- 33.33
- 33.33
-533.33
-666.67
-633.33
-666.67
-600.00
-600.00
-566.67
-433.33
-250.00
-166.67
-100.00
16.67
16.67
233.33
416.67
366.67
366.67
433.33
533.33
466.67
MEOH
500.00
0.0
566.67
333.33
400.00
533.33
366.67
333.33
266.67
66.67
—
-266.67
-466.67
-533.33
-566.67
-533.33
-633.33
-516.67
-433.33
-350.00
-266.67
-183.33
- 66.67
0.0
233.33
333.33
283.33
333.33
350.00
416.67
333.33
1 ppb**
566.67
66.67
633.33
366.67
266.67
466.67
366.67
300,00
200.00
66.67
- 66.67
-400.00
-416.67
-566.67
-733.33
-566.67
-600.00
-566.67
-433.33
-316.67
-250.00
-133.33
- 16.67
16.67
183.33
300 . 00
250.00
283.33
333.33
383 . 33
300.00
10 ppb**
266.67
533.33
500.00
600.00
466.67
433.33
433.33
266.67
266.67
- 66.67
-133.33
-466.67
-600.00
-633.33
-633.33
-600.00
-666.67
-583.33
-450.00
-383.33
- 83.33
- 16.67
0.0
0.0
100.00
200.00
150.00
200.00
200.00
283.33
250.00
100 ppb**
433.33
100.00
233,33
233.33
300.00
200.00
66.67
133.33
0.0
-100.00
-266.67
-466.67
-600.00
-566.67
-500.00
-416.67
-216.67
- 66,67
- 33.33
0.0
- 16.67
0.0
0.0
0.0
0.0
16.67
0.0
0.0
16.67
0.0
0.0
1000 ppb**
133.33
-733.33
533.33
-1100.00
- 66.67
-166.67
-66.67
- 83.33
-116.67
33.33
-266.67
- 66.67
- 33.33
0.0
0.0
0.0
0.0
0.0
- 16.67
0.0
0.0
0.0
0.0
- 10.00
10.00
0.0
0.0
0.0
0.0
16.67
0.0
* Time - midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
106
-------
TABLE C5.23. GUINEA MARSH DOME SET, 14 JULY 1980
mg 0; m"2 hr"1
Date Time* Control MEOH 1 ppb** 10 ppb** 100 ppb** 1000 ppb**
7/14/81 0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
233.:^
400.00
433.33
400.00
400.00
400.00
200.00
166.67
133.33
-200.00
166.67
466.67
433.33
300.00
133.33
66.67
100.00
-200.00
-166.67
-366.67
300.00
433.33
466.67
400.00
400.00
400.00
200.00
133.33
0.0
-266.67
400.00
ftOO.OO
600.00
733.33
166.67
366.67
166.67
- 33.33
-167.67
-400 . 00
400.00
133.33
200.00
133.33
133.33
200.00
266.67
-333.33
-166.67
-333.33
-200.00
-333.33
-300.00
-216.67
-166.67
-150.00
- 83.33
-100.00
- 50.00
0.0
* Time • midpoint of hour, e.g. 0700-0800
** Nominal dissolved atiazine concentrations
107
-------
TABLE C5.24. GUINEA MARSH DOME SET, 18-19 JULY 1980
mg 0? m~2 hr~l
Date
7/18/80
7/19/80
Time*
0730
0830
0930
1030
1130
1230
1330
1430
1530
1630
1730
183C
1930
2030
2130
2230
2330
0030
0130
0230
0330
0430
0530
Control
100.00
233.33
366.67
200 . 00
333.33
433.33
500.00
300 . 00
200.00
- 66.67
-266.67
-333.33
-633.33
-566.67
-783.33
-316.67
-100.00
-433.33
-133.33
- 16.67
- 16.67
- 16.67
0.0
MEOH
100.00
366.67
433.33
283.33
200.00
633.33
400.00
333.33
2C3.00
0.0
-233.33
-366.67
-633.33
-600.00
-816.67
-450.00
-533.33
-250 . 00
- 66.67
16.67
- 33.33
0.0
16.67
1 ppb**
166.67
300.00
333.33
200.00
300.00
433.33
400.00
233.33
200.00
- 33.33
-366.67
-533.33
-633.33
-800.00
-766.67
-400.00
- 50.00
- 83.33
0,0
0.0
0.0
0.0
0.0
10 ppb**
33.33
233.33
266.67
66.67
200.00
300.00
233.33
166.67
0.0
-133.33
-433.33
-600.00
-650.00
-600 . 00
-183.33
0.0
- 16.67
0.0
0.0
0.0
0.0
0.0
16.67
100 ppb**
166 67
233.33
233.33
66.67
100.00
233.33
166.67
66.67
- 66.67
-133.33
-366.67
-366.67
-450.00
-450.00
-366.67
-133.33
- 16.67
0.0
0.0
0.0
0.0
0.0
0.0
1000 ppb**
-200.00
-233.33
-200.00
-166.67
-133.33
-100.00
- 33.33
0.0
-33.33
0.0
0.0
0.0
- 16.67
- 16.67
.- 16.67
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
108
-------
TABLE C5.25. GUINEA MARSH DOME SET, 29 JULY 1980
Date Time*
7/29/80 0730
0830
0930
1030
1130
1230
1330
1430
1530
1630
1800
1930
2030
2130
Control
-126
- 50
183
383
166
393
210
306
173
20
-111
-1036
-326
-430
.67
.00
.33
.33
.67
.33
.00
.67
.33
.00
.67
.67
.67
.00
Control
51Z
Shade
-266.67
-113.33
16.67
133.33
- 23.33
90.00
0.0
13.33
- 80.00
-116.67
- 85.00
- 36.67
26.67
23.33
MEOH
tng 02
m-2
MEOH
512
hr-1
10 ppb**
Shade
-230
- 56
173
413
150
0
580
296
143
- 86
-148
-1210
-286
-270
.00
.67
.33
.33
.00
.0
.00
.67
.33
.67
.33
.00
.67
.00
- 46
-126
23
133
- 33
106
- 3
0
-116
-180
- 66
- 0
21
11
.67
.67
.33
.33
.33
.67
.33
.0
.67
.00
.33
.67
.67
.67
-233
- 60
203
420
116
343
156
260
- 40
-126
-541
-386
-156
- 20
.33
.00
.33
.00
.67
.33
.67
.00
.00
.67
.67
.67
.67
.00
10 ppb**
51Z
Shade
-290
-130
0
103
- 36
210
-143
16
-116
-113
- 66
10
10
- 6
.00
.00
.0
.33
.67
.00
.33
.67
.67
.33
.67
.00
.00
.67
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
109
-------
TABLE C5.26. GUINEA MARSH DOME SET, 30 JULY 1980
Date Time*
7/30/80 0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1745
Control
30.00
276.67
286.67
346.67
370.00
443.33
- 43.33
303 . 33
156.67
-193.33
-580.00
Control
51 Z
Shade
-230.00
-100.00
- 20.00
56.67
93.33
86.67
- 33.33
60.00
- 46.67
-280.00
-280.00
MEOH
13.33
410,00
313.33
336.67
340.00
360.00
- 53.33
196.67
126.67
-256.67
-933.33
mg 02 m"2
MEOH
512
Shade
-266.67
-136.67
- 60.00
40.00
63.33
40.00
- 10.00
60.00
-110.00
-153.33
-126.67
I hr-l
10 ppb**
- 3.33
126.67
193.33
200.00
256.67
266.67
120.00
123.33
103.33
-270.00
-1306.67
10 ppb**
512
Shade
-150.00
-130.00
- 30.00
13.33
80.00
253.33
-150.00
- 20.00
- 90.00
-190.00
-433.33
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
110
-------
TABLE C5.27. GUINEA MARSH DOME SET, 31 JULY 1980
Date Time*
Control
Control
512
MEOH
Shade
7/30/80 0800
9900
1000
1100
1200
1300
1400
1500
1545
1630
1730
1830
1930
73
286
293
280
1073
-663
80
273
-800
-100
-433
1233
-2350
.33
.67
.33
.00
.33
.33
.00
.33
.00
.00
.33
.33
.00
466
830
470
203
66
146
96.
723
-1700
-
—
.67
.00
.00
.33
.67
.67
67
.33
.00
-
"~
50
303
460
940
-836
143
- 80
-217
-
-300
-766
-500
- 16
.00
.33
.00
.00
.67
.33
.00
.80
-
.00
.67
.00
.67
mg 03 m~'
MEOH
512
Shade
-120.00
-330.00
- 20.00
23.33
- 66.67
- 20.00
340.00
-284.40
—
-116.67
- 33.33
- 16.67
- 16.67
I hr-l
10 ppb** 10 ppb**
512
Shade
3
156
216
163
76
76
443
-510
-880
-233
600
-1350
- 16
.33
.67
.67
.33
.67
.67
.33
.00
.00
.33
.00
.00
.67
-236
- 43
- 40
3
- 43
- 40
320
173
-1033
0
16
- 33
- 16
.67
.33
.00
.33
.33
.00
.00
.33
.33
.0
.67
.33
.67
* Time " midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
111
-------
TABLE C5.28. GUINEA MARSH DOME SET, 1 AUGUST 1980
Date Time*
8/1/80 0830
0930
1030
1130
1230
1330
1430
Control
100.00
126.67
220.00
153.33
163.33
123.33
113.33
mg 05
Control
30Z
Shade
-186.67
- 63.33
33.33
- 33.33
- 43.33
- 20.00
- 86.67
m-2 hr-1
10 ppb**
116 67
200.00
130.00
60.00
16.67
- 10.00
- 90.00
10 ppb**
30%
Shade
-320.00
- 56.67
- 56.67
-130.00
-166.67
-106.67
-163.33
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
112
-------
TABLE C5.29. GUINEA MARSH DOME SET, 12 AUGUST 1980
mg 03
Date Time*
Control
Control
302
1 ppb**
Shade
8/12/80 0900
1000
1100
1200
1300
1600
1500
1600
1700
1800
1900
-453
-166
3
76
60
100
243
30
-153
-430
-540
.33
.67
.33
.67
.00
.00
.33
.00
.33
.00
.00
-426
-203
-123
- 73
- 80
- 30
156
6
-193
-320
-266
.67
.33
.3?
.33
.00
.00
.67
.67
.33
.00
.67
-333
-103
- 3
113
90
96
296
126
- 96
-356
-553
.33
.33
.33
.33
.00
.67
.67
.67
.67
.67
.33
m-2 hr-1
1 ppb**
30%
Shade
-523.33
-270.00
-116.67
- 3C.OO
- 63.33
6.67
153.33
- 16.67
-180.00
-320.00
-206.67
10 ppb**
10 ppb**
302
Shade
-346
-110
23
100
43
90
280
53
-143
-410
-513
.67
.00
.33
.00
.33
.00
.00
.33
.33
.00
.33
-676
-333
-176
- 73
- 80
26
150
13
-126
-246
-153
.67
.33
.67
.33
.00
.67
.00
.33
.67
.67
.33
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
113
-------
TABLE C5.30. GUINEA MARSH DOME SET, 13 AUGUST 1980
«ng 02
Date Time*
Control
Control
302
1 ppb**
Shade
8/13/80 0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
190
713
200
500
366
466
350
383
200
-533
-766
-1333
.00
.33
.00
.00
.67
.67
.00
.33
.00
.33
.67
.33
-206
100
66
116
383
200
200
66
-200
-583
-516
-416
.67
.00
.67
.67
.33
.00
.00
.67
.00
.33
.67
.67
-163
1000
200
333
866
200
833
233
66
-466
-600
-1033
.33
.00
.00
.33
.67
.00
.33
.33
.67
.67
.00
.33
m-2 hr-1
1 ppb**
302
Shade
-173.33
120.00
0.0
233.33
400 . 00
166.67
200.00
0.0
-166.67
-583.33
-583.33
-416.67
10 ppb**
10 ppb**
302
Shade
150
723
166
433
733
333
616
183
66
-466
-633
-1033
.00
.33
.67
.33
.33
.33
.67
.33
.67
.67
.33
.33
10
360
50
133
316
166
200
- 33
-100
-466
-616
-633
.00
.00
.00
.33
.67
.67
.00
.33
.00
.67
.67
.33
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
114
-------
TABLE C5.31. GUINEA MARSH DOME SET, 14 AUGUST 1980
rag Oj
Date Time*
Control
Control
302
1 ppb**
Shade
8/14/80 0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
0.00
- 33.33
100.00
250.00
266.67
283.33
-166.67
33.33
-266.67
-333.33
-400.00
-400.00
-383.33
0.00
- 16.67
-233.
-20T.
- 33.
166.
166.
66.
-150.
-116.
-100.
-283.
- 83.
—
- 83.
- 16.
- 16.
33
00
33
67
67
67
00
67
00
33
33
33
67
67
- 50
- 33
133
166
216
66
-250
-266
-283
-483
-191
-
0
33
- 50
.00
.33
.33
.67
.67
.67
.00
.67
.33
.33
.67
-
.00
.33
.00
m-2
hr-1
1 ppb**
30X
10 ppb*'
Shade
-233
-166
0.
233
200
83
-250
-233
-250
-156
-
- 16
0
0
.33
67
00
.33
.00
.33
.00
.33
.00
.56
-
.67
.00
.00
-166
- tt
lo"
200
183
66
-366
-283
-366
-
-144
-
0
0
- 16
.67
.07
.00
.00
.33
.67
.67
.33
.67
-
.44
-
.00
.00
.67
ppb**
*
j!:,, '
-300.
-200.
- 50.
133.
133
16.
-266.
0.
-383.
—
- 58.
- 50.
16.
0.
- 16.
£
00
00
00
- •»
•»
) 1
67
i7
00
33
33
00
67
00
67
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine ctncentrations
115
-------
TABLE C5.32. GUINEA MARSH DOME SET, 15 AUGUST 1980
Date Time*
8/15/80 080C
0900
1000
1100
1200
1300
1400
1500
1600
Control
-120.00
33.33
133.33
233.33
100.00
33.33
66.67
-100.00
- 66.67
Control
30Z
Shade
-466.67
-266.67
-100.00
- 50.00
- 33.33
- 66.67
- 50.00
-100.00
-100.00
mg 02
1 ppb**
-300.00
-166.67
-100.00
- 16.67
-116.67
-133.33
-150.00
-100.00
- 66.67
m-2 hr-1
1 ppb**
302
Shade
-550.00
-333.33
-200.00
- 16.67
—
- 25.00
-133.33
- 50.00
- 50.00
10 ppb**
-550.00
-233.33
- 83.33
16.67
- 16.67
-133.33
-116.67
- 50.00
-100.00
10 ppb**
30*
Shade
-516.67
-333.33
-166.67
- 50.00
- 66.67
- 83.33
- 83.33
- 16.67
- 83.33
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved acrazine concentrations
116
-------
TABLE C5.33. GUINEA MARSH DOME SET, 8 SEPTEMBER 1980
Date
9/8/80
Time*
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
Control
33.33
266.67
280.00
400 . 00
400.00
333.33
- 33.33
0.00
-266.67
-400 . 00
-566.67
Control
20%
Shade
-100.00
33.33
120.00
142.86
266.67
200.00
-100.00
-200.00
-233.33
-266.67
-466.67
mg 02
1 ppb**
- 33.33
233.33
280.00
428.57
400.00
300.00
- 33.33
-100.00
-400.00
-466.67
-600.00
m-2 hr-1
1 ppb**
202
Shade
- 66.67
- 66.67
320.00
114.29
166.67
100.00
-233.33
-200.00
-366.67
-300.00
-233.33
10 ppb**
133.33
200.00
360.00
342.86
633.33
266.67
66.67
- 66.67
-333.33
-466.67
-500.00
10 ppb**
20Z
Shade
66.67
333.33
160.00
285.71
366.67
333.33
- 66.67
-133.33
-366.67
-466 . 67
-600.00
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
117
-------
TABLE C5.34. GUINEA MARSH DOME SET, 9 SEPTEMBER 1980
Date Time*
9/9/80 0800
0900
1000
1100
1200
130J
1400
1500
1600
Control
- 66.67
- 66.67
100.00
333.33
300.00
233.33
200.00
- 33.33
-433.33
Control
20Z
Shade
-166.67
-133.33
33.33
166.67
100.00
133.33
66.67
-166.67
-333.33
mg 02
1 ppb**
- 66.67
- 66.67
100.00
300.00
300.00
300.00
166,67
-133.33
-400.00
m-2 hr-1
1 ppb**
20Z
Shade
-133.33
-133.33
33.33
166.67
100.00
100.00
0.00
-200.00
-333.33
10 ppb**
- 33.33
-100.00
100.00
266.67
266.67
200.00
- 33.33
-366.67
-500.00
10 ppb**
20Z
Shade
-133.33
-100.00
33.33
166.67
66.67
133.33
0.00
-166.67
-266.67
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
118
-------
TABLE C5.35. GUINEA MARSH DOME SET, 10 SEPTEMBER 1980
rag 02
Date Time*
Control
Control
202
1 ppb**
Shade
9/10/80 0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
1800
-233
- 66
-16b
-120
-200
0
- 66
- 66
- 66
-133
- 66
.33
.67
.67
.00
.00
.00
.67
.67
.67
.33
.67
-233
-133
-100
-200
-114
-100
-133
-100
- 66
-133
- 66
.33
.33
.00
.00
.29
.00
.33
.00
.67
.33
.67
-200.
-133.
-200.
-160.
-200.
- 66.
-100.
-100.
-133.
- 33.
0.
00
33
00
00
00
67
00
00
33
33
00
m-2 hr'l
1 ppb**
20Z
Shade
-233.33
-100.00
- 66.67
-120.00
-171.43
- 33.33
-100.00
-100.00
-133.33
- 66.67
- 33.33
10 ppb**
10 ppb**
20Z
Shade
-233
-100
-166
-120
-114
- 66
- 66
-100
- 66
-100
-100
.33
.00
.67
.00
.29
.67
.67
.00
.67
.00
.00
-266
-133
-200
-200
-142
-100
- 66
-100
- 66
- 33
0
.67
.33
.00
.00
.86
.00
.67
.00
.67
.33
.00
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
119
-------
TABLE C5.36. GUINEA MARSH DOME SET, 11 SEPTEMBER 1980
Date
9/11/80
Time*
0800
0900
1000
1100
1200
1300
1400
1500
1600
1700
Control
- 33.33
0.00
100.00
133.33
133.33
200.00
200.00
366.67
100.00
0.00
Control
202
Shade
- 66.67
-133.33
0.00
- 33.33
0.00
- 66.67
166.67
166.67
566.67
-733.33
mg 02
1 ppb**
-100.00
- 66.67
33.33
66.67
100.00
66.67
166.67
233.33
66.67
-333.33
m-2 hr-1
1 ppb**
202
Shade
-133.33
-133.33
0.00
0.00
33.33
- 33.33
133.33
'33.33
0.00
-200.00
10 ppb**
- 33.33
- 66.67
166.67
66.67
200.00
133.33
233.33
266.67
100.00
-200.00
10 ppb**
20Z
Shade
- 66.67
-166.67
- 33.33
33.33
66.67
-100.00
200.00
133.33
- 66.67
-200.00
* Time - Midpoint of hour, e.g. 0700-0800
** Nominal dissolved atrazine concentrations
120
-------
SECTION 6
GREENHOUSE STUDIES
INTRODUCTION
The greenhouse experiments were designed to accomplish longer term
exposures of Zostera marina to atrazine than w« could accomplish with field
experiments.
The results of the field surveys, particularly the Severn River survey,
indicated herbicides were carried into the estuary by runoff and subsequently
be found subsequently in th«> water over SAV beds for periods of several days.
In an effort to evaluate the potential effects a long-term, low-level exposure
to atrazine might produce in Zobtera, we undertook a series of three week
chronic dosing experiments. The three week period was selected to be longer
than we believed a typical exposure in tne lower Chesapeake Bay might be.
(This was based on sampling in the Severn River system, and a general
assumption about flushing times in other subestuaries.) The dosage levels
were the same as those used in the dome studies. The range of concentrations
was suggested by our 1978 survey of concentrations in the lower Chesapeake
Bay.
METHODS
Zostera marina plants collected from the lower York River were exposed to
atrazine in a flow through dosing system. T'.ie aboveground morphology of the
plants was monitored in an effort to detect effects of the exposure.
The dosing apparatus (see Figure 6.1) utilized 37.8 liter glass aquaria
as test chambers. Water from the York River at Gloucester Point
(approximately 20 ppt salinity) was pumped into the greenhouse and filtered by
10 Gaflo (trade name) polypropelene bag filters. Filtered water was
collected in a storage tank from which it was continuously pumped to a
constant level header tank. Calibrated siphons delivered the water to
individual glass mixing chambers. Stock solutions of atrazine (in either
methanol or acetone) were also delivered to the mixing chambers by a
peristaltic pump. The water with the added atrazine was then delivered by
glass tubing to a glass flow splitter which was designed as a secondary header
tank. Calibrated siphons delivered the water-herbicide to duplicate dosing
tanks for each test concentration. Water entered the top rear of each dosing
tank and exited from the bottom front by a constant prime siphon. The
peristaltic pump was connected to a float switch which prevented dosing if
diluent water flow ceased.
121
-------
01
0)
4J
CO
4J CO
>> <0
iH
C CO
o a
01
4-1 CQ
U ^
•H C
O.
-------
The entire system was allowed to fill with the appropriate atrazine
concentration prior to initiation of an experiment. The flow rates of all
calibrated siphons and the toxicant delivery rates were monitored daily.
Maximum and minimum water temperatures were also monitored daily, although no
effort is made to regulate them. No effort was made to regulate the
photoperiod. A 50% shading cloth was placed over the greenhouse durii.g summer
months to prevent photoinhibition and to help minimize inside air
temperatures.
Each experiment utilized fifteen individually potted plants per dosing
tank. The plants were placed in small peat pots filled with subtidal mud.
Plants were measured at the beginning of each experiment and, depending on the
experiment, at weekly intervals or at the end of the dosing. Each plant was
measured for the height of the longest leaf, total number of leaves, and total
number of shoots. A shoot was defined as any leaf group separated by more
than one centimeter from other groups. All plants were harvested, rinsed, and
divided into aboveground and belowground tissues at the termination of an
experiment. Plant tissues were pooled for each dosage tank and subjected to
analysis for atrazine content.
The data from each experiment was analyzed by calculating a mean percent
change in the test parameters over the course of the experiment. The mean
percent change was based on the initial measurements, and calculated as
XA -X0 (100)
mean percent change •
X0
where: XQ * mean of parameter at time zero
XA = mean of parameter after time A
This index varies between +1COZ and -100% with 0 indicating no change over the
time interval. A -50% value indicates a 50% reduction in the parameter
measured. Twenty one day LCso's and EC50's for each test parameter were
determined by the graphic method. Dead plants were not included in the data
analyses used to determine the £€50*8.
RESULTS
The data for experiments conducted in 1980 are reported in the appendix
to this section, Tables D6.1 through 06.28. Experiment 5 (Tables D6.14
through D6.20) is omitted from further data analysis because of the
unacceptable mortality in control treatments.
A twenty-one day LC5Q was determined by the graphic method to be 0.07
mgl~* (70 ppb). Data from the experiments were pooled for this analysis (see
Figure 6.2).
The effect of atrazine on plant height, number of leaves, and number of
shoots is graphed in Figure 6.3. Again data from all the experiments were
pooled for this analysis. The £050 for atrazine effects on plant height was
0.41 mgl~l (410 ppb). The £€50 for atrazine effects on number of leaves was
0.06 mgl~l (60 ppb). The £650 for atrazine effects on number of shoots was
123
-------
100 -i
90-
80 -
70-
» so^
>•
t son
_i
<
I 40-
z
o 30-1
UJ
a.
20-1
Y= 14.5+ 6.65
r = 0.74
LC50 = 0.072 mg/l
/
CONTROL
I l I I ~~
0.0001 0.001 0.01 O.I
ATRAZINE CONCENTRATION (mg/l)
1.0
Figure 6.2. Graph of percent Zostera mortality in test chambers vs. atrazine
concentration. Linear regression line is plotted,
by extrapolation using the regression equation.
124
,.^
determined
-------
r"~l
1 | *JI 0| 1 1 1 1 1
1 1 '
(0
JJ ll I
e ~
0) 0) O
s e
00 3
CO C
01
E •
U Ol
•H C
o .
01
I
e
•o •
c «-»
CO 01
c
z .*
O 01
r~ *
Ol
>-
60
01
la
.Q
O
01
e c
•H O
i—I -H
CO
C CO
O CU
•H Vl
CO 60
-H 73 CO 01
•H 01 )-i
01
01
ui 73
u
z c
8 -S
U 0) 4J
oc o
3 *
T3
c c
O (0
O r;
UJ
z
N
CO 01
u u
C t-
01 i-l
U U
B V^"
o
O 4J
Q) 00
C -H
0)
N £
OC.C
0) CO
>-. CO
•a i
01 4J
£ o
ca -a
co
,-g
T3 18
CO CO
01
CO i-l
01 M
>- G
CO CO
3 -H
cr M
ca 4->
O
O
O
O
£ CO
a
« •
w o>
0)
*U 00 CO
3 01 4-1
i-t > O
U CO o
d o) js
O
CM
I
O
I
o
CO
I
U > t-t iH M
vO
01
SiN3W3«nSV3W Oldl3WOHdaOW X33M 33dHl Nl 39NVHO !N30«3d
125
-------
0.27 mgl""1 (270 ppb). Confidence limits foi these values have not been
developed due to the highly variable nature of the morphometric data.
The effects of the six atrazine concentrations on the morphological
parameters through time are graphed in Figures 6.4, 6.5 and 6.6. Mean height
of the Zostera plants was decreased 502 during the test period by only the
highest concentration, 1.0 mgl"1 (1000 ppb). All concentrations except the
control and the 0.1 mgl"1 (100 ppb) level produced negative slopes for linear
regression lines fitted to the data. The 0.1 mgl'1 (100 ppb) data produced a
regression slope of 0.314. This positive slope appears to be caused by the
marked reduction in height recorded on day 16. The only clearcut effect of
atrazine on plant height was achieved by the 1.0 mgl"1 (1000 ppb)
concentration which produced a 50% reduction in mean height within
approximately 14 days.
The effect of atrazine on the mean number of leaves per plant was similar
to the effects on mean height. Linear regression analyses demonstrated that
the 1.0 mgl"1 concentration (1000 ppb) produced the most marked effects,
resulting in a 50% reduction in number of leaves within approximately 13 days.
Other concentrations also produced a decrease in leaf number, according to the
regression analysis, but none effected a 50% decrease in numbers within the
test period.
The number of shoots per plant was reduced markedly by only the 1.0 mgl"1
(1000 ppb) concentration of atrazine. A 50% reduction in the mean number of
shoots was produced within approximate 16 days according to the regression
analysis. Other concentrations of atra me effected little change in the
number of shoots during the test period.
In each of the morphometric data sets, it is ->ificant to note that the
control treatment resulted in an increase in mean -ght, mean number of
leaves, and mean number of shoots over the course of the test period. Tests
of the statistical significance of differences between control treatments and
atrazine treatments are inconclusive, however, because of the highly variable
nature of the morphometric data.
During the experiments reported here, the minimum water temperature
averaged 22.2*0 and the maximum water temperature averaged 27.3*C.
Temperature usually fluctuated between these values daily.
DISCUSSION
The long-term dosing experiments reported here clearly demonstrated that
atrazine at high concentrations (approximately 1 mgl"1 or 1000 ppb) can reduce
the productivity of Zostera marina. The regression analysis utilized in this
study, suggests major changes in morphology of Zostera may be produced by
long-term exposure to atrazine concentrations as low as 0.06 mgl"1 (60 ppb).
We believe the twenty one day EC50 are actually much higher than this value.
A review of Figure 6.3 indicates that the trend established by the data points
appears sigmoidal rather than linear. We have attempted more sophisticated
analyses of the data, unfortunately, we do not have enough data points at high
concentrations to allow a more rigorous determination of the twenty one day
126
-------
CM
CM
00
o
o
It
o<
O>l*» —
Eoo
->:
a
(D
00
a>
CM
X
00 (O
i
o
(O
e (* en
5??
o ., ,.
I
O
00
CJ
(O
CM
CM
CM
o
-1*-
i 1
J.H5I3H Nl 3DNVHD !N30y3d
s §
CM
_O
— 00
(a oc
> e
o oc
tH O
O •
M —
4-1
C T3
O C
U CO
O
OJ—
4-J C I
J3 -H i— I
00 N OC
•H ce E
01 |^
4S 4J —
CO •
C O
•H U-
O -
41 —
oo ca i
c c «H
n o oc
£ •* e
u M O
C -u •
V C O
U OJ
t- o •
oj c—
a o i
O i-H
a o
to i- o
la O •
o
-------
00
10
-X.O
00
10
I
X
CM
I
X
'
o
o
O.Q q
c i o
0
I
00
CM
CM
_l~
M*
oil •:
m
O
CM
> i i
I ill
— CMO
O I I
O „ .,
o
00
• t\J
(O
t\J
CVJ
CM
o
• t\J
_ 00
<"
-2!
/
/
/
/
1 ' 1 « ' 1 ' 1 '
3 § 8 00 C
1 1 1 •
_N
_g
-00
I
o
o
oo
• E
F-l
O O
U '
O T3
V-4 O C
O ^ CO
CO 01 •
cu c —
> -H I
CO N i-l
0) CO OO
pH ^ E
4-1
<4-i CO •-»
o o
14-1 •
vi o o
01
XI CO »
e c-i
3 O I
C -H i-l
4-1 OO
C CO E
E
in
M3
cu
M
s,
S3AV31 JO 838WnN Nl 39NVHO !N3Dd3d
128
-------
o
n
4J
O
o
U)
o
o
o
• *-x
o —
• l-t
rH 60
2 e
o —
u
E
- 00
>o
•
vO
ID
Id
DO
S100HS dO
Nl 39NVHO iN30«3d
129
-------
LC5Q or EC5o's. The data points on Figure 6.3 suggest the £€50*8 for the
morphometric parameter are all somewhere over 0.1 mgl~l (100 ppb). The same
suggestion is made by the data points used to establish the l*C$Q (Figure 6.2).
With either interpretation of the data several observations are
significant. First, the effective concentrations of atrazine for production
of a 50Z decrease in selected morphological parameters are much higher
concentrations than either of our survey programs found in Bay waters.
Additionally, these experiments exposed Zostera to atrazine concentrations for
longer periods of time than we believe occur in natural conditions. Finally,
our experiments do not indicate whether the effects of atrazine exposure
persist after Zostera plants are returned to unstressed conditions.
It is obvious from these studies that efforts to define atrazine £059*8
and LC5o's for Zostera marina will need to focus on concentrations between
0.1 mgl'1 (100 ppb) and 1.0 mgl"1 (1000 ppb). These studies were not designed
that way because our interest was principally in the very low concentrations
found by the survey work to be typical of lower Bay waters. It should also be
obvious from these studies that gross morphology is not sufficiently
responsive to detect effects at the levels of replication we have employed.
Either much larger numbers of plants will be required or an alternative, more
sensitive test parameter must be employed.
130
-------
G SYSTEM
z
CO
g
X
o
g
H
U.
z
Ul
1
o
H
a
u
CO
o
PH
W
U
Z
CO
o
Cs)
U.
o
CO
f-l
EASUREMEN
*
u
as
H
. MORPHOMl
1-4
NO
Q
J
CO
o
a
OQ
01 ^
d* QJ C
C <0
U) -J H
O (0
.C 4-1
in co
>4-l rH
00
i- e
41 «5 <
E f-4 .*
3 C
C (0
„ H
0 OT
00
' 4J
en o
1 a
in M
a CQ
a.
S M 0) C
5|
-------
RAZINE IN A FLOW THROUGH DOSING SYSTEM
H
<<
8
o
LC
cn
O
Id
Z
*— i
oe
:£,
^
06
td
H
O
N
CJL.
0
cn
z
id
ad
cn
H
z.
U
at
E-i
£
O
i
i
CM
a
4J
&
01
a
CO
4J
0
o
CO
M-l
O
l-i
V
XI
J
II
cn
o
00
T i
I U*
14
CO
41
r-l
M-l
O
-4 41
** "i
H 3
Z C
til
3C H
M
Id
>< E"
[d U
C
•**
O
0
.c
ca
4-1
co
41
i— 1
CO
M-l
O
&
•H
V
J=
X
41
C
N
CO
M
U
CO
r-l
eo
o
rl
41
C
N
CO
CO
r-4
"So
1— 1
•
0
41
C
N
CO
M
CO
!—i
00
0
en i i i i i i i i i i i i i i i
C >-) 1 1 1 1 1 1 1 I 1 1 I I I 1 I
CO
H
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
en i i i i i i i i i i i i i i i
Jrf rJ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
ca
H
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
l-l
"
ca m
*4" *
•M »J ON in m oo I-H ^ «^ m in m en oo m CTN \o \o
c
CO
H cMOf»oo-j-inr>-!-iooinr^r^ao»3iONoo
X ocMcMr^oooNOONvomvfNOOcnooo>
CMCMCMr^t^r-ICMiHCMi-li-li-4^«MCMT-4^icMeM4cMcM^
BO •*»
i-t i-i t-* m IH OONOO
c
ca
t^ ^D ^^ f^ ^^ ^0 ^^ ^J CO vO PO ^^ ^^ ^^ f^ ^^ ^i
PM ^^ i^ ^^ 1^ ^J ^D CO ^J (3 *^ OO CO CO ^^ ^3 \O
cneNeNCMCMCMCNCMCNCNCMcncMCMmeN
en CMr-li— Ir-lr-ICMl-lr-ICMr-lrHr-lt-ICXICni— 1
CO
00
•
0
(^
•
J
10
m
o
CM
•
CO
•
8
m
m
m
r-l
>t
^
r^
^
i-t
in
i-H
m
m
1-4
in
m
1-4
in
1-1
C
•o
41
•a
CO
JJ
G
-------
NG SYSTEM
rH
cn
8
X
o
§
06
X
H
3
<
F
£
"
2
M
QC
H
"*
8
o
Id
W
O
§3
W
^,
Z
M
otf
2
A
^C
Ed
H
w
s
Cb
0
C/5
H
35
Id
£
Ed
OS
a
en
•<
s
oc
H
Ed
2C
O
X
ft.
ai
i
CO
NO
o
id
DQ
?S
O
00
1
CM
m
(d
H
^1
Q
CM
^
r-i
Z
Ed
i-l
as
Id
a.
(d
^•s
1 1
O
a
0)
a
01
o
tt
M
3
-H
U-l
o
IJ
0)
XI
E
C
n
J
E
C
"**
4J
O
o
x:
CO
4-1
0]
4)
eg
14-1
o
4J
60
^ ON ON m «^ in NO CO NO NO in ^ NO i-H rH NO CO rH
-^CONOCO ^CM r»r~.r~mcMCMNom
X CONOrH^t "rHON •-^•OOOCMNOONOP** »in
rHCMCMrHOOCMrHmi-HrHCMCMrHi-HCNIrHinrH
CO NO
• • in
W i-ICOi-ICMi-li-4i-« O CM rH • • in
>J inrHrHi-HNO-^NONOONinrHr^inr^NO^COrH
•JrHt^OOr^r^COCMrHi^inNOCMCOCMr*
X ONcoi-4ONr».incooNco^cMCMooooNOON «m
i-H ^>
. . in
m oo
o co . . m
^3 NO rH in in NO rH 00 NO rH 00 in ON «^ in ^9 if> (^| r>H
coONrHcMini^NotMinocMinoNNOcocM
co «n
• • in
t/3 rHi-HrHi-HCMi-HCMrHi-ICMCMrHi-li-ICMrHO'-1
r^ <^
COrH rH
-------
G SYSTEM
z
l^
en
g
X
THROUG
2
*
X
1— 1
z
M
N
S
O
H
Q
en
O
H
w
z
H4
OS
*c
-C
s
H
en
o
N
It.
0
en
z
sg
U3
K
J^
en
tft
§
O
rH
OS
E-
hj
32
o
X
CL,
OS
§
-a-
•
SO
Q
O
00
1
ON
1
vO
•»
S
^
CM
•4it
H
Z
i— i
tt
Ct)
CL,
X
w
4J
o
Q.
M
4)
Q.
shoots
o
01
a
c
n
en
.^
o
• ^
01
a
to
>
01
1-4
0
)»4
01
JO
s
3
II
g
C
o
o
.e
4J
<8
r-l
,^4
ca
>4-i
o
4J
JZ
oo
0)
jt
it
s
01 C
5 S
CO
u
CO
oo
1 <
c
(D
H
CQ
rsg
0) C
c
rH
^s^
oo
e
rH •<
<5 J^
ca
H
«
*±^
OJ C
C CO
"H H
N
a
u
u
a
rH
•*s^.
00
e
o <
o ^
c
H
i-i cocococM-a-coiiii'iii
co m o CM
• • • -31 • 00
X rH O O\ • CM •
CMrHi-icni-ir-i i i i i i i i
en i i i i i i i i i i i i i i
i-J i i i i i i i i i i i i i i
K
1 1 1 1 1 1 1 1 1 1 1 1 1 1
\o ^**
hJ Ov f^> -^ CO Os -^ f^» CO P>> -^ ^O r* ^^ «»J
CNeyvrH^OCMi-lrMCMrHrHCMCMrHfM
Cn rHrHrHCMrHrHCMrHrHrHrHCMrHCO
CM rH -voooi^oin
rH
f\
•
r-l
r-l
!>'
vO
rH
CM
CO
CO
•
f*»
1-4
CO
M
in
1-4
ir\
•
c^
00
m
CM
1 X
vO
•
0
rH
m
•
o
m
CO
.
m
vo
•
o
f"4
«n
•
-*
m
o
o
CO
^
CO
r—
•
O
f**»
*
^J
•
CO
D
vO
vO
-
m
^^
m
rH
m
'"
m
i— i
m
in
rH
•*
""*
^
rH
-.
rH
m
m
rH
in
rH
C
TJ
11
.^4
"O
to
C
n)
/-N
1
134
-------
SYSTEM
o
Z
C/J
g
u
3
0£
=,
3
3
f*4
^«
Z
1— (
Ed
Z
N
^j*
o2
L^
^J
O
H
a
(d
O3
o
X
[d
^t
Z
M
as
2
2
(d
H
CO
s
U,
O
c/]
£~l
Z
Id
OS
V]
u
£
CJ
H4
H
SH
o
a,
Qg
i
in
vO
a
3
oa
o
ao
1
CO
CM
vO
.•
(d
Q
co
H
Z
52
M
0£
Cu
X
U
/— N
4-1
O
O.
P
01
a
CO
4J
2
O
n
0
M
0)
,0
e
g
II
yj
*«
^
0
a.
^4
CU
a
CO
01
CO
01
i-H
M-l
O
M
01
,0
c
1
»J
.»
B
u
C
•H
4J
O
O
co
4J
CO
i-l
^
14-
O
£
00
•H
0)
£
II
X
PQ
^
c
ca
jj H
c
•1^
N
2
4J
Q
^
t>o
B <
O
•-1 ^!
d g
H
CQ
X
C
S 5
•f^
N
ft
Id
4J
«8
l-l
M
E <
O X
C
O CO
Ei
J^
« e
B to
-4 t-l
N
(1)
P
CD
^.
e
O <
O
O £
n)
H
i-i ro
• . m
•» r^
co CM . • in
hJ vo to <"^ iO vo cr\ in *— * Q
X cooooicooo^p^ocMvoe^cMOOoo • »/•»
»-^ «-^ ^^
-------
z
w
H
CO
co
O
3
h.
<
Z
W
CM
O.
w
o
M
.!-i
<
Q
H
o
< a) « «
Q § 5
CMCMCMCMi^CM'-''-*'-''-''— I CM CM «^ CM 1^
(U
co
"g
£ II O «
SB cMOOOvOi-ii-ICM'-i'>>rOOs>-00
-------
Ed
H
CO
CO
O
§
O
o
a
i-i
u
a
03
CO rHi—ICNi-li-!i-!«d-f*>«»»3'-*'">»>-1
a) c
52 i^ rx o\ PI
N . • . \O
W
Z
M
M
s
s
o
H
a
u
CO
2
u
H
cn
s
u.
o
CO
H
Z
0
oo
B
g
fi£
U
a,
x
ta
VM
o
m
at
A
c
ii
CO
o
a
v
a.
ll-li-li-li-li-lpgcv»cor»r»r»c»ioovocM 1-1 c\ ^o 1-1
Id
Dfi
CO
<
£
u
02
H
O
a.
OA
%
vO
a
3
03
0
O
.£
in
PQ
eo
4)
II
X
0)
c
N
«
e\j
CO OO
~-» • « m
60 CO i-4i-li-li-4i-(r-ICNCNi-4i-l'-4r-4i-Hr-li-4i-lO'-l
0
•
o
«
H
• •*
i x o e
137
-------
X
w
H
CO
>>
CO
o
z
M
CO
g
THROUGH
S
O
gJ
<
Z
U
z
hH
^
Ofi
f-i
^
g
a
EXPOSE
z
OS
si
OSTERA
N
Cb
O
M
Z
fd
£
s
3
CO
^
(d.
•"•
U
1-4
05
f-
O
(X
CC
s
•
00
ta
*3
CO
O
00
1
1
ON
• •
Cd
H
a
^
atrazine
^4
00
a
§
o
irazine
CO
1-1
E*
O
o
o
o
4)
C
1-1
N
CO
4J
CO
iH
f^
t
0
^
c
CO
H
C
H
«
i^>
S
1
CO
c
ca
H
**•
c
CO
H
• in
i-4i-li-Hi-Hi—l^-li—Ir-li—Ii-Hi-Hi—(i-li—li-lr-IOi—I
o oo
. . (T|
in^sjmin^-^-enenen-*-^-enin-tf«*Oi-'
m oo 01 ON
X in r^ ^ «^
CN |H CN CN
00
m
m
CO l-ll-ll-li-4i-li-4i-4i-li-li-lr-li-li-li-li-ll-IOi-l
O 1*^
• • m
hJ •*mcnen! r>! " ' '*
1-icNi-ieNCNeMCNeMi-ii-ii-ieNf-ii-if-iiH.^i-i
• in
CO I—If-lrHi—li-li-Jr-li-l!—Ir-li-li—IrHi—li-li—IOr-(
00 1*^
• • in
m3-eMcninooeMinOi-4cno<-i
cn«-icNCNeNi-4r-ii-ifMrN|cNeNi-»cni-ieN
oo
in
m
o ^
en
^•in\oenm^in«*^
-------
z
Id
H
en
>-
en
o
z
l-l
en
g
0
5»
as
g
0
El
<
z
w
z
h-l
N
2
H
o
H
a
Cd
en
O
u
z
1—4
1
**
STERA
o
N
b.
O
en
H
Z
W
w
™
en
i
O
00
1
1
ON
..
w
H
a
^j
H
Id
i^
OS
u
X
u
^-«
o
a
i4
o.
to
4-1
§
0)
o
|H
cu
1
c
II
en
..
0
a.
M
a.
aves
01
14-1
O
0)
e
c
H
_
*—•
e
c
i-i
o
o
Si
01
4J
cnoum* en O
rHi-lf-ti-li-li-li-li-li-li-li— IrHrHi-li-li-IO'-*
C
CO
H
. . o
t O •
IX D
139
-------
Cd
H
CO
§
|
H
0
a
0)
a.
O
o
01
c
•H
N
R)
l-l
4-J
m
CB
H
o
oo
in
'in
u,
w
N
O
00
CO
m
O
number
n
j
* M
c*
r*
^*
c
0)
t-l
N
«
4-1
f— 1
ff
IH
O
o
o
•
0
CO
o
• m
00 CT>
in CM oo
CM CM i-l
o
« in
O r^
. «m
C
«
H CTI cJ CM in
X 00 O CJN w >•*
O 60
jj O
oo o
«
j:
in
a
H
^J CO •
s
U
ta
I X D C
140
-------
r
Ld
CO
>•
CO
o
o
o
OS
H
O
a
• •
U
*^
^
H
Z
W
Q£
W
ft.
X
U
|^
0)
e
3
C
H
CO
• VI
JJ
o
a.
^
-------
:NG SYSTEM
1-^
en
2
X
CJ
o
06
H
|
z
cd
r-4
H
O
H
Q
O
te.
X,
cS
td
en
O
N
0
en
H
Z
cd
at
en
<
g
H- 1
an
id
RPHOM
g
tM
•H
NO
0
u
03
O
CU
eu
a
|
U)
O
"e
c
§ in
i
5X1 o
1 O
ON o.
1)
td CU
< ">
a «>
eg
CJ
U-l
0
* S
£ 1
S c
r-l H
£ .j
a.
x •"
w S
c
o
o
Si
rH
CO
IM
0
4J
BO
eu
II
C
oa
c c
MJ *0
N H
CO
eg
rH
00
1 <
o ^
o §
H
oa
<" «
c -|
2 ^
C3
lb
E
rH
§ <
O _^
<= s
eg
H
03
>•?
V C
C eg
"" C"" '
N
eg
Vj
a
! <
o ^
eg
0
IV.
1-3 rHrHCOCMCM'HI 1 1 1 1 1 1 1 IrH
CM •» st m 00 CM -tf
XCMeTNOVOCMO f"»
rHrHrHpOrHrHI I I 1 I 1 I 1 |r-4
O
i_] COCOCOCM-JCOCOCOCM'-'I 1 1 1 ICM
intv.vOcOCMOOCOCOrHO CO
X CO O CM ^ NO NO 4
f*^ r^ co co CM co r*v &\ m r*» o
3; inNOOuoeMOoooNincM I 1 I 1 ICM
rHrHrHcMCMCOrHrHCMCO CM
O
tn rHrHrHrHrHrHrHrHI 1 I 1 1 1 lt-4
J rHCOrHCOCMCOCOCOl 1 1 1 1 1 ICNl
ps» fsj \@ P*. co i—* SN CO
SCO-rHCMin^rvO 00
iNOOrHCMCMrHrHCMI 1 1 1 1 1 IrH
1 X
CO
CO
•
00
CO
CJN
O
in
Q
rH
rH
ON
"*
°i
0
•
in
c
ON
o
CO
NO
t>
-
0
s
o
rH
^
3
OO
00
00
o
o
rH
O
rH
00
OO
oo
c
•o
CU
•o
•j)
to
rH
CU
II
^T
^"
-------
SYSTEM
o
z
CO
Q
X
o
o
X
H
3
u.
<
z
M
b)
Z
»-*
N
2
H
8
Q
U
CO
O
£3
U)
1
M
oc
^*
j£
^j
Ob
u
H
CO
O
N
tb
0
CO
H
z
z
u
CO
<
i
dU
as
H
O
O,
06
i
.
f^
•
s
ca
H
0
00
m
41 C
C CO
**4 f^
H
a
u
4J
a)
-
60
E
0 <
_J j<
C
CO
H
09
V
V c
C CO
N
CO
**
CO
1-4
*^»
60
E
•
O *•*
C
CO
H
03
0) «
5 <=
tl "
S H
CO
^
JS
o <
•
0 j^
c
CO
H
CO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
i-3 1 l 1 1 1 1 1 1 l 1 1 1 1 1 1
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
CO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
I-J 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
CO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
bJ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
o
•
CO fli-lllllllllllllli-IICN
O
f
»•! r*4 fH Illllllllllll ^4 Q CN|
(S| f\ f^
. . .in
x Is- ON oo •
i-liHIIIIIIIIIIIIli-li-ICM
O
1-4 00
1-3 i-«eN-lrt<--l«N(M(N 1 1 1 1 1 IIN-JON
0
fl
at
ON ON -a
' • 1— 1
l-J *^ ^" fO f> CN| ^ C*> CN »^ C^ CO 1 1 1 1 CN O *™^ CO
u
C
X aOaOt-«r-»O-l/1OO\O-*NO f-l^r-l I
CM«HcNtNi^CMCMtN>-|f-ll 1 1 1 CM ^ r-l ^»
IX 0 C ^
143
-------
£
W
H
CO
CO
z
t>*4
i
ROUGH
X
H
3
3
U.
<
^•4
111
z
rH
• • m
OO
o oo
CM CM
m
rHCMrHCMCMeslCMCMCMrHrHCMinrH
i-(rHrHrHrHrHi-4rHi-lrHrHi-4i-4rHi-HO rH
o
• m
CM CM
m vo
CM f-<
en en
in
in
oo
!-4rHrHcMCMrHt-4rHCMCMrHrH
O
m en st «*
CM rH CM O
O CO
i lO ^J" PO ^f CO
CM OO -* O
CO
r^vtoOf-iinenvoenooininoooovrcnrH .in
CMCMrHCMrHCMrHcMCMrHCM'—irHrHCMCMstrH
rHrHrHrHrHrHrHrHrHrHrHrHrHrHrHOrH
00 O
• *in
en in en en rH rH
\X D C
144
-------
w
H
CO
>•
CO
CJ
g
x
u
o
H
3
u.
O
a
-lrHi-^i-lrHi-li-li-4f-lt-4iHOi^
•*cN4 bd
O ft.
X
CO U
H
CO
iT
o
a
^
0)
a
(0
01
>w
0
0)
c
*""^
Q
^
c
1-1
01
c
N
CO
CO
1— 1
oo
E
1-1
d
oa
c
CO
H
,,.
CO
H
CO
,_! rH r-l t-l O
PI P»
X r-» NO 1-1 ts
i-t >H tM CM ON CN
. . . . (M
CN tN •-< i-l -*
{/) r-
-------
X
w
H
CA
>
M
O
z
en
s
X
3
S
3C
H
2
ta.
^*
Z
»-i
w
z
H4
N
^
5
8
Q
U]
en
O
X
U
<
5
as
J?
Cb
U
H
CO
O
N
b.
O
C/5
H
Z
M
1
3
en
<
£
o
H^
MORPHOME1
vO
i
a
03
0
00
1
CM
ON
w
H
Q
m
H
i
ft
at
u
eu
X
U
^
4J
a
M
01
ex
01
I-l
^4
HH
o
4J
60
01
II
X,
01
iH
N
CO
fa
U
(I)
rH
60
6
rH
O
O
0
01
•iH
M
(0
M
4J
CO
rH
"x*.
60
e
rH
O
O
o
0
01
N
IH
4J
CO
rH
60
e
o
o
pa
^yj
eg
H
^
jtf
c
eg
H
pa
(0
H
^
^
C
CO
H
P3
C
fl
H
<
H
I I I I I
lrH
CQ
U
«
?
-------
Cd
H
CO
CO
o
M
CO
g
X
o
§
06
H
to
llllllllllllrHQCO
C
CD CO
C H
1-1
N
CO
(-1
iJ 1-4 i-H rH I |
•-( O •
• • CO
m vo i-i i i i i i i i i
o
I I I I i-l o f""1
f- CO
i i i » CT> r*. ro
Cd
Z
N
•* O
o2 co
H I
CN
60
E <
o
i-l C
I?
CN CN i-l I I I I I I I I
I I I I r-l Q CO
Is* VO
I I I I rH O CO
00
vO
i-l i-l O
vO vT* i-4
CNrHrHllllllllilllrHr^CO
O
I I I I rH o I
a
w
CO Cd
O H
Cd
Z
t—i
OS
i-4vOr-ICN I I
O CM
* *
I I I I CN rH
i i i i 1-4 in i>«
o
I I I I 1-4 O **
VO !•»•
I I I I r-4 O Cft
vO
• in
m •
i i i i r-i in o>
s
Cd
CO
o
rH CN CM r-l CM CM r-l I I I
147
i i i i CM r^ r-
IX O C
-------
THROUGH DOSING SYSTEM
rj
3
Eh
^
Z
w
^
rs3
^;
(X
H
O
H
XPOSED
ta
^
z
06
*
^t
2
Id
H
OO
s
s
CO
H
Z
w
s:
M
at
1
i
^
H
til
|
OS
K
™
,
00
>—
vO
Q
U
OQ
4-1
0
a
a
in
4J
0
0
«]
M-l
0
)J
IU
§-°
c
n
o w
CO
*• -
i °
o "*
— »H
(11
Pu
H g
Q >
CO
01
rH
UH
O
M
m u
as. J3
* B
£ §
^ »
M ,
^ g
w o
i_i
O
(0
in
0)
^H
(d
^
0
.c
oo
•H
41
X
X
trazine
Tank B
t«
rH
00
E
l-H <
8 .*
• c
O CO
H
CO
,
f
i-H
O <
°. c
O cd
H
CQ
n r>> I-H .
rHcMIIIIIIIIIIIIICMOOCM
0) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
_; i i i i i i i i i i i i i i i
X
i i
1 X t> C
•o
•f^
•o
CO
4J
C
i-H
a.
n
i
148
-------
JE
w
H
cn
cn
CJ
z
1-4
cn
§
X
u
o
on
X
H
3
a
<
z
1— 1
id
z
1— 1
M
rf
a2
H
8
Q
Id
cn
o
CL,
X
u
CO
CU
*— i
0
Wl
(U
"1
3
C
II
^J
• M
B
u
C
»
4-1
CO
r-l
ff
i— I
O <
o ^
rt
H
cn i i i i i i i i i i i i i i i
>J i i i i i i i i i i i i i i i
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
cn i i i i i i i i i i i i i i i
J i i i i i i i i i i i i i i i
X
i i i i i i i i i i i i i i i
cn i i i i i i i i i i i i i i i
rJ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
cn i i i i i i i i i i i i i i i
rJ i i i i i i i i i i i i i i i
X
i i i i i i i i i i i i i i i
cn i i i i i i i i i i i i i i i
1-3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X
i i i i i i i i i i i i i i i
0
Cn t-llllllllllllllli— llr-4 T;)
•rl
0 TJ
*
|J r-llllllllllllllll-Hll-t (O
4J
ts
CO
• 'a.
X 0 ( 1 1 1 ° 1 rH "
IX D C T
>.x
149
-------
u
H
CO
O
Z
M
CO
8
X
o
p
o
as
15
*
3
U4
^
z
H-t
U
Z
H-*
ts)
Qfi
H
*t«
O
H
Q
td
CA
O
&
JjJ
^£
z
1-1
os
1
.rf
QB
ti3
H
en
8
u.
O
en
E*4
z
u
£
OS
3
en
si
2
cj
OS
H
£
o
OK
(£
2
o
CM
s
I
<
H
/^
4J
a
.^
s.
01
u
0
o
CO
«!_!
o
M
01
J3
g
3
c
II
CO
0
CD U
1 O
-* o.
^^
1 Vl
O (!)
-< o.
p
a)
w >
H C9
< 01
a -i
tw
O
j^
E
in 3
* C
H H
Z
g J
1-1
of.
[•3 E
X
u c
1-4
u
o
o
SI
01
4J
CO
^4
r^
ca
u
U-l
0
4J
Si
31
c
C CO
•«•! r i
N
CO
eg
-•^
60
0 <
C
eg
H
CO | 1 1 1 1 1 1 1 1 1 1 1 1 1 1
-4|r-l <-•<
<-> r-l
• •
X O O
CO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
iJ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
4
4J CO
eg c_i
O 2
* |~4 *5
-^^
en \o 1-1 j
* * * H^
J mcNCNIIIIIIIIIIIIINOfO O<
^J ^^ -f\ \fr ^4
• • • • \f*\
X m o i-i CM •
CMCMrMIIIIIIIIIIIIC4CN
-------
bJ
W
>•
CO
O °- m
Z /-•» CO — I — I^H-H— <— .-H^H-H^rf — -H-HrtrtO'-"
1-1 4J
to o
CM CM 00
t-i -H • • in
X »3'rocNO —
O ex to C
3 H (fl
O 01 4J H oc7NONfMC'M'-n
H O i-l CN-Hrt-*{N{N-HCSCMCMCS-H(NICNCNCMf>-H
3 M 60 O
3
• • m
00
• m
O
-in
................ o
X rovO^OoodropotMr-.i^np-.OfSfn-
CMCMCMrM-^OCNJCMtSCMCNICMi-tCMCMtN^
O
\o
• m
X tvJIM-- — CMfMCNPJ--f^tMCNtNr"-3--H
« X I X D C
a ^
-------
£
H
W
O
M
i
g
s
w
a
W
CO
§
Cd
,0
O
00
m
8 2
Id
H
0
a
01
ex
o
o
•g
M
IV
c
n
CO
O
a
01
a
(0
• . m
o
• in
i —i o "*
. m
•H O
c
cd
H
vo -m
|(M>3--<
ix G e
BQ
H
152
-------
£
U
CO
>•
CO
o
s
O
as
H
3
0
a
CD
a
O
O
CO
fc
c
It
o
in
fl i-t i-H rH r-l O ••*
0)
c
N
CO
i1
I-H
O
o
CO
I
O
p* i-l »-i o ""•
C
CO
H
<; o
o2 oo
H 1
< cs
O V
H O
a
en
o w
§3 <
M a
^,
z
**2
1
•<
06
H
CO
s
vO
^fc
H
Z
CK« 32
O W
a.
CO X
H W
Z
u
as
CO
r,l
VJ
• ^
o
Q.
.^
01
a
01
01
U-l
o
(U
•§
3
C
H
J
B"
y
C
O
O
U]
cu
c
N
CO
u
cd
r-4
^^
00
1-1
o
0
o
o
01
c
pa
•^
H
<
C
CO
H
PQ
c:
CO
o
at
u
O
O.
i
ca
01
•a
u-i
1-4 1-H I—* I-* f-^ O ^
O r^
• • m
CN ON
. . in
m
0 r-
oo
to
o
0
o
• m
-------
X
w
H
CA
CA
U
Z
CA
§
rf THROUGH
<
z
HH
W
Z
CM
^
<
8
Q
EXPOSE
z
HH
Jfi
3»
ZOSTERA
b.
O
H
Z
i
CA
U
a
U
hH
H
W
O
MORPH
^
CM
vD
a
Ul
ca
?5
o
CO
i
CN
O
H
O
3fc
H
i
at
U}
cu
X
•^
o
a
M
cu
shoots p
o
^4
H
<
C
cd
H
CO
c
H
C
CO
H
CO
c
CO
H
c
H
00
i-lf>l I f-l lH N r4 01 CM 1*1 ICMfO
• • 01
iJ CMCM-* I^Oli-H IfncMOl I^CMCMrH^H
C3 ^D ^\ c^ ^^ ^H ^| co O4 fO CO ^D
... . . . ^. o
CMCMCO ICM^JCM ICOO|i-< ICMCMCMUIi-H
•J -H
. 1/1
C/5 i-4 r-l r-( i—I i-t i-H
I I-H I-H Q «
\O C7^
X OOcnvOCMOItSOOOOOCMCJ>CM
CMCMCMCMO1OJCM01CSCM04CM
o 1-1
i-» -*
CO CM
X ff\i-ir>-eNvOCMOCM'-ICMi-ICMOOvOvO
o
« U"l
rH Q i-H
1^ CO
OlCMCMCNCMCMCMCOCMOICOCMCMi-HOI^J-i-1
IX G C
•o
(U
(d
1-1
IP"
154
-------
£
fad
H
en
>>
0
z
w
S
0
1
X
H
3
Lu
<-
z
^^
[jj
z
HH
N
2
H
<
O
H
EXPOSED
<
z
{£
2
ZOSTERA
u.
O
en
H
Z
bJ
£
U
OS
=l
en
2
C3
CJ
1—4
{A
H
UJ
2
O
X
Ou
at
g
.
U"l
l i-lrHrH I Ir^Or-l
o w
.r^
CMCMMi-lCNi-ICMi-ICMCNi-ICMCN I ICMfO'-l
• • -a-
1 (N O <-<
in
... .ro
(Nt-ICMCStv|CMO4CMi-leNCMeM I ICMCOt-l
i-( i-l I i—i O i—i T3
CU
* • ^J
CO PO I O4 ^D ^^ W
c
• f- C
co • vr H
'i^< t? c T
155
-------
SYSTEM
o
i— (
CO
g
X
o
3
o
Oi
X
f_
3
3
u.
<
z
h— *
u
2=
1— 1
N
g
<
_
r""|
O
tlj
CO
o
u
^
z
2
<
*
^£
as
H
co
0
NJ
CL.
O
co
H
Z
a:
g
CO
i
o
X.
f-
ij
O
£
a:
i
»
vO
r>*
S
_i
03
t-
****
4J
O
a.
u
c c
N H
£
•J
•3
1-1
5C
E
O
O C
o
•
tO «||||.|||||||||_|,-,
c
•
-J — 'Illlllillll'll— >l-
sO vO
Z miiiiiiiiiiiiiiini
CN CN — i
O
CO — Illlllllllllll— '1— '
0
•J — i l i i i l i i i i l l i i " l —
m m
= vriliiiiiiilliii| | 1 1 1 1 ICNOOO
^inmr^oo^^in O
X oo — oOxOi i | l i i (""> .
CN O-l C^4 ~ f*> CN C^J CJ CM U", CO
O
„ . ,_lo-
— vO
i-l mri~3--?vfnr"mrNr^r^CNr^ l |f^o-"
r^ — oOCNr^CNr--vOvrcNOOr-o CN
^4 CN fM CNJ r*j fN CM r^j r*"» CN c^j CM cj CM f^ -^
O
* O "^
• • m
cMvomvrco^cNOoor^cNCN'^'^JOf^'--'X)r'imf-.r-o-3'CrNOONCNr~ON
X r~r~inri-^r^omoOf*>
-------
y-
\i3
i—i
-n
>•
en
§
o
a
s
0
s
o
at
X
3
3
u.
z
UJ
z
N
2
H
<
Q
f-<
O
u
en
O
Si
u
<
5
<
S
w
H
en
O
N
6u
Q
en
H
z
UJ
z:
u
a:
a
en
<
£
^j
,_<
at
H
z:
as
a,
at
i
.
r-»
CM
a
UJ
T
CO
o
1
yO
CM
|
O
..
UJ
H
a
^
H
z
r
1-4
DC
U
a.
X
U]
^1
cu
a.
(A
^j
o
o
CO
1*.
o
u
HI
XI
E
3
C
II
t/2
• «k
u
O
C.
1-
41
a.
(A
111
>
C5
a
o
u
cu
A
C
II
pjj
• «
E
U
C
•**
u
o
o
Si
ca
^
CO
D
t-H
i-H
C9
4J
u^
O
J=
6C
0)
£
n
X
01
J2
•H
N
CO
t.
U
CO
i— i
oc
E
— «
O
O
o
0)
C
•ft
N
14
CO
-H
OC
E
^
O
o
o
•
o
01
•H
N
cd
i-
4J
«8
^^
6C
E
O
•
O
V!
C
C9
H
<-
^
C
fS
H
PQ
v
C
CO
^
w
C
CO
H
aa
v
c
ra
8-
"*
»<*
c
C3
H
CO fH CO CM I CM O f*
m — oo
CN CM — CM CM
en
-* O O m I o* • •&
CM CM CM CM —1 CO —
o
I I —' O —
CM «M CO I I CM O -*
en
*>O CJ> vO r^ 00 C^ I I • ^ • CO
—• -« CM — — CM CMC1~H
o
• CM
^ _4 — — -H I I I — O -<
vO 00 I
*-> CM
en
• CM
CM m
o
— o
CM 00
CM O
— CJ> 0-1 CM
O CM er\ — i
CM CO — CM
CM
-------
ATRAZINE IN A FLOW THROUGH DOSING S
o
H
XPOSED
u
^
z
en
"£•
STERA
o
tsl
%
en
H
7i
X
tin
Dti
3
cn
<
£
o
32
H
^
O
0,
Sti
%
^
00
CN
•
vD
O
U
CQ
H
u
o
O.
b
0)
a
in
0
o
.c
CO
IM
o
u
"s
c
II
00 f>
1
vO
7 o
o o.
"" w
0)
0.
£ «
< ^
Q 1
"-*
o
u
s z
£ I
Id „
r "
M ,
OS i-3
W
a. "
x s
u u
C
4.1
O
o
(A
CO
(U
fl
o
OC
at
•^
u
o
ca
01
-I C
N ffl
B H
U
4J
1 1 1 1 1 1 1 1 1 1 1 1 1 , 1
^ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
C" 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
I-J 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0
m
— 1 CNCNCNCNmmlllllllllCN
CN
x c\^^^^^o-^i i i i i i i i ICM
—* CM fO r^ CN CM
0
H>4 c^ f> CO fvj ro »-H «-^ ^^ co fs) I | t | | CN
cy>eNincNin<- r-
CNCNCNCNI— '•^CSICS—CS CM
•— t
~H
•
cNOOoocNAa>l I t I lin
CNCNf^CNCNCN— iCNCNCN CM
o
•
m
,J CN CN •— • f*^ "^ CN CN CM CM CN CN •••< CM
1 X
158
m
0*
00
^
Q
d
00
m
O
O
•
F*4
"^
^3-
^
o
CM
•
^
O
*G
vO
vO
O
O
IP-*
0
o
o
0
*»
^*
-'
^.
*«l
c
•o
cu
•f^
•o
CO
4J
c
flj
I— t
rt.
fr
^v
1
N.'
-------
SECTION 7
ADENYLATE ENERGY CHARGE STUDIES
GENERAL INTRODUCTION
The adenylate energy charge (EC) was first defined by Atkinson and Walton
(ATP) + 1/2 (ADP)
EC
(ATP) + (ADP) + (AMP)
This ratio was proposed as a fundamental metabolic control parameter. As
such, EC represents the metabolic energy state of the cell. Broad
applications of EC include the following:
1) disciplines, ranging from cellular biochemistry (Atkinson, 1977) to
community ecology (Wiebe and Bancroft, 1975);
2) different cellular and organismic types, prokaryote vs. eukaryote ,
autotroph vs. heterotroph, and single vs. mult icellular organisms (Chapman et
al. , 1971); and
3) a range of environments, including marine (Karl and Holm-Hansen,
1978), estuarine (Mendelssohn and McKee, 1981), and terrestrial systems (Ching
and Kronstad, 1972).
Recent application of EC measurement to higher plants is extensive,
primarily involving agriculturally important crop species (e.g. Raymond and
Pradet, 1980; Saglio et al . , 1980; Bonzon et al . , 1981; Quebedeaux, 1981;
Hampp et al . , 1982). In contrast, adenylate literature on seagrasses (Knauer
and Ayers, 1977) is extremely limited. Plants respond to environmental stress
in numerous ways (Levitt, 1972; Cottenie and Camerlynck, 1979; Rabe and Krebb,
1979). Since the metabolic energy state of an organism is sensitive to
environmental variation, both natural and anthropogenic, EC has been advanced
as an index of sublethal stress (Ivanovici, 1980).
Zostera marina (eelgrass), a submerged marine angiosperm, functions as a
food source, habitat, nutrient pump, and sediment stabilizer. The basic
biology (Setchell, 1929; Burkholder and Doheny , 1968; Harrison and Mann, 1975;
Orth et al., 1981) and ecological value (McRoy and Helfferich, 1977; Stevenson
and Confer, 1978; Phillips and McRoy, 1980; Wetzel et al . , 1981) of Z. marina
are well documented.
Historically and more recently, the distribution and abundance of Z_.
marina have undergone large fluctuations in the Chesapeake Bay (Orth and
Moore, 1981). The reduction of eelgrass beds has been attributed to disease
159
-------
(Renn, 1934), temperature increase (Orth, 1976), herbicide input (Stevenson
and Confer, 1978), cownose ray disturbance (Orth, 1975), and to a lesser
extent, dredging and boating activities (Orth, 1976). A reliable method to
assess the metabolic state of eelgrass is, therefore, essential. Application
of energy charge measurement to Z_. marina is a logical choice.
Objectives
1. A major objective of this study was development of a methodology to
quantitatively measure adenine nucleotides and adenylate energy charge (EC) in
Zostera marina (eelgrass). The remaining objectives incorporated these
optimized techniques.
2. Adenylates and EC were compared among "L_. marina tissues, including leaf,
leaf sheath, root plus rhizome, and seed pod. Comparative measurements were
made on eelgrass epiphytes, aboveground Ruppia maritima (widgeongrass), and
abovegruund Spartina alterniflora (saltmarsh cordgrass).
3. Monthly variation of adenylates and EC was assessed in above and below-
ground Z_. marina tissue over a one year period. Associated environmental and
morphemetrie data were collected.
4. Adenylate and EC responses to two atrazine levels over 6 hours, and five
atrazine levels over 21 days, were assessed in Z. marina leaf tissue. Hourly
production rates were measured during the 6 hour experiment. Weekly
morphometric changes and mortality were examined over the 21 day atrazine
exposure period.
160
-------
REFERENCES
Atkinson, D. E. 1977. ' ellular energy metabolism and its regulation.
Academic Press, N. Y. 293 p.
Atkinson, D. E. and G. M. Walton. 1967. Adenosine triphosphate conservation
in metabolic regulation. J. Biol. Chem. 242:3239-3241.
Bonzon, M., M. Hug, E. Wagner, and H. Greppin. 1981. Adenine nucleotides and
energy charge evolution during the induction of flowering in spinach
leaves. Planta 152:189-194.
Burkholder, P. R. and T. E. Doheny 1968. The biology of eelgrass. Lament
Geol. Obs. No. 1227. 120 p.
Chapman, A. G., L. Fall, and D. E. Atkinson. 1971. Adenylate energy charge
in Escherichia coli during growth and starvation. J. BacC.
108:1072-1086.
Ching, T. M. and W. E. Kronstad. 1972. Varietal differences in growth
potential, adenylate energy level, and energy charge of wheat. Crop Sci.
12:785-789.
Cottenie, A. and R. Camerlynck. 1979. Chemical aspects of stress in plants.
Meded. K. Acad. Wet. Lett. Schone Kunsten Belg. K. Wet. 41(4):1-21.
Hampp, R., M. Collier, and H. Ziegler. 1982. Adenylate levels, energy
charge, and phosphorylation potential during dark-light and light-dark
transition in chloroplasts, mitochondria, and cytosol of mesophyll
protoplasts from Avena sativa L. Plant Physiol. 69: 448-455.
Harrison, P. G. and K. H. Mann. 1975. Chemical changes during seasonal cycle
of growth and decay in eelgrass (Zostera marina) on the Atlantic coast of
Canada. J. Fish. Res. Bd. Canada 32:615-621.
Ivanovici, A. M. 1980. Application of adenylate energy charge to problems of
environmental impact assessment in aquatic organisms. Helg. Meers.
33(1-4):556-565.
Karl, D, M. and 0. Holm-Hansen. 1978. Methodology and measurement of
adenylate energy charge ratios in environmental samples. Mar. Biol.
48:185-197.
Knauer, G. A. and A. V. Ayers. 1977. Changes in carbon, nitrogen, adenosine
triphosphate and chlorophyll a in decomposing Thalassia testuc'inum
leaves. Limnol. Oceanogr. 22:408-414.
161
-------
Levitt, J. 1972. Responses of plants to environmental stresses. Academic
Press, N.Y. 697 p.
McRoy, C. P. and C. Helfferich (eds). 1977. Seagrass ecosystems: A
scientific perspective. Marcel Dekker, Inc., N.Y. 314 p.
Mendelssohn, I. A. and K. L. McKee. 1981. Determination of adenine
nucleotide levels and adenylate energy charge ratio in two Spartina
species. Aq. Bot. 11:37-55.
Orth, R. J. 1975. Destruction of eelgrass, Zostera marina, by the cownose
ray, Rhinoptera bonasus, in the Chesapeake Bay. Chesapeake Sci.
16:205-208.
Orth, R. J. 1976. The demise and recovery of eelgrass, Zoatera marina, in
the Chesapeake Bay, Virginia. Aq. Bot. 2:141-159.
Orth, R. J. and K. A. Moore. 1981. Distribution and abundance of submerged
aquatic vegetation in the Chesapeake Bay: A scientific summary. Final
Report, US EPA, Chesapeake Bay Program, VIMS SRAMSOE No. 259.
Orth, R. J., K. A. Moore, M. H. Roberts, and G. M. Silberhorn. 1981. The
biology and propagation of eelgrass, Zostera marina, in the Chesapeake
Bay, Virginia. Final Report, US EPA, Chesapeake Bay Program, Grant No.
R805953, VIMS.
Phillips, R. C. and C. P. MeRoy (eds.). 1980. Handbook of seagrass biology:
An ecosystem perspective. Garland STPM Press, N. Y. 353 p.
Quebedeaux, B. 1981. Adenylate and nicotinamide nucleotides in developing
soybean seeds during seed-fill. Plant Physiol. 68:23-27.
Rabe, R. and K. H. Kreeb. 1979. Enzyme activities and chlorophyll and
protein content in plants as indicators of air pollution. Environ.
Pollut. 19:119-137.
Raymond, P. and A. Pradet. 1980. Stabilization of adenine nucleotide ratios
at various values by an oxygen limitation of respiration in germinating
lettuce (Lactuca sativa) seeds. Biochem. J. 190:39-44.
Renn, C. E. 1934. Wasting disease of Zostera in American waters. Nature
134:416-417.
Saglio, P. H., P. Raymond, and A. Pradet. 1980. Metabolic activity and
energy charge of excised maize root tips under anoxia. Plant Physiol.
66:1053-1057.
Setchell, W. A. 1929. Morphological and phenological notes on Zostera marina
L. Univ. Calif. Publ. Bot. 14:398-452.
Stevenson, J. C. and N. M. Confer. 1978. Summary of available information on
Chesapeake Bay submerged vegetation. USFWS/OBS-78/66. 335 p.
162
-------
Wetzel, R., K. Webb, P. Penhale, R. Orth, J. van Montfrans, R. Diaz, J.
Merriner, and G. Boehlert. 1981. Functional ecology of eelgrass. Final
Report, US EPA, Chesapeake Bay Program, Grant No. R805974, VIMS.
Wiebe, W. J. and K. Bancroft. 1975. Use of the adenylate energy charge ratio
to measure growth state of natural microbial communities. Proc. Nat.
Acad. Sci. 72:2112-2115.
163
-------
METHOD DEVELOPMENT
Introduction
Adenine nucleotides, expressed as the adenylate energy charge (EC) ratio,
regulate cellular energetics (Atkinson, 1977). Problems associated with
methodology for the determination of in situ adenine nucleotide levels may
limit the utility of the EC concept (Pradet and Raymond, 1978; Karl, 1980;
Ivanovici, 1980). Methodology must be tailored to the specific chemical
characteristics of a particular biological material in order to accurately
determine in situ levels of intracelluiar adenine nucleotides. In addition,
ease of operation and reproducibility are essential to any useful analytical
technique.
The most frequently employed methods for determination of adenine
nucleotides involve enzymic conversion of adenosine monophosphate (AMP) and
adenosine diphosphate (ADP) to equivalent amounts of adenosine triphosphate
(ATP), followed by quantitative analysis of the ATP via the firefly
bioluminescent reaction (Karl and Holm-Hansen, 1978). Determination of ATP by
the firefly luciferase reaction, reviewed by Leach (1982), has been widely
applied (DeLuca, 1978; DeLuca and McElroy, 1981). After reviewing Che
literature, Sofrova and Leblova (1970) concluded that the firefly reaction is
the most rapid, sensitive, and specific method for ATP determination in plant
tissue. Several studies which specifically address methodology for adenylate
determination in higher plants utilize the firefly reaction (Pradet, 1967;
Guinn and Eidenbock, 1972; DeGreef et al. , 1979; Mendelssohn and McKee, 1981).
Employing the firefly assay, this study developed a methodology to
optimize determination of adenine nucleotides in Zostera marina (eelgrass), a
submerged marine angiosperm. Z_. marina is an ecologically important
macrophyte species (McRoy and Helfferi.cn, 1977; Stevenson and Confer, 1978;
Phillips and McRoy, 1980; Wetzel et. al., 1981; Orth et al., 1981), occurring
in temperate and subarctic coastal and estuarine waters in the Northern
Hemisphere (den Hartog, 1970). Major analytical procedures were evaluated,
including sample collection and preparation, adenylate extraction, conversion
ol AMP and ADP to ATP, firefly lantern extract preparation, and photometry.
Tissue composition and seasonal patterns of adenine nucleotides were also
assessed in order to provide baseline information on natural adenylate
variability in Z. marina.
Methods
Sampling Sites—
Zostera marina was collected at low tide from an extensive grassbed
(37°15'40" N, 76°23'50" W) off Sandy Point at the mouth of the York River in
the lower Chesapeake Bay estuary. This bed was close to the laboratory and
accessible by land. Epiphytes and Ruppia maritima were also obtained from
Sandy Point. Spartina alterniflora was collected from nearby Indian Field
Creek (37°16'5' N, 76°33'30"W).Locations of these sites are shown in Figure
7.1.
164
-------
-------
Adenine Nucleotide Methodology Experiments—
Assay principles—Adenylate assay reactions have been described by Pradet
(1967), Holm-Hansen and Karl (1978), and DeLuca (1976). ATP is assayed with
the firefly bioluminescent reaction (Figure 7.2). AMP and ADP are first
converted enzymically to ATP (Figure 7.3), which is then analyzed by the
firefly reaction. The equilibrium constant for the PK reaction is
sufficiently large to convert most ADP, and consequently most AMP, to ATP
(Adam, 1965).
Sample collection and preparation—Plants were uprooted with a shovel,
swirled in river water to remove macro-algae and loose sediment, and stuffed
in a 180 or 530 ml plastic bag (Whirl-Pak). Liquid nitrogen was poured into
the bag (within 1 rain of harvest) and the entire bag was submerged in liquid
nitrogen contained in a 4 1 polyethelene dewar flask (Nalgene) for return to
the laboratory.
Liquid nitrogen was drained from the bag and the bag was then placed in a
lyophilizer. The chamber was sealed and vacuum inititated, with condenser
temperature allowed to reach -55°C before sample introduction. Chamber
shelves, were not heated. Samples were lyophilized for 70-90 hrs.
After lyophilization, plant tissue was handled with forceps to prevent
hydration. Brown aboveground tissue was discarded, since this material was
considered dead at time of harvest. Leaves were scraped with a flat spatula
which removes 70-902 of the epiphytes (Penhale, 1977).
For methodology experiments (excluding freeze delay), plants were pooled
to provide a uniform substrate for experimental treatments. For tissue
comparison and seasonal survey experiments, plants within a treatment (i.e.
tissue type or monthly sample, respectively) were pooled in order to minimize
within treatment variation. Leaf tissue was used for methodology experiments.
Leaf, leaf sheath, root plus rhizome, and seed pod tissue were examined in the
tissue comparison experiment. Aboveground (stem plus leaf) and belowground
(root plus rhizome) parts were analyzed in the seasonal survey.
Tissues were ground in a cutting mill to pass a MO (425y) mesh screen.
Scrapings (epiphytes) off lyophilized "L_. marina leaves were ground by hand
with mortar and pestle. Samples were either processed immediately or stored
in a vacuum desiccator (Nalgene) in the dark for up to 5 days. Tissue
preparation was adapted from the method of Mendelssohn and McKee (1981).
Extraction—Tissue was weighed into 20-80 mg aliquots and held in a
desiccator. The extrictant solution was 1 mM ethylenediaminetetraacetic acid
(EDTA) + 5Z (w/v) polyvinylpolypyrrolidone (PVPP) at pH 7.6. Four to eight ml
of extractant are heated to 100°C in a 50 ml beaker on a hot plate (Corning).
Tissue was added (
-------
E + LH2 + ATP ^ =»> E-LH2AMP -f PP,
E-LH2AMP + 02 > E + oxyluciferin + AMP + C02 + hv
E: firefly luciferase (EC 1.13.12.7)
LH2: luciferin
E-LH2AMP: enzyme-bound luciferyl-adenylate
Figure 7.2. Firefly bioluminescent reaction.
1.7
-------
ADP conversion
PK
ADP + PEP
, Mg"
ATP -f pyruvate
K - 2.89 x 103
pH 7.6
(Krimsky, 1959)
Coupled AMP conversion
AK
AMP + ATP
2 ADP
K = 1.2
(Atkinson, 1977)
PK
ADP + PEP,
K ,
ATP + Pyruvate
PEP: phosphoenolpyruvate
PK: pyruvate kinase (EC 2.7.1.40)
AK: adenylate kinase (EC 2.7.4.3)
Figure 7.3. Enzymic conversion reactions.
163
-------
on ice, and processed as soon as possible. Extraction methodology was adapted
from Mendelssohn and McKee (1981).
EDTA extractant solution was stored at 4*C in the dark and used for
periods up to 1 month. A working aliquot of EDTA solution was vacuum-filtered
through a 0.2 y nitrocellulose membrane (Nalgene) for each day's analyses and
discarded immediately after use. PVPP was added to the filtered EDTA solution
approximately 30 min prior to extraction.
Conversion—AMP and ADP were enzymically converted to ATP. Three sets of
reaction mixtures (13 x 100 mm disposable glass tubes) were prepared as
follows:
Tube A (ATP Reagents): 400 yl blank (extractant),
standard (ATP in extractant), or
sample extract
400 yl reaction buffer (45 mM
TRICINE, 18 mM MgS04, pH 7.6)
400 yl distilled water (DW)
Tube B (ADP + ATP Reagents): 400 yl blank, standard, or sample
extract
400 yl reaction buffer
400 yl PK (30 yg), PE? (1.5 mM)
Tube C (AMP + ADP + ATP Reagents): 400 yl blank, standard, or sample
extract
400 yl reaction buffer
400 ul PK, PEP, AK (30 yg).
These tubes were incubated (30"C, 30 min), heat deactivated (100°C, 2 min),
and allowed to re-equilibrate (on ice, 20 min). Composition of conversion
reaction mixtures with ATP standards appears in Table 7.1
Buffer was stored at 4°C in the dark and used for periods up to 2 weeks.
Working aliquots of buffer and DW were filtered (0.2 (0 for each day's
analyses and discarded immediately after use. Fresh solutions of [PEP + PK]
and [PEP * PK + AK] were prepared in filtered DW in glass vials for each day's
analyses, held on ice, and discarded immediately after use.
Firefly lantern extract preparation—One vial of lyophilized firefly
lantern extract(FLE),commercially prepared from 50 mg dried lanterns, was
hydrated with 25 ml filtered (0.2 y) 45 mM TRICINE-18 mM MgSO^ (pH 7.6) and
aged (room temperature, 6-8 hrs) in order to degrade endogenous ATP. After
aging, the insoluble residue was removed by centrifugation at 3000 RPM for 15
min. Whenever a large volume of FLE was required, several vials were pooled
in order to eliminate variation between individual vials (Holm-Hansen and
Karl, 1978).
Photometry—The photometer was allowed to warm up for at least 1 hr prior
to assays. A sensitivity setting of 7.00 was utilized, since best instrument
stability is achieved by using the lowest setting adequate for analysis (SAIT,
169
-------
TABLE 7.1. COMPOSITION OF CONVERSION REACTION MIXTURES
Component
Na2ATP
TR1CINE
buffer
MgS04
Na2EL)TA
PK
AK
Na3PEP
(NH/t)2S041
Units
ng ml"'
mM
mM
UM
Ug ml"1
Ug ml"1
UM
mM
[ATP]
Reagents
13-1333
15
6
333
-
-
-
-
Reaction
[ATP+ADP]
Reagents
13-1333
15
6
333
25
-
500
15
Mixture
[ATP+ADP+AMP]
Reagents
13-1333
15
6
333
25
25
500
30
From PK and AK suspensions
170
-------
1975). Dark current was nulled by adjusting the zero (4.80 - 4.90 at
sensitivity 7.00), just before each standard set was run.
One hundred ul of solution from Tubes A, B, or C were pipetted
(Eppendorf) into a 6 x 50 mm disposable glass tube. Fifty \\l of FLE were
pipetted (Eppendorf) into this tube, while simultaneously initiating the
10 sec delay mode of the photometer timing circuit with the footswitch.
During this delay period, the tube was vortexed (Vortex-Genie) to ensure
thorough mixing, inserted into the photometer, and the shutter opened. Counts
were recorded for the following 10 sec integration period. A chart recorder
was interfaced with the photometer to follow reaction kinetics in order to
detect interferences or instrument malfunction.
For peak height measurements, 100 pi of solution from Tubes A, B, or C
were pipetted into a 6 x 50 mm tube. The tube was placed inside the
photometer, the shutter opened, and 50 yl of FLE injected with the electronic
pipet system which simultaneously activates the photometer. Sensitivity
settings from 7.00 - 10.00 were used. As a check on initial reagent mixing
for peak height measurements, each tube was read, removed from the photometer,
vortexed, and re-inserted into the photometer. If the recorder trace
exhibited continuity, the reading was considered valid (Karl and Holm-Hansen,
1978). If not, the tube was discarded, and the process was repeated until a
continuous trace (i.e. thorough initial mixing) was obtained.
Composition of firefly reaction mixtures with ATP standards appears in
Table 7.2. In addition, pH values for reaction components and mixtures are
presented ''n Table 7.3.
Standards and blanks—A primary standard was prepared with a weighed
amount of ATP dissolved in filtered (0.2 i:), distilled, deionized water. This
primary standard was divided into 1 ml aliquots and stored frozen (-20°C) in
glass vials for a period up to 3 months. A fresh set of working standards was
prepared in glass vials for each day's analyses. An aliquot of primary
standard was thawed and serially diluted with filtered (0.2 u) extractant
solution (1 mM EDTA) to produce a set of standards which bracketed sample ATP
levels. Working standards were held on ice and discarded immediately after
use. Although Holm-Hansen and Karl (1978) reported no significant loss of
these standard adcnylates during an 8 hr period, a standard set was run at
least every 2 hrs. Working standards and blanks were carried through enzymic
conversion and incubation steps to parallel sample processing. This resulted
in similar ionic composition and ATP reactivity, permitting more accurate
adenylate quantification (Holm-Hansen and Karl, 1978). Standards and blanks
were each read in duplicate per reaction Tube A, 5, or C. In cases where a
large discrepancy in duplicate readings occurred, a third reading was taken.
Another primary standard was prepared with weighed amounts of ATP, ADP,
and AMP dissolved in filtered (0.2 u), distilled, deionized water. The
resultant standard, containing equal concentrations of ATP, ADP, and AMP, was
used to calculate recovery and conversion efficiencies.
Data reduction—Net light output was computed by subtracting the
appropriate blank value from each total light emission value. The log of net
171
-------
TABLE 7.2. COMPOSITION OF FIREFLY REACTION MIXTURES
Component
Units Reaction Mixture
[ATP] [ATP-t-ADP] [ATP+ADP+AMPj
Reagents Reagents Reagents
Na2ATP
TRICINE
buffer
M8S041
Na2EDTA
PK
AK
Na3PEP
(NH4)2S042
FLE3
KH2As044
ng ml~l 8-888 8-888
mM 25 25
mM 11 11
uM 222 222
ug ml~l - 17
ng ml"1
UM - 333
mM - 10
yg ml"1 667 667
mM 3 3
8-888
25
11
222
17
17
333
20
667
3
1 1 mM from FLE preparation
2 From PK and AK suspensions
3 Expressed as precursor firefly lanterns
* From FLE preparation
172
A
-------
TABLE 7.3. REACTION COMPONENT AND MIXTURE PH VALUES
Label Component or Mixture pH1
R Distilled water 7.45
S 75 pg ml"1 PK + 1.5 mM PEP 7.05
T 75 pg ml"1 PK + 1.5 mM PEP + 75 pg ml"1 AK 6.90
1 mM EDTA 7.58
U 1 mM EDTA + 1 pg ml"1 ATP 7.66
V 45 mM TRICINE + 18 mM MgS04 7.62
W 2Tube A - 400 pi R + 400 pi U + 400 yl V Conversion 7.55
X 3Tube B - 400 u1 S + 400 pi U + 400 pi V Reaction 7.55
Y 4Tube C = 400 pi T + 400 ul U + 400 pi V Mixtures 7.53
Z 2 mg ml"1 FLE5 + 45 mM TRICINE + 18 mM MgS04 7.43
2Tube A - 100 pi W * 50 pi Z Firefly 7.48
3Tube B - 100 pi X + 50 pi Z Reaction 7.49
4Tube C - 100 pi Y + 50 pi Z Mixtures 7.49
1 pH meter calibrated with .05M (KH2P04 - NaOH) buffer to pH 7.00 at
25°C
2 (ATP] Reagents
3 [ATP+ADP] Reagents
4 [ATP+ADP+AMP] Reagents
^ Expressed as precursor firefly lanterns
173
-------
light emission (dependent variable) is regressed against the log of ATP
concentration (independent variable) for three separate series of standards
(Tubes A (ATP Reagents), B (ATP + ADP Reagents) and C (ATP + ADP + AMP
Reagents)).
Each sample extract was similarly processed in reaction Tubes A, B, and C
(duplicate reading per tube), and tube concentrations were calculated from
corresponding standard regressions. Amounts of adenylates (ATP equivalents
ml"*) and EC were computed from tube concentrations as follows:
ATP
ADP
AMP
AT
EC
Tube A
Tube B - Tube A
Tube C - Tube B
Tube C
Tube A + Tube B
2(Tube C)
An ATP equivalent is the amount of AMP, ADP, or AT, given as the weight of an
equimolar amount of ATP (Pamatmat and Skjoldal , 1979). The formulation used
for EC (Ball and Atkinson, 1975) reduces propagation of errors by using
directly measured quantities. Since standards, blanks, and sample extracts
all underwent identical dilution:
Hg ATP equivalent
yg dry wt tissue
ATP equivalent x ml extraction volume
ml
dry wt tissue
Recovery and conversion efficiencies—Efficiency of adenylate recovery after
extraction was determined by assaying two aliquots: 1) sample with addition
of known amo-mts of ATP, ADP, and AMP (internal standard) immediately before
extraction, and 2) sample without internal standard addition. Recovery was
calculated as follows (Mendelssohn and McKee, 1981):
2 Recovery *
(*%issue_+_Intern_al Standard "^Tissue> Determined by Assay
-------
X Conversion *
^""Standard' Determined by Assay
(ANStandard^Known Amount
where AN - ADP or AMP.
Reagents anc' equipment—The following reagents were obtained from Sigma
Chemical Co.: firefly lantern extract (FLE-50), ATP (A 5394), ADP (A 6521),
AMP (A 1877), GDP (G 6506), PEP (P 7002), PK (P 1506), AK (M 3003), PVPP (P
6755), TRIS-HC1 (T 3253), HEPES (H 3375), and TRICINE (T 0377). Other
chemicals used in this study were analytical reagent grade.
Adenylates were measured with an ATP photometer (Model 3000, SAI
Technology Co.) and, in the case of peak height measurements, with the Enzyme
Kinetics Kit electronic injection pipet (No. 020302, SAI Technology Co.). A
chart recorder (Model 250/MM, Linear) was modified to accommodate an input
voltage from 0.01-10 V.
Other equipment included a lyophilizer (Model 10-100, VirTis), mechanical
analytical balance (Model H31, Mettler), electronic top-loading balance (Model
PL 200, Mettler), drying oven (Model SW-17TA, Blue M Electric Co.),
refrigerated centrifuge (Model PR-2, International Equipment Co.) with high
capacity attachment, high speed angle centrifuge (Model SS-1.Sorvall),
Thomas-Wiley intermediate mill (Model 3383-L10, Arthur H. Thomas Co.), water
bath (Model MW-1110A-1, Blue M Electric Co.), vacuum pump (Millipore), and
digital pH meter (Model 610, Fisher Scientific Co.), equipped with a
glass-body combination electrode (No. 13-639-90, Fisher Scientific Co.).
Disposable tubes, vials, filters, pipets, and pipet tips were routinely used.
Reusable glassware was acid washed, rinsed 3 times with DW, and oven-dried to
minimize contamination.
Adenine nucleotide methodology experiments—Differences between
adenylates, subjected to various analytical treatments, were detected and
located by the procedure diagrammed in Figure 7.4. Dependent variables are
ATP, ADP, AMP, AT, and EC. Independent variables are treatment levels. The
null hypothesis states no difference in adenylates between k treatments (i.e.
H0 : Ul M P2 " ••• Wc>-
Standard curves, generated by three different photometer counting modes,
were compared. Homogeneity of these linear regression slopes and intercepts
was tested by analysis of covariance (ANCOVA). Data were log-transformed and
satisfied the assumptions of homoscedasticity and normality. Pearson
correlation coefficients for log-log regressions used in ANCOVA were
calculated. Null hypotheses stated no difference in slopes (Ho : BI " 63 *
63) or intercepts(H0 : 04 » «2 * 03) between regressions. Significant
differences were located by the Student-Newman-Keuls multiple range test.
Tissue comparisons—Differences between adenylates in four tissue types
were detected and located by the procedure diagrammed in Figure 7.4.
Relationships among adenylates were evaluated by Spearman rank correlation.
175
A
-------
w
01
u
B
01
M
i
u
rt
01
§
(0
u
o
o
u
0)
00
•H
-------
Seasonal Survey—Differences between adenylates at monthly intervals were
detected by the procedure diagrammed in Figure 7.4, although significant
differences were not located. Relationships among adenylate, environmental,
and morphometricd data were analyzed by Spearman rank correlation.
Environmental data include water temperature, salinity and pH. Water
samples, collected in brown bottles (Nalgene), were returned to the laboratory
for salinity (induction salinometer, Model RS 7B, Beckman) and pH
measurements. Daylength and low tide time and height data were determined
from tide tables (NOAA, 1979,1980).
At each harvest, one 0.03 m2 plug of eelgrass, 10 cm deep, was collected
with a plexiglass tube (0.10 m radius), placed in a coarse mesh bag, and
washed free of sediment. This sample was returned to the laboratory and
analyzed for total number of shoots, shoot lengths, and above and belowground
biomass, according to Orth (1977).
Statistical Analysis—
The following procedures in the SPSS software package (Nie et al., 1975;
Hull and Nie, 1981) were used: ONEWAY (single factor ANOVA, Hartley F max and
Cochran C tests for homoscedasticity, Student-Newman-Keuls multiple range
test), NPAR TESTS (Kruskal-Wallis single factor ANOVA by ranks and
Kolmogorov-Smirnov one sample test for normality), and NONPAR CORR (Spearman
rank correlation).
Other statistical procedures employed included analysis of covariance
(test for homogeneity of linear regression slopes and intercepts) with an
associated multiple range test (Zar, 1974), nonparametric multiple range
testing by rank sums (Zar, 1974), linear regression, and Pearson correlation.
In standard curve regressions, ATP net count and concentration data were
log-transformed. It was initially determined that log-transformed count data
satisfy the assumptions of homoscedasticity and normality. Pearson
correlation coeficients corresponding to these log-log regressions were
calculated.
Results
Adenine Nucleotide Methodology Experiments—
Overview—Table 7.4 summarizes tested factors and their associated
treatment levels, grouped under the appropriate analytical procedure.
Standards and blanks, used to quantify samples and internal standard recovery
and conversion, were processed in parallel with samples and internal standards
for the following factors: extractant, all conversion factors, all FLE
preparation factors, and photometer counting mode.
Sample collection and preparation—Eight harvest-freeze delay periods
are compared in Table 7.5. The delay period represents the time interval
between uprooting the plants and freezing in liquid nitrigen. ATP, AT, and EC
generally increased as delay period lengthened. These trends are shown
graphically in Figure 7.5. Associated regression statistics are presented in
177
A
-------
CO
OS
o
u
b.
O
bl
H
CO
H
b.
O
•^
.y*
3
B
^
1^
bl
BQ
(-<
0)
t^
0)
>
01
pj
u
o
4J
u
to
b.
OJ
1.
3
•o
01
u
o
LJ
Du
C
• H
B
O
vO
A
O
(^
•»
o
•— »
*
I.A
•»
CM
«
»~4
M
tA
•
•M
m
CM
'
(0
ff*
01
•e
4)
N
01
0)
fc*
1*4
1
4U
01
01
^
H)
X
01
*-4
D.
E
10
CO
d
o
•i^
«J
U
01
f— 4
o
o
X™\ *"N
C C
'a 'a
0 0
l-H •••*
M M
u u
01 01
09 09
O O
en en
to
10
f?
u
X
60
• •4
C
09
>
^
CO
•a
e
o
>*4
^
10
%4
a.
*T3 4)
C 1*
CO Bu
s^
^
in
CM
s.^
•o
c
09 3
> O
t-
x^ 00
B i
i -o
01
m N
^•^ *f^
i— *
•o ••<
oi x
a. a
a o
o x
X ^4
U 1
1 C
X 0)
U) N
oi o
U U
14-1 14-4
01
U
CO
a
01
3
0)
09
••4
"
14-1
CO
01
T3
0)
a
to
^
u
^
r*
a
.^4
D.
01
09
>^
CO
•O
O
CM
•t
in
M
O
1
e
01
14
o
^4
u-c
14-1
O OI
3
01 01
00 CD
CO ••<
o
u tj
m C
3
J* 0
kl kt
CO 00
•a I
i •«
•0 01
0) N
U *»4
03 -4
U —l
0 X
.-. a
CD O
01 X
•0 •-
0
01
r-. N
35 ^
a c
tx **•'
^^ «£ "^
- > H C
x-x ^. a o
\O S W -H
• ^S M 4.4
r^ W 35 tj
t^C 4) B co
X m 4-1 i*
a. 4j
— + » X
+ 0)
H H 4> >» k4
a a -> o oi
b] W *~ * to *J U
.H CM*** 0)
£ je 4j x co o)
B B to
••* Z t»N O
^ — -O • CM
*O r*« 1-4
00 00 00 • 1
c c e *o
.* .H -^ O • O
^-i _ i —4 » r~ en
•»4 .H -i^ ^
O O O 1 X
JQ ja ^ o a. m
• ^4
4-t
*J C
C 0
a) -rf
u u
U U
to to
Li Li
*J 4J
X X
OI 01
d
o
,p4
4J
U
CO
14
X
bl
OB
4J
d
CO
a.
79
^4
o
o
a.
ao a
> X
<0
-* 13
CO
3 O
t3 CM
If4
^ •*
•M in
•w
d -
• P4 O
«
op
CO
Ll
O
4J
0) 09
3
09 4J
a u
.-1 CO
«J W
4J
•o x
01 01
u
u d
CO 01
Li N
*•» O
X Li
01 M-l
^
•o
01
3
d
•r*
d
o
u
••^
178
-------
•Q
4)
3
•r*
u
c
O
u
>-x
.
»}•
r».
U
J
BO
<
?
CO
^*
01
>
41
J
hi
O
4-1
U
eg
PL,
41
U
3
•a
41
u
o
hi
0*
*-* '•^
MS -«
r» oo
SB X
CL. ft.
>-x s«^
•* •*
o o
en eo
CM eN
tt u
x-T G ^£ £
MS - • MS B B
. <~* F*
I** MS r» m in
. -c • •
X r- (X 33 r» r-
D. >— ' Q.
^ a -^ + +
a -*
-*^ O --t •* -»
O to O O O
en si- oo en e/j to
oo o Z « oo oo oo
Z en >~* z s £
_ *° 5 >°
|Z% r,'iii
S MS T3
MS B X MS MS MS 41
+ O. 4J
+. MS -^ + + + eg
— • 41
td + O (dbJUId £
Z X Z Z Z Z
i-i en i M M M M 4J
o w en u u u u o
l-4(V,t-l MMMM C
as u a: as as a: at
HZH HHHH
T3
ill ^i^i 2
eg
inmm mmmin 41
c
m o
hi •-
O 4-1
4^ eg
u >
0} •*
U-l 4J
o o
u eg
41
hi 41 -a
4i i
>4-l X 4J
«-i N eg
3 C 41
J2 1) £
C
0
• lp4
en
hi
41
e
o
o
M
*^ ^^
MS —
r- oo *-N
U
S X •
a a m
•-' ^* eN
-.t -*
O O -*
w en s^
00 00
£ £ «
hi
£ £ £
B 1
^>
CO CO CM
~* i-^
M t
hi +• + ^N _
4) U 1
4-1 [I] W • ••*
eg z z m e
3 l-l l-l CM
u u BO
tJ l-l M « 3.
4i as a! "> •*
a
•f +
o
- X X O
4j eg eg *•*
X ^^ *— '
60 41 41
••* -O -B O
41 in
.COO
41 41
JK eo co O
eg CM
Si. 2 2
V
B
3
O
?*
S
u.
"i
4> 3
•Q t"^
1 5
60 TJ
C hi
•M eg
u T3
e c
3 eg
0 4J
U CO
X
hi
u
eu
O
4J
0
£
a.
S
[X.
r-4
3
O
o
CM
•
o
O
•>
O
m
«
in
CM
•i
m
^^
eu
1
u
60
• *4
01
Si
X.
eg
eu
0.
«tax
179
-------
M
s
»M
s
o
z
u
h-4
Z
u
o
I
N
U]
3
(b
H IN
M I
U] C
is
g"S
U • I-
^•°
60
H 3
er
C** V
o
Ou
H H
O <
U. 60
U. 3-
Ul ^
U
PO
JS
XI
CO
co
•H
U-l
O
CM
co
•H
0>
00
_^
44
T5
—
vO
•H
u
v£>
CM
-.
•H
a
0
-»
m
O
44
18
O
sO
U3
180
m
O
04
'u'
X
C
id
u
C
oo
cu
U-l
• H
•o
o
o
CO
4J
C
BJ
0)
C
Ol
!
1
u
CO
41
a
0)
cu
0)
10 O
4j M
—i CM
-------
•o
'o»
a
4
S
500-
400-
300-
200-
100-
AT
ATP
-.6
•2
L0
10'
IOJ
TIME (sec)
Figure 7.5. Semi-log regressions of ATP, AT, and EC vs. harvest-freeze
delay interval (n = 2).
181
-------
Table 7.6. Results suggest that adenylate concentrations reflect in situ
levels for a period <2 minutes following harvest.
Adenylate levels in plants harvested during the day or at night, each at
two delay intervals, appear in Table 7 7. Results suggest that increases in
ATP, AT, and EC (as delay period lengthens) are light-related, since
corresponding increase was not observed at night. It is of interest to note
that EC was higher at night than during the day for the 30 sec delay.
Fresh-chopped vs. frozen-lyophilized-ground tissue is compared in Table
7.8. Although data show no significant difference, variability (i.e. standard
error) in the lyophilized tissue was considerably lower, reflecting increased
homogeneity of the quick-frozen, lyophilized, and more finely ground tissue.
Fresh tissue was held (4 hrs) in river water at in situ temperature and light
levels prior to processing.
The effect of epiphytes was evaluated with scraped vs. unscraped
lyophilized leaves (Table 7.9). ATP, ADP, AT, and EC were significantly lower
in unscraped tissue than in scraped tissue. The decreases was apparently due
to low epiphyte adenylate levels.
Two modes of sample storage are evaluated in Table 7.10. Desiccated-dark
storge of frozen-lyophilized-ground tissue and frozen extract storage were
both suitable over 5 days, but not 20 days. AT significantly decreased in
both preparations over a 20 day storage period.
Extraction—Four extractants are compared in Table 7.11. The
superiority of boiling 1 mM EDTA + 52 PVPP (pH 7.6) was evident, among those
extractants tested. Without addition of PVPP to the EDTA solution, light
output was reduced and firefly reaction kinetics did not display their
characteristic decay pattern (Figure 7.6). Data on recovery of added
adenylates (internal standard) appear in Table 7.12. Again, the superiority
of boiling 1 mM EDTA + 5% PVPP (pH 7.6) was evident. Standards, prepared in
EDTA, quenched light output to a lesser extent than those prepared in either
distilled water or neutralized acid (Table 7.13).
Duration of three extraction times is evaluated in Table 7.14. No
significant differences were observed for extraction times of 5, 30, or 120
sec.
Extraction of individual plants vs. extraction of multiple aliquots from
a pooled sample was compared in Table 7.15. Adenylates show no significant
difference, although variability (i.e. standard error) in the pooled plant
sample is considerably lower, as would be expected. Pooling masked natural
variability between plants but yielded mean adenylate levels, similar to those
obtained from individually extracted plants. Standard errors, associated with
individually extracted plants, provide information on adenylate variability
between plants in the field.
Conversion—Methodology experiments in the conversion procedure were
tested by calculating conversion efficiency of AMP and ADP (internal standard)
182
-------
TABLE 7.6. SEMI-LOG REGRESSION (N-8) STATISTICS FOR HARVEST-FREEZE
DELAY
Statistic ATP AT EC
Slope .3991 .3431 .0559
Intercept .8294 2.6321 .5188
Pearson Correlation
Coefficient .8507* .8729* .7078*
* P < .05
183
-------
TABLE 7.7. EFFECT OF DAY VS NIGHT HARVEST, AT TWO FREEZE DELAY
INTERVALS, OH ADENiNE NUCLEOTIDES
(ug ATP equiv g"1 dry wt) and EC ( n-4)
Variable
ATP
ADP
AMP3
AT
EC
Day (
30 sec
I73al ± 42
92a t 3
52a ± 1
317a t 5
./Oa ± <.01
1200 hrs)
10 min
227b ± 4
84a ± 4
34 a i 4
344b t 8
.78b t .01
Night (2400 hrs)
30 sec
16<»a ± 6
53b ± 3
33a t 1
254C ± 8
.77bc t .01
10 min
167a ± 3
59b t 1
35a ± 2
260C ± 3
.76C ± .01
• Values with same letter superscripts (between treatments) do not
differ significjntly (P > .05).
2 Standard error.
3 Although the Kruskal-Wallis test shows a signifincant difference,
the nonparametrie multiple range test failed to detect differences
between any pair of means for AMP.
184
j
-------
•V"/ -' : /
TABLE 7.8. EFFECT OF TISSUE STATE ON ADENINE NUCLEOTIDES
( Ug ATP equiv g-1 dry wt) and EC (n-4)
Variable
ATP
ADP
AMP
AT
EC
Fresh-Chopped
(5 mm)
226al
192a
112a
530a
.61a
± 212
t 36
t 14
t 71
t .01
Frozen-Lyophylized-
Ground (425 )
253a
151a
129a
533a
.62a
t 4
± 15
t 4
i 20
± .Cl
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard erro'-.
185
-------
TABLE 7.9. EFFECT OF EPIPHYTE REMOVAL, BY SCRAPING LYOPHILIZED LEAF
TISSUE, ON ADENINE NUCLEOTIDES
ATP equiv g"1 dry wt) and EC (n-4)
Variable
ATP
ADP
AMP
AT
EC
Scraped Leaf
313a2 ± 23
91a ± 1
106a t 5
509a i 7
.71a ± .01
Unscraped Leaf
253b ± 1
84b ± 1
95a ± 4
432b ± 3
.68b ± .01
Scrapings*
(Epiphytes)
43 * 2
33 ± 1
25 ± 1
101 t 2
.59 ± .01
Scrapings excluded from comparison test.
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard error.
186
-------
TABLE 7.10. EFFECT OF TWO STORAGE METHODS AT 5 AND 20 DAYS ON ADENINE
NUCLEOTIDES
(ug ATP equiv g"1 dry wt) and EC (n"4)
Variable Initial
ATP
ADP
AMP
AT
EC
278al ± 42
91a t 5
120a t 3
489a t 10
.66a t <.01
5 Days
Frozen-
Lyophilized-
G round and
Desiccated-
Dark
267a ± 3
96a ± 4
114ab t 2
477a t 4
.66a t <.0
20 Days
Frozen Frozen-
Extract Lyophilized-
(-20°C) Ground and
Desiccateu-
Frozen
Extract
(-20*C)
Dark
277a ± 4
o>7ab ± 2
112ab ± 4
475a ± 8
1 .67a ± .01
248b ± 3
88ab ± 3
107b ± 3
443b ± 6
.66a t <.0
272a ±
75b t
84C t
430b ±
1 .72b t
4
3
3
10
<.01
Values with same letter superscripts (between treatments) do not
differ sig^ificani-ly (P > .05).
Standard error.
187
-------
TABLE 7.11. EFFECT OF EXTRACTANT ON ADENINE NUCLEOTIDES
(wg ATP equiv g~l dry wt) and EC (n-4)
Variable Boiling 1 mM Boiling 1 mM Boiling 0-4°C .6N
EDTA + 52 EDTA (pH 7.6) Distilled H2S04 + 1 mM
PVPP (pH 7.o) Water EOTA (neutralized
to pH 7.6-7.9 with
HaOH after extraction)
ATP
ADP
AMP
AT
EC
144al
102a
108 a
354a
.55*
±
t
t
±
t
22
2
4
6
<.0l
27b ± <1
25b ± <1
61b ± 1
113b 4 1
31b
23b
29C
82C
.52C
±
±
t
±
±
1
1
1
1
.01
38*> t
22b t
27e *
87<= ±
.57a ±
4
2
5
11
.01
Values with same letter superscripts (between treatments) do not differ
significantly (P > .05).
Standard error.
188
-------
EDTA + PVPP
EDTA
60
of
60
TIME (sec)
Figure 7.6. Reaction kinetics, obtained from EPTA extraction of sample
with and without PVPP addition. FLE is injected at time zero,
the tube is vortexed, inserted into the photometer, and the
shutter is opened (indicated by arrow).
189
-------
TABLE 7.12. EFFECT OF EXTRACTANT ON RECOVERY (V OF 200 NG MI/"1 ATP,
ADP.AMP ADDED IMMEDIATELY PRIOR TO EXTRACTION (N-4)
Variable
ATP
ADP
AMP
Boiling 1 mM
EDTA + 52
PVPP (pH 7.6)
82*1 ± 42
83a ± 22
112« t 12
Boiling 1 mM
EDTA (pH 7.6)
17b ± 1
31b ± 4
64b ± 10
Boiling
Distilled
Water
22b l 2
25b t 5
51b 1 7
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard error
-r
190
-------
TABLE 7.13. EFFECT OF EXTRACTANT ON LIGHT OUTPUT (NET COUNTS) (N-2)
ATP [ATP] Reagents JATP+ADP] Reagents [ATP+ADP+AMP] Reagents
Standard
(ng ml"1) XYZXYZXYZ
4000 75096 47370 13760 66377 38828 12841 53072 32408 12682
40 426 287 86 350 252 83 294 204 77
X - 1 mM EDTA (pH 7.6)
Y - Distilled Water
Z - .6N H2S04 ••• 1 mM EDTA (neutralized)
191
-------
TABLE 7.14. EFFECT OF EXTRACTION DURATION ON ADENINE NUCLEOTIDES
ATP equiv g"1 dry wt) and EC (n-4)
Extraction Duration (sec)
Variable
ATP
ADP
AMP
AT
EC
165al
142 a
116a
423a
.56a
5
±
t
±
±
t
42
9
11
5
.01
178a
133a
144 a
455a
.54a
30
±
t
±
±
±
4
4
11
10
.01
120
162a ± 8
141a t 8
llla ± 6
414a ± 22
.57a ± <.01
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard error.
X
192
\
\J
-------
TABLE 7.15. EFFECT OF POOLING PLANTS ON ADENINE NUCLEOTIDES
ATP equiv g"1 dry wt) and EC (n"4)
Variable
ATP
ADP
AMP
AT
EC
Individual Plants
369al
65 a
66 a
4993
.80a
± II2
t 5
± 4
± 16
t .01
Pooled Plants
372a ±
62a ±
68a ±
501a ±
,81a t
4
1
3
6
<.01
* Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
^ Standard error.
193
-------
to ATP. Concentrations given for treatment levels refer to the conversion
reaction mixture.
Three buffers are evaluated in Table 7.16. AMP and ADF conversion
efficiencies show no significant difference among the three buffers. TRICINE
yielded the highest light output (Table 7.17).
Conversion enzyme cofactors are compared in Table 7.18. Results 'ndicate
that MgSC>4 is abolutely required, but that l^SO^ is not. Furthermore, K2i»04
addition may decrease conversion accuracy. MgSC-4, 1(2804, and higher pH all
quenched light emission (Table 7.19).
The effect of heat deactivation on AMP and ADP conversion was assessed in
Table 7.20. It is clear that this procedural step was essential. Without
heat deactivation, ATP was produced in the presence of PEP and PK, presumably
from ADP contained within the crude firefly lantern extract (Figure 7.7).
With heat deactivation, ATP was not produced, and firefly reaction kinetics
displayed their characteristic decay pattern.
Firefly lantern extract preparation—Three solutions to reconstitute
lyophilized firefly lantern extract (FLE) are compared in Table 7.21. One
vial of Sigma FLE-50 was trisected by weight to minimize FLE variability.
Specified MgSC<4 concentration is exogenous, since Sigma FLE-50 also contains
MgSC<4. The buffer solution at pH 8.1 resulted in significantly lower AMP and
AT than either of the other reconstituents tested.
FLE aging times and temperatures are evaluated in Table 7.22. Sample
extract was frozen between 6 and 24 hr assays, in order to minimize adenylate
degradation. Although the 24 hr ATP levels were significantly higher than the
6 hr levels, the magnitude of the increase was slight. Significance resulted
from the low variability within treatments. No other adenylate differences
were observed. As both aging time and temperature increase, light output was
reduced (Table 7.23).
The effect of guanosine diphosphate (GOP) addition to FLE was examined.
ATP may be produced from ADP in the presence of guanosine triphosphate (GTP),
or any other nucleoside triphosphate (NT?), and nucleoside diphosphokinase
(NDPK). Results show no difference in adenylate levels (Table 7.24), however
firefly reaction kinetics differ markedly (Figure 7.8). With GDP addition,
light output was reduced and decay was more rapid in both standards and
samples.
Photometry—Three photometer counting modes are evaluated in Table 7.25.
Although ATP levels differed significantly among the three modes, the
magnitudes of these differences were not large. No other adenylate
differences were observed. Log-log standard regressions, derived from the
three counting modes, were compared for ATP Reagents (Table 7.26). Slopes
show no difference, but intercepts were significantly higher for the 30 sec
integration. Correlation coefficients vere highly significant. These
regressions are plotted in Figure 7.9.
194
-------
TABLE 7.16. EFFECT OF BUFFER ON AMP AND ADP CONVERSION EFFICIENCY (Z),
USING 80 NG ML"1 ATP,ADP,AMP (N-3)
Variable
ADP
AMP
15 mM TRICINE
+ 6 mM MgSO^
(pH 7.6)
106al ± 22
83a t 5
15 mM HEPES
+ 6 mM MgS04
(pH 7.6)
103a ± 2
75a ± 5
15 mM TRIS-HC1
* 6 mM MgSO^
(pH 7.6)
104a ± 3
84a ± 4
Values with same letter superscripts (between treatments) do not differ
significantly (P > .05).
Standard error.
195
-------
TABLE 7.17. EFFECT OF BUFFER ON LIGHT OUTPUT (NET COUNTS) (N-l)
ATP [ATP] Reagents [ATP+ADP] Reagents [ATP+ALP+AMP] Reagents
Standard
(ng ml'1) XYZXYZXYZ
4000 59163 54416 53769 46227 45020 43242 38650 37885 37091
40 367 343 323 303 283 269 294 264 258
X - 15 raM TRICINE * 6 tnM MgSO^ (pH 7.6)
Y - 15 mM HEPES + 6 mM MgS04 (pH 7.6)
Z • 15 mM TRIS-HC1 + 6 mM MgS04 (pH 7.6>
196
-------
TABLE 7.18. EFFECT OF ENZYME COFACTORS ON AMP AND ADP CONVERSION
EFFICIENCY U), USING 80 NG ML"1 ATP,ADP,AMP (N-3)
Variable
ADP
AMP
15 mM
TRICINE
(pH 7.6)
-1-1 * 12
15 mM
TRICINE
+ 6 mM MgSO^
(pH 7.6)
108b t 4
110b ± 3
15 mM
TRICINE
f 6 mM MgS04
+ 7.5 mM K2S04
(pH 7.6)
114b ± 2
85ab i <1
15 mM
TRICINE
+ 6 mM MgSO^
* 7.5 mM K2S04
(pH 8.1)
114b ± 1
82ab ± 4
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard error.
Group 1 shows no difference with groups 3 and 4 for AMP conversion,
because the nonparametric multiple range test uses ranks.
197
-------
•
z
^
to
3
o
H
W
Z
e^
«^
0*
H
8
r-
O
^^
"
_
O
C/3
1
^
u.
O
0
8
y^
Ul
U.
0
H
U)
u.
bk
Ul
t
ON
,
•*•
W
oa
.
e
01
00
(0
QJ
OS
^^
gj
5
+
ft.
0.
H
^•^
SJ
X
3
in
C
00
41
ac
a.
a
*
Q.
N
>.
X
3
1
u
C
U
CK
<*
t>
06
^
O.
H
<
N
X
3
^^
•o— •
U 1
ro -•
0. T3 E
% 5 eo
CM -<
ON
IV ~t>
(*i
CM
O CM
^5 ^^
•—4
in -ft
in <£>
CO "^
O
m
in en
r- CM
r* -^
CM
ON f-
ON —
f^
*"*
g\ "*
en ^j
CM CM
f^»
r- r-
CM CM
00
«n
en CM
00 — •
00
in r^.
^ f^
— O
in ^^
en CM
m
^
tn m
m ao
f»- CM
00
o o
o * -*
c o
en en
CM CM
* :*
r~ in in
3; r- r-
^ •«• +
to en in
oc oo ob
~ s: r r
^9
* ? ? 5
**•• E c n
a.
V— + * +
UJ U Ul U"
2 z z *:
cj o o u
S Otf BC OS
H H H H
_ _ 3. _
B t ^ B
in m in ^^
NIDI
3 x >• N
198
-------
TABLE 7.20. EFFECT OF HEAT DEACTIVATION ON AMP AND ADP CONVERSION
EFFICIENCY (2), USING 80 NG ML-1 ATP,ADP,AMP (N-4)
Variable Heat No
(2 min, 100*C) Heat
ADP lllal ± 42 55b ± 13
AMP 102a ± 7 185b ± 14
* Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
2 Standard error.
199
-------
5
0) tJ
w C
o *
V
.1-1 •
c t-
• H 4)
"0-0
Q. - «
t- * X
< Q. C
c
o
r
bo
e £
O U
'u O
a u
> c
o -a
a « •
« -u -^
•o i- ^
w o
4J CD h
w c i-
4) -i-( <8
* ->,
4J -O Ji
3 «
O K -O
X at v
u .*
-o -o
c « c
o
O . «•
00.0
U
w
a.
O
2
W «
W «.H
-S 2 w
J* V 41
N u
e <->
O g 3
.* e j:
4J >M 0
O «•'
10 «l
«J u f.
£ ta u
LO
ea
200
-------
TABLE 7.21. EFFECT OF FLE RECONSTITUENT ON ADENINE NUCLEOTIDES
(yg ATP equiv g~l dry wt) and EC (n-4)
Variable
ATP
ADP
AMP
AT
EC
Distilled
Water
92 a 1 ± 1 2
65a ± 1
65a ± 3
221a ± 4
.57a ± .01
45 mM TRICINE
+ 18 mM MgS04
(pH 7.6)
93a ± 1
66a ± 2
67a ± 3
226a ± 2
.56a ± .01
45 mM TRICINE
+ 18 mM MgS04
(pH 8.1)
92a ± 1
62a ± 1
54b ± 1
208b ± 1
.59b ± <.01
Values with same letter superscripts (between tieatments) do not
differ significantly (P > .05).
Standard error.
201
-------
TABLE 7.22. EFFECT OF FLE AGING TIME AND TEMPERATURE ON ADENINE
NUCLEOTIDES
(pg ATP equiv g"1 dry wt) and EC ( n-4)
Variable
ATP
ADP
AMP
AT
EC
88a*
72 a
48 a
208 a
.60a
6
4'C
* 12
t 1
± 4
± 3
± .01
hr
25 "C
89a * 1
70a ± 2
60a ± 1
218a ± 2
.57a * <.01
4'C
91b ± 1
67a t 3
56a ± 3
214a ± 2
.58a ± .01
24 hr
25'C
93b t 1
68a ± 2
55a ± 3
215a ± 2
.59a t <.01
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05)
Standard error.
202
-------
, I
I ,
I
/•N
CM
II
Z
^s
*~*.
CO
H
Z
o
u
H
Id
Z
H
^
ft.
H
3
o
H
X
O
M
»J
Z
o
i
H
OS
Id
OH
£
B-
O
1
s
H
O
Z
1-4
Q
bd
£
O
H
td
Cx
U.
Id
•
»• F
CM
r-.
Id
03
H
to
C
01
60
ID
41
at
•f
CU
5
DM
H
i_^
N
^4
>^
3
to
4_l
C
41
00
CO
Ou
^^
BK
^
eu
H
N
^4
X
3
09
C
41
00
C/5 >--
m
O
m
CM
m
^
^^
CM
5
sO
o
l/^
p*»
fx.
o
iTt
fs.
r*^
pSh
^H
m
CM
00
O
in
^^
m
00
O
m
CM
O
<£>
^
m
00
vD
•*
en
CO
m
r^
ON
vD
CM
O
vO
i*^
00
o
o
o
vO
*^
-H
^
CM
CM
O
CM
vO
0
^
00
f— t
CO
00
CM
•_!
Is*.
CM
-a-
00
en
vO
vO
CM
0
00
00
CO
vO
ro
m
o
U U
0 0 • •
• • Wi £ JS
*c ^s
•41 *^
\O VO CM CM
* » » »
3 X >« N
203
-------
TABLE 7.2b. EFFECT OH GDP ADDITION TO FLE ON
ADENINE NUCLEOTIDES
(yg ATP equiv g~^ dry wt) and EC
(n-4)
Variable
ATP
ADP
AMP
AT
EC
No
104al
62a
35 a
202 a
.67a
GDP
±
±
±
t
t
6.85 ug ml"1 GDP
12
2
2
2
.01
104a
65 a
38 a
206 a
.66a
± 2
± 6
± 1
± 1
*<.01
Values with same letter superscripts (between
treatments) do not differ significantly
(P > .05).
Standard error.
/
204
-------
90400
GDP = 0
ATP=4000ng ml"
60
579
X
ATP = 40ng ml"'
O* 60
TIME (sec)
18938
SAMPLE
0< 60
46519
GDP= 6.85 ug ml'
lATP = 4000ng ml"
399
ATP=40ngml"
60
Of 60
TIME (sec)
10095
\
SAMPLE
"I' '
Of 60
Figure 7.8. Reaction kinetics with and without GDP addition. FLE is
injected at time zero, the tube is vortexed, inserted into
the photometer, and the shutter is opened (indicated by
arrow). Counts represent a 10 sec integration period,
immediately following a 10 sec delay from time zero.
205
-------
TABLE 7.25. EFFECT OF PHOTOMETER COUNTING MODE ON ADENINE
NUCLEOTIDES
ATP equiv s"1 dry wt) and EC (n»4)
Variable
ATP
ADP
AMP
AT
EC
10 Sec Delay
10 sec Integral
162al ± 22
161 a * 7
230a ± 2
552a ± 5
.44a ± <.01
followed by:
30 sec Integral
171b ± 2
145a ± 4
235a ± 17
551a ± 14
.44a t .01
Peak Height
150C ± 2
141» ± 6
238a ± 7
529a t 7
.42« ± <.01
Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
Standard error.
206
-------
TABLE 7.26. COMPARISON OF LOG-LOG REGRESSION (N-O STATISTICS,
OBTAINED FROM THREE PHOTOMETER COUNTING MODES WITH [ATP]
REAGENTS
Statistic
Slope
Intercept
Pearson Correlation
Coefficient
10 Sec Delay
followed by:
10 Sec
Integral
1.1150al
10.9392a
.9989*
30 Sec
Integral
1.12898
11.4838b
.9985*
Peak
Height
1.0699a
10.6016s
.9999*
1 Values with same letter superscripts (between treatments) do not
differ significantly (P > .05).
* P < .001
207
-------
-\
.0%
.05H
O
o
UJ
to
10
o 10 Sec Delay 4 10 Sec Integral
a 10 Sec Delay + 30 Sec Integral
A Peak Height
10
STANDARD ATP (g ml"1)
10'
To5
Figure 7.9. Comparison of photometer counting modes with ATP reagents
(n - 2). 208
-------
Mixing kinetics with the peak height mode, using a 6 x 50 on tube, were
examined by varying the ATP standard/FLE volume ratio (Table 7.27). Proper
mixing was evaluated, as described in Figure 7.10. Although several
standard/FLE volume ratios mixed properly (i.e. 20/15, 50/15, 100/50), samples
would not consistently mix well. Therefore, whenever peak height was used,
proper mixing was evaluated.
Standard curve—Six standards and one blank were routinely run for each
reaction Tube A, B, and C. Using a 10 sec delay followed by a 10 sec
integration, representative standards, net mean counts, and
regression-calculated ATP concentrations appear in Table 7.28. Log-log
regression plots are shown in Figure 7.11. The associated statistics are
presented in Table 7.29. Correlation coefficients were highly significant.
Differential quenching of light output was apparent among the three
regressions.
Analytical variability—Optimized recovery and conversion efficiencies
were presented in Table 7.30. Since these efficiencies were near 100Z with
relatively low variability (i.e. small standard error), no correction factors
were applied in data reduction.
Photometer variability, expressed as coefficient of variation, appears in
Table 7.31. Coefficients were A -nerally <2Z, with the exception of blank
readings. Higher coefficients for blanks were the mathematical result of
division by a small mean rather than multiplication by a large standard
deviation. These data were based on a 10 aec delay, followed by a 10 sec
integration.
Tissue Comparisons—
Zostera marina—Adenylate levels in four types of tissues from Z_. marina
are presented in Table 7.32. Leaf tissue clearly had the highest level of
ATP, ADP, AT, and EC, while root plus rhizome tissue showed the lowest
measured level? of ATP, ADP, AMP, and AT. An adenylate correlation matrix was
derived by pooling values from all four tissues (Table 7.33). ATP was
positively correlated with ADP, AT, and EC, while ADP was positively
correlated with AT and EC. Environmental and morphometric data, associated
with this eelgrass sample, are presented in Table 7.34.
Other species—Adenylate levels in "L. marina epiphytes, aboveground
Ruppia~maritima (widgeongrass), and aboveground Spartina alterniflora
(saltmarsh cordgrass) appear in Table 7.35 for comparative purposes. _Z.
marina leaf tissue and aboveground jl. maritima had comparable adenylate
concentrations. Both were higher than either the epiphytes or aboveground S_.
alterniflora tissue. Environmental data, associated with collection of these
samples, are presented in Table 7.36.
Seasonal Survey—
Monthly mean aboveground adenylates (Figure 7.12), belowground adenylates
(Figure 7.13), and resultant EC values (Figure 7.14) in Z. marina are plotted.
Each of these time series contained significant differences (p X.05) over the
one year period. Adenylates and EC were generally higher in aboveground
tissue.
209
-------
TABLE 7.27. EXAMINATION OF REAGENT MIXING IN PEAK HEIGHT MODE
(COUNTS) tN-5)
ATP
Standard
Volume
(ul)
20
50
100
Statistic
X
8/X
Z PM1
X
s/x
Z PM
X
s/x
Z PM
15
4532
.08
100
3625
.04
100
1287
.33
0
FLE
25
8314
.11
80
9776
.11
80
6077
.08
0
Volume
50
13310
.19
60
19896
.06
0
24241
.03
100
(ul)
100
8452
.28
0
22097
.22
0
40259
.78
20
200
10038
.07
0
26449
.15
0
38310
..13
0
Properly Mixed Tubes.
210
-------
N
PROPER
MIXING
IMPROPER
MIXING
60
60
TIME (sec)
Figure 7.10. Mixing kinetics in peak height mode. The sample tube is
inserted into the photometer, and FLE is injected at time
zero with the electronic pipet system which simultaneously
activates the photometer. After 15 sec, the tube is removed,
vortexed, and re-inserted into the photometer. Continuity
in decay kinetics indicates proper initial mixing.
211
-------
a,
H
^
C3 ^J
u *^
si
S z
K ••*
OU
U
a w
Z CO
^
O
^ ""*
m <;
H
z >-
^•^ Oil
en O
gz o
J
U -1
o
z o.
UJ ^*
2 ^
wJ
t^ dj
u o
z
u
* till
Q
a: o
o
5
H 0
CO Z
(-4
2 CO
0 =>
CO -
CO CO
w z
B! 0
0 H
X ^1
3C
U H
5 z
1 U
Q O
-5 u
.
oo
.
r*»
U]
BQ
H
U9
C
01
00
4
X
X
^*
<^s
^4
|
cu — •
H e
§c
c
s«r
m 00 «S CN Ov 00
(*^ ^H t/^ CN ^^ r^
r-«. o O -^
r-» CN — •
00 CN (S ^ O\ \f\
•^ \f\ r""* •—* op QO
O ^ -^ *^ O PO
00 ON -^ \f\ ^^
iA es — *
^h ^^ ^^ r^ ^^ oO
-^ o i/> c-4 ^ rn
r^ ^5 ^3 ^j
m O rn
r^ a» O "3" ^«
00 u^ CN OO ON ^
m «^ r*» *n r^ in
Os m CM oo — «
00 •* X
212
-------
•O4^
to
H-
Z
r>
o
•o3H
10
10
•8
O [ATP] Reagents
O [ATP + ADP] Reagents
a [ATP + ADP + AMP] Reagents
10
.7
10'
10"
STANDARD ATP (gmf
Figure 7.11. Standard curve regressions, using a 10 sec delay followed
by a 10 sec integration (n - 3).
213
-------
TABLE 7.29. LOG-LOG REGRESSION (N-6) STATISTICS, USING A
10 SEC DELAY FOLLOWED BY A 10 SEC INTEGRAL
Statistic
Slope
Intercept
Pearson
Correlation
Coefficient
[ATP]
Reagents
1.0932
10.8890
.9994*
[ATP+ADP]
Reagents
1.0957
10.7902
.9995*
[ATP+ADP+AMP]
Reagents
1.0963
10.7144
.9996*
* P < .001
214
-------
TABLE 7.30. RECOVERY AND CONVERSION EFFICIENCY (Z) WITH OPTIMIZED METHOD
(N-4)
Variable
ATP
ADP
AMP
Recovery:
200 ng ml"1 ATP , ADP , AMP
added immediately prior
to extraction
109 ± 91
96 t 5
97 ± 4
Conversion:
ATP.ADP.AMP Standard (ng ml"1]
1000 80
-
102 t 1 104 t
108 ±2 96 ±
>
1
2
* Standard error.
215
..-r
-------
TABLE 7.31. PHOTOMETER VARIABILITY (COEFFICIENT OF VARIATION) WITH
OPTIMIZED METHOD (N-5)
Standard ATP [ATP] Reagents [ATP+ADP] [ATP+ADP+AMP]
(ng ml"1) Reagents Reagents
Blank .250 .026 .057
4000 .010 .006 .010
2000 .006 .012 .005
1000 .010 .012 .015
400 .007 .008 .019
100 .010 .011 .016
40 .015 .022 .003
216
-------
TABLE 7.32. ADENINE NUCLEOTIDES ( G ATP EQUIV G"1 DRY WT) AND EC IN FOUR
TYPES OF TISSUE FROM Z. MARINA (N-4)
Variable
ATP
ADP
AMP
AT
EC
Leaf
245al ± 22
95a t 1
47a ± 4
387a ± 5
.76a ± .01
Leaf Sheath
72b ± <1
49b ± 2
55a t 3
175b ± 5
.55b ± .01
Root •*• Rhizome
34C ± <1
13C ± 1
27b ± <1
74C ± 1
.55b t <.01
Seed Pod
129d ± 3
63d ± 2
108C ± 14
299d t 13
.54b ± .03
Values with same letter superscripts (between treatments) do not differ
significantly (P > .05).
Standard error.
217
-------
TABLE 7.33. SPEARMAN CORRELATION COEFFICIENTS
AMONG ADENINE NUCLEOTIDES AND EC,
OBTAINED BY POOLING VALUES FROM
FOUR TISSUE TYPES (N-16)
ATP
ADP
AMP
AT
ADP AMP AT
.9512* .4490 .9608*
.4240 .9594*
.5018
EC
.6206*
.5871*
-.2724
.4682
* P < .05
218
-------
TABLE 7.34. ENVIRONMENTAL DATA AND MORPHO-
METRICS FOR Z. MARINA. USED
IN TISSUE STUDY
1981 Harvest (mo) May
Low Tide
EST (hr) 1214
Height (m) .1
Salinity (°/oo) 22.58
pH 8.00
Water Temp. CO 23.8
38*N Daylength
(hr-min) 14-15
Density (shoots m~2) 1333
Shoot Length (cm')
x ± SE (n) 25.8 ± 1.4 (40)
Live Dry Wt (g m~2)
Aboveground 291
Belowground 109
Total 400
219
-------
TABLE 7.35. ADENINE NUCLEOTIDES (yG ATP EQUIV G~l DRY WT) AND
EC IN £. MARINA EPIPHYTES (N-4), ABOVEGROUND
RUPPIA MARITIMA (N-2), AND ABOVEGROUND SPARTINA
Variable
ATP
ADP
AMP
AT
EC
ALTERNIFLORA
Epiphytes
43 ± 21
33 ± 1
25 ± 1
101 ± 2
.59 t .01
(N-4)
R. maritima
215 ± 5
137 ± <1
41 l 8
39A i 3
.72 t .02
S. alterniflora
87 * 1
69 ± 1
33 t 1
189 ± 2
.64 * <.01
Standard error.
220
-------
./
TABLE 7.36. ENVIRONMENTAL DATA FOR COLLECTION OF EPIPHYTES, 1R. MARITIMA,
AND S. ALTERNIFLORA
Variable Epiphytes jl. maritima S_. alterniflora
1981 Harvest (mo) Jul Jun Apr
Low Tide
EST (hr) 0951 1336 1702
Height (m) .1 -.1 0
Salinity (°/oo) 20.87 20.42 22.89
pH 7.86 8.12 8.02
Water Temp. CO 28.0 27.1 19.5
38*N Daylength
(hr-min) 14-39 14-47 13-24
221
-------
\
o
o
o
o
m
o
o
o
o
(O
o
o
(M
o
o
1-
o
14
w
e>
<
«a
e
• »4
M
«0
8
•O
3
O
t-
M
V
O
|
cr
a.
IT
<
O
O
UJ
2
•o u
.rt o
u w
o u
01 01
f—I
o -o
3 U
C eg
•O
41 C
c a
•f-l U
C O
O CO
41
hi
3
bO
IM
222
-------
• o
o
o.
'UJ
V)
o
• o
<
.5
-5
UJ
1
cc
<
2
m
• UJ
o
• UJ
o
t-i
O
1-1
w
c
<8
c
•H
u
10
a
•O
C
o
o
I— I
41
c
E 41 •
— -o g
UJ 'w ki
S o fc
— 41 41
O T3
3 ^
C (0
T3
41 C
C «
•H U
C »
41
•8-
41
X Wi
—I <0
u
-------
u
LU
.9-
.8-
7-
6-
.5-
4-
3-
2"
H
NOV DEC JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT
I960 1981
TIME (month)
Figure 7.14. Monthly EC from above and belowground £. marina (n-4). Error bars are
1 standard error.
224
-------
Correlation matrices for aboveground (Table 7.37) and belowground (Table
7.38) adenylates were derived by pooling values from all 12 monthi. For both
above and belowground adenylates, AT was positively correlated with ATP, ADP,
and AMP, while EC was negatively correlated with AMP. Correlation
coefficients between above and belowground aaenylates, using monthly means,
are presented in Table 7.39. Above and belowground AMP were positively
correlated. Weaker positive correlation (0.05 situ adenylate levels in
Spartina patens leaves (Mendelssohn and McKee, 1981). The longer time
interval required for dry ice freezing may allow for more transphosphorylase
and ATPase activity. Even after plant tissue is frozen, enzymic activity
persists (Bieleski, 1964).
Lyophilization of frozen tissue (e.g. Bomsel and Sellami, 1974; Wilson,
1978) effectively maintained in situ adenylate levels, and homogenization by
grinding lowered variability in replicate aliquots. Advantages of
lyophilization include adenylate stabilization by enzyme deactivation
(dehydration) and direct determination of tissue dry weight (Mendelssohn and
McKee, 1981). It is critical that the sample remain frozen below its lowest
eutectic point during the time interval required by the lyophiiizer to reach
sufficient vacuum. Freeze-thaw treatment increases ce]l permeability to ATP
(Rhodes and Stewart, 1974) and may dislodge ATPases from thy lake id membranes
(Garber and Steponkus, 197C), reducing ATP content in plant tissue,
(Mendelssohn and McKee, 1961).
225
-------
\
TABLE 7.37. SPEARMAN CORRELATION COEFFICIENTS
AMONG ADENINE NUCLEOTIDES AND EC, FROM
ABOVEGROUND Z. MARINA USED IN SEASONAL
SURVEY, OBTAINED BY POOLING ALL VALUES
(N-48)
ADP
ATP .094""
ADP
AMP
AT
AMP AT
.2622 .8475*
.4282* .4806*
.6121*
EC
.2641
-.5100*
-.7952*
-.2106
* P < .05
226
-------
/
L. /
TABLE 7.38. SPEARMAN CORRELATION COEFFICIENTS AMONG
ADENINE NUCLEOTIDES AND EC, FROM
BELOWGROUND £. MARINA USED IN SEASONAL
SURVEY, OBTAINED BY POOLING ALL VALUES
(N-48)
ATP
ADP
AMP
AT
ADP AMP AT
.6150* .3280* .8416*
.3414* .7160*
.7078*
EC
.3846*
.1548
-.6160*
-.0263
* P < .05
227
-------
TABLE 7.39. SPEARMAN CORRELATION COEFFICIENTS BETWEEN
ABOVE AND BELOWGROUND ADENINE NUCLEOTIDES
AND EC, FROM Z. MARINA OBTAINED IN
SEASONAL SURVEY, USING MONTHLY hfcANS
(N-12)
Variable Correlation
Coefficient
ATP .0420
ADP -.1961
AMP .6364*
AT -.0490
EC .5845
* P < .05
228
-------
,..r
TABLE 7.40. MONTHLY ENVIRONMENTAL DATA FOR
COLLECTION OF Z. MARINA, USED
IN SEASONAL SURVEY
Harvest
(mo)
Nov 1980
Dec
Jan 1981
Feb
Mar
Apr
May
Jun
Jul
Aug
Sept
Oct
Low
EST
(hr)
1022
0855
1730
1706
1301
1359
1214
1336
0951
1604
0603
0727
Tide
Height
(m)
.1
0
-.2
-.2
-.1
-.1
.1
-.1
.1
0
.1
.2
38 *N
Daylength
(hr-min )
10-5
9-31
9-44
10-16
11-23
12-37
14-15
14-47
14-39
14-10
12-56
11-44
229
-------
8.0-
7.9-
78-
o
o
40
1
30H
20-
£ ioH
o
25-
24-
J 23-
>1
| 22-
"o
" 21-
20-
1 1 I 1 1 1 T I I ...
NOV DEC JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT
1980 1981
TIME (month)
Figure 7.15. Monthly environmental data for collection of Z. marina,
used in seasonal survey.
230
-------
o
o
o
40-
30-
20
10-
0-
(16) (20)
(20)
O)
J3
E
2000
1
M
I I500H
j:
co
o IOOOH
500-
0J
E
o>
300-
200-
100-
Totol
Aboveground
Belowground
NOV DEC JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT
i960 1981
TIME (month)
Figure 7.16. Monthly morphometrics for Z_. marina, used in seasonal survey.
Shoot length error bars are 1 standard error and numbers in
parentheses are n.
231
-------
m
O
V
a.
232
-------
CM
X — t
H «
3 N-
U en
u z
^j
|Sg
>4
C/5 iJ
§15
M Z
H O
0 £
U
U Z
•3 m
Z en
Ul
z -
z w
W >
q os
3 3
en
Q
Z -J
o z
OS O
O en
3 ^
O U
J en
A 2S
t-(
*jj
Z 0
OS Z
2 2
H
N|O
O <
H
en <
H Q
Z
w u
ej OS
M H
U. U
u. 2:
r,-\ Q
o x
U CL,
Z O
0 X
SI
W
OS i-3
OS <
0 H
U Z
Z £
2 z
2 o
2 a:
u >
a. z
en u
.
CM
f—
a
a
»*4
W
4J
O
H
•^5
e
a
0
U hri
3 M)
3
>> O
U ,-t
a 01
to
01
J T»
C
3
O
U
00
01
o
.Q
•*!
4J jC
O u
O 00
£ C
en D
X
O -H
O (ID
£ C
en 41
Q
£
00
c
01
0
V4 •
C*4 00 O CS »A
>j ^^ ro "J O
• • • • •
i
CM i— i \O *D f~
-H oo in O co
ON cs oo in r-.
O O -H -< -.
1
00 ^O \O U^ "™^
"•^ *^ VO 00 00
IT\ sO i/*\ >O ON
^j ^"^ ^^ ^J CD
• * • • •
I
* -X
O r*» f^* ^ m
r» O CN — < CN
ON ON ^ O in
\O CM CO NO O
oo NO
-------
A 30 sec extraction was selected for routine use, although no significant
differences in adenylate levels were obtained from 5-120 sec. Mendelssohn and
McKee (1981) found no significant difference with boiling EDTA plus PVPP
extraction over 5-180 sec. However, Karl et al. (1978) caution against
prolonged extraction which may hydrolyze nucleoside triphosphates . When using
a boiling extractant, it is essential that the temperature be maintained at
100°C in order to deactivate ATPases (Holm-Hansen and Karl, 1978).
Leaf tissue extraction from either individual plants or a pooled plant
sample masks adenylate variation on a cellular or organellar level. When
multicellular tissue is extracted, mass-weighted mean adenylate values are
determined. Cellular compartmentation and tissue heterogeneity may actually
permit a range of co-existing metabolic states (Pradet and Raymond, 1978;
Karl, 1980).
Conversion — TRICINE buffer (25 mM in firefly reaction) was selected for
routine use, since this buffer yielded the highest light output. Webster et
al . (1980) have also reported maximum light production with 25 mM TRICINE.
Apparently, luciferase has the most favorable conformation in TRICINE.
Cofactor requirements have been specified for conversion enzymes,
pyruvate kinase (PK) and adenylate kinase (AK) , by Kayne (1973) and Noda
(1973), respectively. Both PK and AK require a divalent cation (e.g. Mg**).
Without MgSC>4 addition, essentially no conversion of AMP or ADP occurs.
Although the PK reaction also requires a monovalent cation (e.g. K*), ^SO^
addition is not necessary. NH^* (present in commercial PK and AK suspensions)
and/or Na* (present in commercial EDTA and PEP salts) meet this requirement.
K2S04, and pH 8.1 quenched light output in the firefly reaction.
DeLuca et al . (1979) report that SOf inhibits the reaction. Generally,
cations a; id anions reduce light emission (Karl and LaRock, 1975). Apparently,
sufficient Mg*+ is contained in the FLE preparation to meet the luciferase
divalent cation requirement (DeLuca, 1976). Additional MgSO^ inhibits light
output, but Mg** is needed in conversion reactions. The pH optimum for the
firefly reaction is in the range 7.4 (Strehler, 1968) to 7.8 (Webster and
Leach, 1980). pH 7.6 was selected for routine use, since it falls within this
range and yielded higher light output than pH 8.1.
The heat deactivation step is essential when using integral measurement.
Heating denatures PK, preventing ATP production from reaction of PK and PEP
with ADP contained in the crude FLE preparation. Karl and Holm-Hansen (1978)
report that heat deactivation is not required when using peak height
measurement with in situ AT >50 ng ml~^ , since PK interference is overwhelmed
by the magnitude of the ATP-dependent peak light emission.
When ATP is <30 ng ml"* , AMP conversion to ATP may be incomplete, since
ATP is required to initiate the AK reaction (Karl and Holm-Hansen, 1978). An
increase in ATP lowers the apparent K^ of AK for AMP. Since all sample
extracts in this study contained >50 ng ml~^ ATP, addition of ATP was
unnecessary.
235
-------
Firefly lantern extract preparation—Reconstitution of lyophilized
firefly lantern extract (FLE) with TRICINE buffer plus MgS04 (pH 7.6) was
selected for routine use in order to stabilize pH. This procedure results in
a final buffer concentration of 25 mM (firefly reaction mixture), the optimum
prescribed by Webster et al. (1980). MgS04 addition complies with the
recommendation by Karl and Holm-Hansen (1976) to add Mg** when final FLE
volume (25 ml) exceeds 5 ml, specified for Sigma FLE-50 by the manufacturer.
A 6-8 hr aging period at room temperature (Mendelssohn and McKee, 1981)
was chosen as the routine procedure for FLE preparation. FLE was aged in
order to degrade endogenous adenine nucleotides. Prolonged aging and high
temperature result in loss of luciferin-luciferase activity. Karl and
Holm-Hansen (1976) demonstrated that loss of Sigma FLE-50 activity over 36 hrs
at 25*C was due to luciferin rather than luciferase degradation.
Although firefly luciferase is specific for ATP, transphosphorylases
(e.g. NDPK) contained within crude luciferase preparations, regenerate ATP
from other NTP's (DeLuca, 1976). Karl and Holm-Hansen (1978) reported that
GDP addition to the FLE preparation (400 ng ml"1) effectively inhibits ATP
production from GTP, uridine triphosphate (UTP), inosine triphosphate (ITP),
and cytidine triphosphate (CTP). Christensen and Devol (1980) observed no
reduction in light emission with GDP addition.
In the present study, a greater amount of GDP (6.85 yg ml~*) reduced
light output in both standards and samples. Since standards contain no NTP
(other than ATP), reduced light output with GDP addition reflects ATP
consumption by mass-action adjustment via the NDPK reaction. Apparently, NDPK
does not compete with luciferase for ATP (10 ng ral~l) with GDP addition under
1 yg tnl~l (Karl and Nealson, 1980). Since sample adenylate levels showed no
difference with or without GDP,GDP addition to the FLE preparation (6.85 g
ml~l) appears unnecessary.
Photometry—Since the time course of light production resulting from
non-adenine NTP's is slower than in situ ATP-dependent light emission
kinetics, interference is minimized with peak height measurement (Holm-Hansen
and Karl, 1978). However, DeLuca et al. (1979) have stated that no single
method of measuring light production is adequate for all conditions.
Parallel and linear log-log standard regressions between net light output
and ATP (40-4000 ng ml'1) were obtained with peak height (2 sec delay, I sec
count) and integration (10 sec delay, 10 or 30 bee count). Webster and Leach
(1980) demonstrated parallelism between peak height and integration (15 sec
delay, 60 sec count) over C.2-200 ng ml~* ATP. A 10 sec delay, followed by a
10 sec integration, was selected as the routine counting method for two
reasons: 1) mixing problems with peak height were avoided, and 2) after
thorough mixing during a 10 sec delay, the shortest machine-available integral
(10 sec) minimized time-dependent interferences.
Standard curve—Three standard curves, prepared with reagents for
determination of [ATP] (Tube A), [ATP+ ADP] (Tube B), and [ATP + ADP + AMPl
(Tube C), allow more accurate sample adenylate measurement than single curve
determinations (Holm-Hansen and Karl, 1978). Use of multiple standard curves
236
-------
ensured ionic composition and ATP reactivity were similar in both standards
and samples. All three log-log standard regressions between net light output
and ATP (40-4000 ng ml"1) were highly linear.
In this study, separate regressions were specifically required, due to
(NHit^SO^ addition and heat deactivation. Quenching was lowest in Tube A and
highest in Tube C. Commercial preparations of PK (Tubes B and C) and AK (Tube
C) contain (^4)2804, which reduced light production. The heating step
appeared to effectively denature PK but not AK. Selective PK deactivation
causes the AK reaction to re-equilibrate with backproduction of ADP from ATP
in solution (Tube C), reducing light emission (Karl and Holm-H«nsen, 19'/8).
Christensen and Devol (1980) reported a 152 reduction in peak height due to
this re-equilibration.
Tissue Comparisons—
Zostera marina—Since leaves contain the highest adenylate level* among
four tissues examined, it is suggested that leaf material be routinely sampled
as the test tissue for adenylate analyses in Z_. marina. Low adenylate levels
in Z. marina root plus rhizome tissue are attributed to the presence of
structural or metabolically inert material (Pamatmat and okjoldal, 1979), as
well as lowered aerobic respiration in reduced sediments (Mendelssohn et al.,
1981). Tissue adenyidte distribution in Z_. marina contrasts with that
observed for Spartina alterniflora (cordgrass), where leaf sheath and roots
contained higher levels of ATP than leaves (Mendelssohn and McKee, 1981).
This is presumably due to actively dividing raeristernatic tissue in leaf sheath
and roots.
Tissue ATP level reflects ATP generation, utilization, and translocation.
Light and oxygen availability permit both photo- and oxidative
phosphorylation, respectively (Sellami, 1976), in aboveground tissue.
Belowground tissue in reduced sediments must rely on limited oxidative
phosphorylation, substrate phosphorylation in glycolysis (Mendelssohn et al.,
1981), and possibly translocation (Thigpen, 1981) to maintain an adequate
supply of ATP.
Mathematically, EC should be positively correlated with ATP and
negatively correlated with AMP. AT should correlate positively with ATP, ADP,
and AMP. All of these correlations were observed.
Other species—Although adenylate analytical techniques were
specifically adapted to Z_. marina, the methodology was applied to epiphytes
of Z. marina, Ruppia maritima (a seagrass), and Spartina alterniflora (a
marshgrass) for comparative purposes. As previously suggested, relatively low
adenylate levels in epiphytic algae may result from metabolically in«»rt
material in epiphyte preparations. Adenylate content of Jt. maritima
aboveground tissue was similar to that of Z_. marina leaf tissue.
Differences in methodology and environment preclude strict comparison
with the following values reported in the literature. Thalassia testudinum. a
tropical seagrass, contained 703 ng ATP per leaf disc dry wt (485 yg ATP g~*
dry wt) one day after excision (Knauer and Ayers, 1977). This value
represents about twice the amount observed for seagrasses (Z_. marina and II.
237
-------
maritime) in the present study. In a tissue study with _§_. alterniflora,
Mendelssohn and McKee (1981) report a cotnpartively high leaf concentration of
980 nmol ATP g-1 dry wt (495 vg ATP g"1 dry wt).
Seasonal Survey—
Although temperature, light, salinity, and nutrient regimes all exert an
influence on growth (Setchell, 1929; Biebl and McRoy, 1971; Backman and
Barilotti, 1976; Orth, 1977), temperature appears to be dominant in regulating
the seasonal growth pattern of Z. marina in the Chesapeake Bay (Orth et al. ,
1981). In the present study, maximal shoot density and biomass occurred
during spring. At a nearby site (inshore Guinea Marsh), peak shoot density
and biomass were observed during June-July for the preceeding two years (Orth
et al., 1981).
Aboveground tissue ATP levels were highest during winter and summer and
lowest during spring and fall. Winter and summer correspond to periods of
slow growth and senescencs, respectively, with decreased rates of ATP
utilization. In contrast, spring and fall correspond to periods of more rapid
growth with increased rates of ATP utilization. Seasonal ATP levels in
aboveground Z_. marina contrasted vith those reported for Populus gelrica
(poplar) twigs, which contained greatest amounts of ATP during active growth
and lowest amounts during the no growth season (Sagisaka, 1981).
Sexual reproduction in 7_. marina occuis during spring in the Chesapeake
Bay (Stevenson and Confer, 1978). This expenditure of energy may reduce ATP
content. Low adenylate levels are also observed in Corbicula fluminea
(freshwater clam) during periods of reproductive activity (Giesy and Dickson,
1981).
Belowground tissue ATP levels were highest during sunnier and fall and
lowest during winter and spring. Belowground levels were generally much lower
than corresponding aboveground levels. As p-eviously suggested, low
belowground adenylate levels may be attributed to metabolically inert material
(Pamatmat and Sk'oldal, 1979) or lowered aerobic respiration in reduced
sediments (Mendelssohn et al., 1981).
Although amounts of adenine nucleotides are routinely reported, there is
an important metabolic distinction between amount and turnover rate. The ATP
turnover rate or energy flux through the adenine nucleotide pool is actually
the more important quantitative assessment of cellular energetics (Weiler and
Karl, 1979).
In both above and belowground £. marina tissue, the following expected
correlations were observed: 1) EC positively correlated with ATP and
negatively correlated with AMP, and 2) AT correlated positively with ATP, ADP,
and AMP. In aboveground tissue over the one vear survey, ATP, ADP, and AMP
comprised approximately 41-74%, 12-322, and 7-312, respectively, of the total
adenylate pool. AT fluctuation demonstrates net synthesis and degradation of
nucleotides.
Between month variability in EC was damped relative to individual
adenylate concentrations. This was also observed in a seasonal study of
238
-------
adenine nucleotides in freshwater clams (Giesy and Dickaon, 1981). Lower EC
variability has both biochemical and mathematical rationales. EC is not only
regulatory but is also regulated within narrow limits by enzymes, controlling
rates of reactions which are coupled to the use and regeneration of ATP
(Atkinson, 1977). It has been suggested that AMP removal by adenylate
deaminase serves to buffer the cell against a sharp transient decrease in EC
(Chapman and Atkinson, 1973). The presence of ATP and ADP in both numerator
and denominator of the EC ratio further reduces variability.
Conclusions—
Due to the lability of adenine nucleotides, precautions must be taken
throughout the analysis in order to quantify adenylates at their in situ
levels. Freezing plants within 2 min after harvest, prevention of thawing,
and lyophilization minimized adenylate change. Prolonged desiccated or frozen
storage should be avoided, and hydrated extracts must be held on ice during
the assay. High recovery rates of internal standards, added immediately prior
to extraction, indicated minimal adenylate loss after extraction during the
remainder of the assay. An additional methodological step is unique to
aquatic macrophytes. Z. marina leaves shoi'ld be scraped free of epiphytic
algae after lyophilization, sjnce substantial epiphytic biomass obscures leaf
nucleotide content.
The tissue comparison and seasonal survey provide baseline information on
natural adeny^te variability in "L. marina. Since leaf tissue contained the
highest adenylate levels, leaves appear most suitable as a test tissue for
routine adenylate analyses. Seasonal ATP levels in aboveground tissue reflect
energy expenditures associated with growth patterns.
The method presented for the determination of adenine nucleotides in Z_.
marina has several limitations. Tissue adenylate measurement results in a
mass-weighted mean value and provides no information on intercellular
heterogeneity or intracellular compartmentation. Adenylate levels determined
in metabolic or environmental studies with this technique should be
interpreted in this context. Direct application of this methodology to other
species may be inappropriate. With slight modification, however, the
technique should prove suitable to other important macrophyte species.
239
-------
REFERENCES
Adam, H. 1965. Adenosine-5'-diphosphate and adenosine-5'-monophosphate . Pp.
573-577 In: Methods of enzymatic analysis (H. U. Bergmeyer, ed.),
Academic Press, N. Y.
Atkinson, D. E. 1977. Cellular energy metabolism and its regulation.
Academic Press, N. Y. 293 p.
Backman, T. W. and D. C. Barilotti. 1976. Irradiance reduction: Effects on
standing crops of the eelgrass Zostera marina in a coastal lagoon. Mar.
Biol. 34:33-40.
Ball, W. J. and D. E. Atkinson. 1975. Adenylate energy charge in
Saccharomyces cerevisiae during starvation. J. Bact. 121:975-982.
Biebl, R. and C. P. McRoy. 1971. Plasmatic resistance and rate of
respiration and photosynthesis of Zostera marina at different salinities
and temperatures. Mar. Biol. 8:48-56.
Bieleski, R. L. 1964. The problem of halting enzyme action when extracting
plant tissues. Anal. Biochem. 9:431-442.
Bomsel, J. L. and A Sellami, 1974. In vivo measurement of the rate of
transfer of -* from adenylate through the chloroplast envelope. Pp.
1363-1367 In: Proc. Int. Congr. Photosynthesis 3rd, Elsevier,
Amsterdam.
Chapman, A. G. and D. E. Atkinson. 1973. Stabilization of adenylate energy
charge by the adenylate deamin*se reaction. J. Biol. Chem.
248:8309-8312.
Christensen, J. P. and A. H. Devol. 1980. Adenosine triphosphate and
adenylate energy charge in marine sediments. Mar. Biol. 56:175-182.
Cockburn, W. 1974. Simultaneous measurement of oxygen evolution and
endogenous ATP levels in isolated intact chloroplast suspensions. J.
Exp. Bot. 25:111-120.
DeGreef, J. A., E. Moereels, E. Spruyt, and J. P. Verbelen. 1979. Problems
'• associated with kinetic ATP measurements in crude plant homogenates. Pp.
350-362 In: Int. Sym. on Analytical Applications of Bioluminescence and
Chemiluminescence (E. Schram and P. Stanley, eds.), State Printing and
Publ. Inc., Westlake Village, CA.
240
-------
DeLuca, M. 1976. Firefly luciferase. Adv. Enzymol. 44:37-68.
DeLuca, M. A. (ed.). 1978. Bioluminescence and chemilumineacence. Methods
in Enzymolugy, Vol. 57.
DeLuca, M. A. and W. D. McElroy (eds.). 1981. Bioluminescence and
chemiluminescence. Basic chemistry and analytical applications.
Academic Press, N. Y. 782 p.
DeLuca, M., J. Wannlund, and W. D. McElroy. 1979. Factors affecting the
kinetics of light emission from crude and purified luciferase. Anal.
Biochem. 95:194-198.
den Hartog, C. 1970. The seagrasses of the world. North Holland Pub. Co.,
Amsterdam. 275 p.
Garber, M. P. and P. L. Steponkus. 1976. Alterations in chlorplast
thylakoids during an in vitro freeze-thaw cycle. Plant Physiol.
57:673-680.
Giesy, J. P. and G. W. Dickson. 1981. The effect of season and location on
phosphoadenylate concentrations and adenylate energy charge in two
species of freshwater clams. Oecologia 49:1-7.
Guinn, G. and M. P. Eidenbock. 1972. Extraction, purification, and
estimation of ATP froir leaves, floral buds, and immature fruits of
cotton. Anal. Biochem. 50:89-97.
Hampp, R., M. Goller, and H. Ziegler. 1982. Adenylate levels, energy charge,
and phosphorylation potential during aark-light and light-dark transition
in chloroplasts, mitochondria, and c/tosol of mesophyll protoplasts from
Avena sativa L. Plant Physiol. 69:448-455.
Holm-Hansen, 0. 1973. Determination of total microbial biomass by
measurement of adenosine triphosphate. Pp. 73-89 In: Estuarine
Microbial Ecology (L. H. Stevenson and R. R. Colewell, eds.), Univ. of S.
Carolina Press, Columbia, S. C. 536 p.
Holm-Hansen, 0. and D. M. Karl. 1978. Biomass and adenylate energy charge
determination in microbial cell extracts and environmental samples.
Methods Enzymol. 57:73-85.
Hull, C. H. and N. H. Nie (eds.). 1981. SPSS Update 7-9, McGraw-Hill, N. Y.
402 p.
Ivanovici, A. M. 1980. Adenylate energy charge: An evaluation of
applicability to assessment of pollution effects and directions for
future research. Rapp. P. V. Reun. Cons. Int. Explor. Mer. 179:23-28.
Karl, D. M. 1980. Cellular nucleotide measurements and applications in
microbial ecology. Microbiol. Rev. 44:739-796.
241
-------
Karl, D. M., J. A. Haugsness, L. Campbell, and 0. Holm-Hansen. 1978. Adenine
nucleotide extraction from multicellular organisms and beach sand: ATP
recovery, energy charge ratios, and determination of carbon/ATP ratios.
J. Exp. Mar. Biol. Ecol. 34:163-181.
Karl, D. M. and 0. Holm-Hansen. 1976. Effects of luciferin concentration on
the quantitative assay of ATP using crude luciferase preparations. Anal.
Biochem. 75:100-112.
Karl, D. M. and 0. Holm-Hansen. 19/8. Methodology and measurement of
adenyTate energy charge ratios in environmental samples. Mar. Biol.
48:185-197.
Karl, D. M. and P. A. LaRock. 1975. Adenosine triphosphate measurements in
soil and marine sediments. J. Fish. Res. Bd. Can. 32:599-607.
Karl, D. M. and K. H. Nealson. 1980. Regulation of cellular metabolism
during synthesis and expression of luminous system in Beneckea and
Photobacterium. J. Gen. Microbiol. 117:357-368.
Kayne, F. J. 1973. Pyruvate kinase. Pp. 353-382 In: The enzymes, Vol. 8
(P. D. Boyer , ed.), Academic Press, N.Y.
Knauer, G. A. and A. V. Ayers. 1977. Changes in carbon, nitrogen, adenosine
triphosphate and chlorophyll a_ in decomposing Thalassia testudinum
leaves. Limnol. Oceanogr. 22:408-414.
Leach, F. R. 1982. ATP determination with firefly luciferase. J. Appl.
Biochem. In press.
Loomis, W. D. and J. Battaile. 1966. Plant phenolic compounds and isolation
of plant enzymes. Phytochem. 5:423-438.
McRoy, C. P. and C. Helfferich (eds.). 1977. Seagrass ecosystems: A
scientific perspective. Marcel Dekkar, Inc., N. Y. 314 p.
Mendelssohn, I. A. and K. L. McKee. 1981. Determination of adenine
nucleotide levels and adenylato energy charge ratio in two Spartina
species. Aq. Bot. 11:37-55.
Mendelssohn, I. A., K. L. McKee, and W. H. Patrick, Jr. 1981. Oxygen
deficiency in Spartina alterniflora roots: Metabolic adaptation to
anoxia. Science 214:439-441.
Mendelssohn, I. A., K. M. Mendelssohn, and W. H. Patrick, Jr. 1978. A
potential indicator of the cumulative impact of sublethal stress in
coastal plant communities. Fianl report to the Louisiana Dept. of
Transportation and Development, Office of Coastal Zone Management. 50 p.
Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H. Bent. 1975.
Statistical Package for the Social Sciences (SPSS), 2nd Ed. McGraw-Hill,
N. Y. 675 p.
242
-------
NOAA. 1979. Tide tables 1980: East Coast of North and South America. U. S.
Dept. Commerce, NOAA, National Ocean Survey, 293 p.
NOAA. 1980. Tide tables 1981: East Coast of North and South America. U. S.
Dept. Commerce, NOAA, National Ocean survey, 285 p.
Noda, L. 1973. Adenylate kinase. Pp. 279-305 In: The Enzymes, Vol. 8 (P.
D. Beyer, ed.), Academic Press, N. Y.
Orth, R. J. 1977. Effect of nutrient enrichment on growth of the eelgrass
Zostera marina in the Chesapeake Bay, Virginia, USA. Mar. Biol.
44:187-194.
Orth, R. J., K. A. Moore, M. H. Roberts, and G. M. Silberhorn. 1981. The
biology and propagation of eelgrass, Zoste.a marina, in the Chesapeake
Bay, Virginia. Final Report, US EPA, Chesapeake Bay Program, Grant No.
R805953, VIMS.
Pamatmat, M. M. and H. R. Skjoldal. 1979. Metabolic activity, adenosine
phosphates, and energy charge of belowground bionass of Juneus
roemerianus Scheele and Spartina alterniflora Loisel. Est. Coast. Mar.
Sci. 9:79-90.
Penhale, P. A. 1977. Macrophyte-epiphyte biomass and productivity in an
eelgrass (Zostera marina L.) community. J. Exp. Mar. Biol. Ecol.
26:211-224.
Phillips, R. C. and C. P. McRoy (eds.). 1980. Handbook of seagrasss biology:
An ecosystem perspective. Garland STPM Press, N.Y. 353 p.
Pradet, A. 1967. Etude des adenosine-5'-mono, di, et triphosphates dans les
tissus vegetaux. I. Dosage enzymatique. Physiol. Veg. 5:209-221.
Pradet, A. and P. Raymond. 1978. Cellular energy control: The importance of
methodology. Trends Biochem. Sci. 3:39.
Rhodes, D. and G. R. Stewart. 1974. A procedure for the in vivo
determination of enzyme activity in higher plant tissue. Planta
118:133-144.
Sagisaka, S. 1981. Adenosine triphosphate levels in the poplar during one
year growth. Plant Cell Physiol. 22:1287-1292.
SAIT. 1975. Model 3000 Integrating Photometer Instruction Manual. Science
Application Inc. Technology Co., San Diego.
Santarius, K. A. and U. Heber. 1965. Changes in the intracellular levels of
ATP, \DP, AMP, and P{ and regulatory function of the adenylate system in
leaf cells during photosynthesis. Biochim. Biophys. Acta 102:39-54.
Sell ami, A. 1976. Evolution des adenosine phosphates ei' de la charge
energetique dans les compartiments chloroplastique et non-chloroplastique
des feuilles de ble. Biochim. Biophys. Acta 423:524-539.
243
-------
Setchell, W. A. 1929. Morphological and phenological notes on Zostera marina
L. Univ. Calif. Publ. Bot. 14:389-452.
Sofrova, D. and S. Leblova. 1970. Determination of ATP in plant tissue.
Photosynthetica 4:162-184.
Stevenson, J. C. and N. M. Confer. 1978. Sunmary of available information on
Chesapeake Bay submerged vegetation. USFWS/OBS-78/66. 335 p.
Strehler, B. L. 1968. Bioluminescence assay: Principles and practice. Pp.
99-181 In: Methods of Biochemical Analysis (D. Click, ed.),
Interscience Pub., J. Wiley i Sons, N. Y.
Thigpen, S. P. 1981. Adenylate metabolism in relation to floral induction in
Pharbitis nil. Ph.D. Dissertation, Univ. Calif. Davis. 102 p.
Webster, J. J., J. C. Chang, E. R. Manley, H. 0. Spivey, and F. R. Leach.
1980. Buffer effects on ATP analysis by firefly luciferase. Anal.,
Biochem. 106:7-11.
Webster, J. J. and F. R. Leach. 1980. Optimization of the firefly luciferase
assay for ATP. J. Appl. Biochem. 2:469-479.
Weiler, C. S. and D. M. Karl. 1979. Diel changes in phased-dividing cultures
of Ceratium furca (Dinophyceae): Nucleotide tripnosphates, adenylate
energy charge, cell carbon, and patterns of vertical migration. J.
Phycol. 15:384-391.
Wetzel , R., K. Webb, P. Penhale, R. Orth, J. van Montfrans, R. Diaz, J.
Merriner, and G. Boehlert. 1981. Functional ecology of eelgrass. Final
Report, US EPA, Chesapeake Bay Program, Grant No. R805974, VIMS.
Wilson, J. M. 1978. Leaf respiration and ATP levels at chilling
temperatures. New Phytol. 80:325-334.
Zapata, 0. and C. McMillan. 1979. Phenolic acids in seagrasses. Aq. Bot.
7:307-317.
Zar, J. H. 1974. Biostatistical analysis. Prentice-Hall, Inc., Englewood
Cliffs, N. J. 620 p.
244
-------
ATRAZINE EXPERIMENTS
Introduction
The role of adenine nucleotides in cellular bioenergeti.es, including
adenylate energy charge (EC) theory, has been presented by Atkinson (1977).
Adenine nucleotides are strategically adapted to metabolic regulation, since
they are operationally linked with nearly all metabolic sequences. The EC
ratio, [ATP] + 1/2 [ADP]/( [ATP] *-[ADP]-i-[AMP]), represerts a linear measure of
the metabolic energy stored in the adenylate pool, ranging from 0 (all AMP)
to 1.0 (all ATP). EC regulates metabolic sequences by controlling enzymic
rates of reactions which are coupled to the use and regeneration of ATP.
Since the metabolic energy state of an organism is sensitive to
environmental variation, EC has been proposed as an index of sublethal stress
(Ivanovici, 1980) and has been widely applied in this context (e.g. Romano
and Daumas, 1981; Giesy et al., 1981; Mendelssohn and McKee, 1981). The
present study evaluates effects of herbicide on adenylate response patterns
in Zostera marina (eelgrass), a submerged marine angiosperm. "L_. marina is an
ecologically important mactrophyte species (McRoy and Helfferich, 1977;
Stevenson and Confer, 1978; Phillips and McRoy, 1980; Wetzel et al., 1981;
Orth et al., 1981), occurring in temperate and subarctic coastal and
estuarine waters in the Northern Hemisphere (den Hartog, 1970).
Atrazine, a triazine herbicide, is widely used for selective control of
broadleaf and grassy weeds in tolerant crop species, including corn, sorghum,
and sugarcane (WSSA, 1974). As an inhibitor of the Hill reaction in
photosynthesis (Ebert and Dumford, 1976; Gardner, 1981), atrazine is expected
to impair photoevolution of oxygen, net photoreduction, and noncyclic
photophosphorylation in the chloroplast and may adversely affect the
adenylate pool. Several factors, which may eliminate or offset atrazine
toxicity, are reduced herbicide uptake and translocation (Ebert and Dumford,
1976), detoxication (Shimabukuro et al., 1971), or compensatory
phosphorylation potential. Since neither cyclic photophosphorylation
(Thompson et al., 1974) nor oxidative phosphorylation (Davis, 1968) are
appreciably altered by atrazine, these processes along with substate
phosphorylation may regenerate adequate amounts of ATP.
Several studies have investigated ATP response to atrazine exposure in
higher plants. Atrazine, administered through leaves (500 ppra) or through
roots (0.5 ppm), generally decreased ATP content in Cucumia sativus
(cucumber) leaves and roots over 1-3 days (Decleire and Decat, 1981). In
contrast, Gruenhagen and Moreland (1971) have reported slightly elevated
levels of ATP in Glycine max (soybean) hypocotyls with atrazine exposure
(43 ppm) over 6 hrs. These inconsistent results may reflect differences in
exposure time or differences between species in atrazine metabolism or
phosphorylation potential.
Nontarget effects of atrazine have been implicated in racent declines of
submerged aquatic macrophytes in the Chesapeake Bay (Stevenson and Confer,
1978). Agricultural runoff, leaching, and aerial transport processes
introduce atrazine into the Bay (Wu, 1981). Forney and Davis (1981) have
245
-------
reported 3-6 week Ij values (the concentration inhibiting growth 1Z) of a few
ppb atrazine for several submerged aquatic macrophyte species. Although
atrazine levels in the Chesapeake Bay are generally below 1 ppb (Correll et
a!., 1978; Wu et al., 1980; this study, Section 3), results presented in
Section 4 suggest that Z_. marina beds in the lower Bay may experience
atrazine concentrations, ranging from 1-10 ppb, for several days over the
growing season.
Assuming "l_. marina is susceptible to atrazine toxicity, decreased ATP
and EC levels with atrazine exposure are expected. This study investigates
adenylate response patterns in Z. marina over short-term (6 hr) and long-term
(21 day) atrazine exposure. Production, morphometric, and mortality data
were collected in order to facilitate interpretation of adenylate response to
atrazine.
Methods
Field Collection and Transplanting—
Location of the Zostera marina sampling site in the lower Chesapeake Bay
is described in Method Development of this chapter. Clumps of eelgrass were
uprooted with a shovel, swirled in river water to remove macro-algae and
loose sediment, transported in a bucket of river water to the laboratory, and
acclimated in a flow-through system. Clumps were then divided into
"individual" plants (i.e. single shoot with the attached leaf cluster and a
2-5 cm rhizome segment) for transplanting. Transplants were planted in
natural sediment (obtained from the VIMS beach) in Jiffy Pots. All
transplants were submerged in a flow-through system.
Adenine Hueleotides—
Samples were processed, as described in Method Development Section of
this chapter, with the following specifications:
1) transplants were uprooted by hand,
2) for each treatment, plants were pooled in order to minimize
within treatment variation and spotlight between treatment
variation,
3) leaf tissue was assayed at the end of short-term (6 hr) and
longterra (21 day) atrazine experiments, and
4) photometry was performed entirely in the integration mode.
Environmental Data—
Environmental data included water temperature, salinity, dissolved
oxygen (DO), and photosyntheticalLy active radiation (PAR). Minimum and
maximum temperatures were recorded with a min-max thermometer (Taylor
Instruments). Salinity was measured with an induction salinometer (Model RS
?B, Beckman). DO was monitored polarographically (Hitchman, 1978) with an
oxygen meter (Model 2604, Orbisphere Corp.). This meter was calibrated in
water-saturated air at specified temperature and pressure. Because it was
not salinity-correctred, DO values are relative and not absolute. PAR was
measured with a light meter (Model LI-185B, Lambda Instruments Corp.),
equipped with a quantum sensor (Model LI-1905, Lambda).
246
-------
Short-Term (6 Hr) atrazine Experiments—
Design—Effects of atrazine exposure over 6 hrs were tested in two
sealed 37 1 glass tanks, one control and one dosed chamber. The flow-through
system inside a greenhouse is diagrammed in Figure 7.17. Nominal atrazine
concentrations of 10 and 100 ppb were evaluated in two separate experiments.
Design specifications are presented in Table 7.43.
Atrazine stock solution was prepared with technical grade atrazine
(97.2Z, CIBA-GEIGY Corp.), dissolved in glass-distilled methanol (Burdick and
Jackson Labs). This solution was metered in with a peristaltic pump (Model
600-1200, Harvard Apparatus Co., Inc.), so that dilution yielded the desired
atrazine concentration (0.07Z v/v methanol). Flow rates were checked hourly.
Short-term experiments did not incorporate a methanol control.
Atrazine—Water samples were collected, filtered, extracted, and assayed
for atrazine by gas chromatography, as described in Section II. The gas
chromatograph (Model 560, Tracor) was equipped with a nitrogen-phosphorus
detector (Model 702, Tracor). Samples were collected to spot-check nominal
atrazine concentrations.
Productivity—Z^ marina productivity measurements are obtained, using
the flow through system. Water was pumped through a 1 cartridge filter, as
shown in Figure 7.17. Potted plants were placed in tanks, which were tightly
sealed with glass tops, leaving no air space. After the tank water had
turned over one time, DO was monitored hourly at both inflow and outflow
ports. Dry weight of aboveground biomass in each tank was obtained at the
end of the experiment.
Productivity was calculated from the following formula:
«8 QZ 8"1 hr~1 * < mS °2 ^^ (1 tank) (g dry wt)~l (hr turnover)""1
where A " outflow DO - inflow DO
The ratio, tank volume/turnover time, is simply the flow rate. These
production rates represent net productivity, cince photosynthesis and
respiration operate simultaneously during daylight hours.
Long-Term (21 Day) Atrazine Experiments—
Design—Effects of atrazine exposure over 21 days were tested in six
pairs (each pair consists of A and B replicates) of 38 1 glass tanks,
corresponding to the following nominal atrazine concentrations: 0, 0.1, 1.0,
10, 100, 1000 ppb. Each tank initially held 15 potted plants. The
flow-through system insiJe a greenhouse is diagrammed in Figure 7.18. Mean
tank turnover times ranged from 7.3-13.5 hrs. This experiment was replicated
four times. Replicate Experiments 1-4 were analyzed separately, as well as
together, in some cases. Spot-check atrazine measurements are listed in
Table 7.44.
Atrazine stock solutions were metered in with a peristaltic pump, so
that dilution yielded the desired atrazine concentrations (0.07X v/v
247
-------
u
0)
a
x
4)
-------
TABLE 7.43. DESIGN SPECIFICATIONS FOR SHORT-TERM (6 HOUR) ATRAZINE
EXPERIMENTS
Specification
Measured
Exposure
atrazine (ppb):
period (hrs)
Tank turnover time (hrs)
Aboveground dry wt (g):
Nominal Atrazine (ppb)
10
Initial 15.77
Final 9.39
1000-1630
1.74
Control 15.04
Test 12.48
100
97.86
91.33
1030-1700
1.74
9.23
9.51
249
-------
-------
Table 7.44. SPOT-CHECK ATRAZINE MEASUREMENTS IN LONG-TERM (21 DAY)
ATRAZINE EXPERIMENTS
Experiment
Exposure Time
(days)
Atrazine (ppb)
Nominal
Measured
1 21
2 7
14
21
3 21
4 21
100
1
10
100
1000
1
1000
1
1000
.1
10
1
100
108.60
2.91
22.49
113.53
1051.08
1.06
1038.69
1.26
1072.86
.70
11.12
1.27
116.09
251
-------
tnethanol). Flow rates were monitored daily. Long-term experiments
incorporated a methanol control.
Atrazine — Atrazine measurement was performed according to the procedure
described for short-term experiments.
Morphometrics — Shoot length and number of leaves were obtained on all
living plants from each tank at 0, 7, 14, and 21 days. Measurements from
replicate tanks A and B were combined to calculate means. Weekly
morphometric changes were calculated from the following formula:
xt - xo
Z Change - -- x 100
where X£ » me&n at time t
XQ " mean at time zero
Mortality — Mortality was recorded in each tank at 7, 14, and 21 days.
Dead plants were removed from the system. Visual criteria for plant death
were loss of green pigmentation (i.e. chlorophyll degradation) and loss of
structural integrity. Mortality observations from replicate tanks A and B
were combined in tabulations.
Statistical Analysis —
The following procedures in the SPSS software package (Nie et al . , 1975;
Hull and Nie, 1981) were used: ONEWAY (single factor ANOVA, Hartley Fmax
test for homoscedasticity, Student-Newman-Keuls multiple range test),
SCATTERGRAM (linear regression and Pearson correlaiion) , NPAR TESTS
(Kruskal-Wallis single factor ANOVA bv ranks and Kolmogorov-Smirnov one
sample test for normality), and NONPAP CORK (Spearman rank correlation).
Other ctatistical procedures employed included nonparametric multiple
range testing by rank sums (Zar, 1974) and dose-effect analysis with
log-probit transformation (Litchfield and Wilcoxon, 1949).
Short-Term (6 Hr) Atrazine Experiments — Differences between adenylates ,
resulting from exposure to atrazine, were detected by the procedure
diagrammed in Figure 7.19.
Long-Term (21 Day) Atrazine Experiments — Differences between adenylates,
resulting from exposure to atrazine, were detected and located by the
procedure diagrammed in Figure 7.19. Morphometric change was regressed
against time for a control and five atrazine concentrations. Relationships
between adenylate and atrazine data were evaluated by Spearman rank
correlation. Median and 1% lethal atrazine concentrations (LC 50 and LC 1,
respectively) and slope function (S), together yith their 95X confidence
limits, were estimated by log-probit analysis. Differences between these
mortality statistics from replicate experiments were evaluated.
252
-------
01
v
u
Wi
41
01
u
o
•a
o
u
HI
r-
41
3
00
153
-------
Results
Short-Term (6 Hr) Atrazine Experiments—
Flow-through system data—Mean temperature and salinity are presented in
Table 7.45. Similar and stable temperature and salinity prevailed over the
course of the two experiments.
Productivity—Hourly net production rates in both control and test tanks,
along with surface PAR readings, are plotted in Figures 7.20 and 7.21 for 10
and 100 ppb atrazine experiments, respectively. At 10 ppb atrazine, net
productivity was positive and relatively similar in both control and test
tanks. At 100 ppb atrazine, net productivity was positive in the control but
generally negative in the test tank. These results indicate that 10 ppb
atrazine had little effect on net productivity over 6 hrs, whf.reas 100 ppb
exerted a marked negative effect.
Adenine Nucleotides—Adenylate and EC values in both control and test
tanks are shown in Figures 7.22 and 7.23 for 10 and 100 ppb atrazine
experiments, respectively. Results at both 10 and 100 ppb were the same. EC
values in control and test tanks show no significant difference, whereas ATP,
ADP, AMP and AT in test tanks w°re all significantly lower than their controls
at both 10 and 100 ppb atrazine over 6 hrs.
Long-Term (21 Day) Atrazine Experiments—
Flow-through system data—Mean temperature and salinity, in four
replicate experiments, are presented in Table 7.46. Mean minimum and maximum
temperatures in Experiment I were considerably lower than corresponding
temperatures in Experiments 2-4. Salinity was similar in all replicate
experiments.
Morphometrics—Mean shoot length and number of leaves, obtained at the
start of each experiment, appear in Table 7.47. Mean changes in shoot length
and leaf number at 7, 14, and 21 days, for each atrazine concer.ti ation, were
pooled from replicate experiments and regressed against time (Figures 7.24 and
7.25, respectively). Statistics associated with these regressions are
presented in Table 7.48. Negative slopes and correlation coefficients for
shoot length change at 1000 ppb atrazine and for leaf number change at both
100 and 1000 ppb have clearly demonstrated a negative effect of atrazine on
growth over 21 days.
Mortality—Twenty-one day mortality, expressed as percent dead, is
presented in Table 7.49 for replicate Experiments 1-4. Mortality in controls
was <72, which is acceptable in acute bioassays (Sprague, 1973). Mortality
was 100% at 1000 ppb atrazine over 21 days in all replicates, with the
exception of Experiment 1.
Results, derived from log-probit analysis of 21 day mortality data,
appear in Table 7.50. Estimates of mortality statistics in Experiments 3 and
4 were very similar. The relatively large slope function, (S) in Experiment
1, due to incomplete mortality at 1000 ppb, was significantly higher than that
obtained in either Experiment 3 or 4 and was reflected in the wide confidence
limits, associated with LC 1 and LC 50 values in Experiment 1. The LC 50
254
-------
TABLE 7.45. TEMPERATURE AND SALINITY DURING SHORT-TERM (6 HOUR)
ATRAZINE EXPERIMENTS
Nominal Temperature Salinity
Atrazine (ppb) (*C) (n-52) (°/oo) (n»l)
x SE
10 20.5 .1 21.97
100 22.5 .2 22.56
255
-------
rlOOO
-500
o
(SI
UJ
a
o
-0
"r- 2i
« 'j=
o *
o ~
o
a o"
°- o.
5^ OJ
Test
Turnover Tim« Log
Control
1000 1100 1200 1300 1400
TIME (hours)
1500
1600
1700
Figure 7.20. Surface PAR and net production rates Curing short-term (6
hour) 10 ppb atrazine experiment. Tank sealed at 1000 hrs.
25fc
-------
4-
3-
o
o>
E
Z
O
O
o
cr
a.
LU
Z
OH
-I
H500
o
«
-1000 u
-500
IU
o
12
cc
Turnover Tim« Log
1000
MOO
1200
1300
1400
1500
1600
1700
TIME (hours)
figure 7.21. Surface PAR and net production rates during short-term (6
hour) 100 ppb atrazine experiment. Tank sealed at 1030 hrs.
257
-------
25Ch
•; 200^
er
o>
150-
1 100
o
3
C
a>
•o
50-
|Tj CONTROL
11 TEST
I
a a
-10
-.8
-6
u
UJ
-4
-2
ATP
ADP
AMP
AT
EC
Figure 7.22. Adenlne nucleotides and EC after 6 hours in the short-term
10 ppb atrazine experiment (n =4). Control-test pairs with
same letters do not differ significantly (P >.05). Error
bars are 1 standard error.
258
-------
30CH
CONTROL
TEST
250-
"5
^^
w
•o
'o»
> 20°"
'5
er
V
0.
<
o>
^
~~ 150-
.-05). Error
bars are I standard error.
259
-------
TABLE 7.46. TEMPERATURE AND SALINITY DURING LONG-TERM (21 DAY)
ATRAZINE EXPERIMENTS
Experiment
1
I
3
4
Temperature
n
9
9
14
9
Minimum
X
6.3
13.4
15.2
18.7
SE
.5
.9
.6
.7
CO
Maximum
x SE
16.0
25.3
24.6
28.4 1.
9
5
6
3
Salinity (°/oo)
(n-1)
21.96
20.14
20.14
19.13
260
-------
TABLE 7.47. INITIAL Z. MARINA MORPHOMETRICS IN LONG-TERM (21 DAY)
ATRAZINE EXPERIMENTS
Experiment
1
2
3
4
n
180
165
180
180
Shoot Length
X
12.9
13.2
20.0
31.0
(cm)
SE
.3
.3
.4
.8
Number
X
3.4
4.2
4.5
5.3
Leaves
SE
.1
.1
.1
.2
261
-------
20-,
10-
0-
— -10-
CJ
o
z
UJ
o
o
r
CO
-20-
-30-
-40-
-50-
-60
1000 ppb
14
TIME (days)
Figure 7.24.
Regressions of shoot length change vs. time for control and
five atrazine concentrations in the long-term (21 day)
atrazlne experiments. Data from replicate experiments are
pooled. O
-------
„ 1-
30-1
UJ
o
z
<
I
o
IT
UJ
CD
20-
10-
0-'
-10-
-20-
Ippb
Oppb (Control)
— lOppb
-.1 ppb
100 ppb
<
iLl
-30-
-40-
-50-
.1000 ppb
-60-
14
21
Figure 7.25.
TIME (doys)
Regesslons of leaf number change VB. time for control and
five atrazine concentrations in the lon^-term (21 day)
atrazine experiments. Data from replicate experiments are
pooled. 263
-------
^4
^J
O
v>M
CN
jr
2
B
1
o
z
3
a
f 1 f-1!
X 3
g8
Z Ou
u
co as
o <
M
H co
co H
f-> Z
S£
H i-"
co at
bj
Z 0.
0 X
l-H M
CO
CO U
o o
2 j
Du
[ij W
0 06
§ £
x o
U DC
u.
w <
§ <
3 a
u, •
< CO
3S
u
a z
5S
u
x a.
H X
O UJ
z
as
^-»
H M
82
X H
•CO <
.
oo
^
j^
U
CO
H
U)
01
M
CO U
fMl **4
* IS
U 01
o o
0 0
w
a
0)
u
4)
C
H- 1
a.
o
CO
^
a.
a
^
Ol
c
N
ia
<
4 £
f» CM >» •£> <*> (••
•—4 <^ r^ oo r*> ^
O -< m ao vo O
^ .* m ^ CM *o
\ 1
o o in o o o^
in O r^ in o "^
r*» co ^J CM ^^ ^^
CM CM CM r~
1 1 1
O >n r> ^ —> —•
in CM m m vO r~
CM PO rx CT* ^^ vfi
00 *^ CT^ >O ^ CM
1 CM
t
* *
£^ ^^ #^ ^^ ^^ ^^
O O 00 -^ OO ^
— * -^ — t CT* f**> OO
r^ ^4 %O ro ^^ "^f
1
O O O »A u-t O
O O O O ^O
1
^ ^ ^ vC NO —
o -• — o o o
— 0 0
••^ O
•™4
m
o
•
V
a»
*
264
-------
TABLE 7.49. MORTALITY (X) AFTER 21 DAYS IN THE LONG-TERM ATRAZINE
EXPERIMENTS (N0-30)
Experiment
1
2
3
4
0
6.7
3.3
3.3
6.7
Nominal
.1 1.0
13.3 10.0
O1 10.0
0 0
3.3 3.3
Atrazine
10
10.0
10.0
0
0
(ppb)
100
20.0
46.7
3.3
10.0
1000
50.0
100.0
100.0
100.0
n0 - 15
265
-------
TABLE 7.50. TWENTY-ONE DAY 1Z (LC 1) AND 50X (LC 50) LETHAL CONCEN-
TRATION, ALONG WITH SLOPE FUNCTION (S), IN THE LONG-TERM
ATRAZINE EXPERIMENTS. LC 1, LC 50, AND ASSOCIATED
CONFIDENCE LIMITS (CL) ARE EXPRESSED AS PPB ATRAZINE.
S AND ASSOCIATED CL ARE UNITLESS
Experiment LC 1
LC 1
95X CL
LC 50 LC 50
952 CL
si
S
95Z CL
1 1.9ab2 .1-35.0 540a 229-1274 11.02s 3.37-36.03
2 2.6b .4-16.4 100b 45-221 4.78«b 2.35-9.70
3 38.7a 16.5-90.9 365a 220-606 2.74b 2.0&-3.67
4 35.5a 16.8-74.9 367a 221-609 2.71b 2.02-3.63
1 Slope function - .5(LC 84/LC 50 t- LC 50/LC 16)
2 Values with same letter superscripts (between experiments) do not
differ significantly (P > .05).
266
-------
estimate for Experiment 2 was significantly lower than those obtained for
other experiments, due to higher mortality at 100 ppb in Experiment 2.
Overall, results conservatively estimate the 21 day LC 1 and IX 50 at 1 and
100 ppb atrazine, respectively.
Adenine Hueleotides—Adenylate and EC values, in replicate Experiments
1-4, are presented in Tables 7.51-7.54, respectively. These data were pooled,
and mean values are displayed in Figure 7.26. In this figure, each experiment
was weighted equally and adenylates at 1000 ppb atrazine were excluded, sir.ce
data at this concentration were obtained in Experiment 1 only.
In this pooled analysis, EC was reduced at 0.1, 1.0, and 10 ppb atrazine
over 21 days, but higher ATP at 100 ppb elevated EC to the control level. ADP
«md AT generally increased with higher atrazine levels. These observations
were reflected in relatively strong and positive correlation of ATP, ADP, and
AT with atrazine (Table 7.55).
Discussion
Short-Terra (6 Hr) Atrazine Experiments—
Productivity—Z. marina net productivity was inhibited at 100, but not 10
ppb atrazine, over 6 hrs. Net productivity of the Z_. marina community,
isolated under large plexiglass domes in the field, was similarly depressed at
100 ppb atrazine during daylight hrs (Section V). Using laboratory
microcosms, Correll et al. (1978) have reported a reduction of net
productivity with 100 ppb atrazine in another submerged aquatic macrophyte,
Zanichellia palustris (horned pondweed), after 1 and 2 week exposures.
Depression of oxygen evolution is expected, since atrazine inhibits the
Hill reaction in photosynthesis (Ebert and Dumford, 1976). Although internal
cycling of gases within lacunar spaces of leaves may have introduced error
into production measurements, based on changes in dissolved oxygen (MeRoy and
McMillan, 1977), both control and test measurements should have contained the
same error.
Adenine nucleotides—Adenylate levels in Z_. marina decreased at both 10
and 100 ppb atrazine over 6 hrs. Since ATP, ADP, ana AMP were reduced
proportionately, EC ratios remained constant. Apparently, EC was stabilized
by removal of AMP with adenylate deaminase (Chapman and Atkinson, 1973). It
appears that ATP or AT serves as a more sensitive index of short-term
herbicide stress than EC in Z. marina.
—• mar'-na adenylates, but not net productivity, were reduced at 10 ppb
atrazine. This indicates that adenylate determinations were a more sensitive
mor; or of short-term ^rbicide stress than net productivity measurements.
Noni-yclic photophosphorylation may have been impaired with lower amounts of
atrazine than photosynthetic oxygen evolution.
Long-Term (21 Day) Atrazine Experiments—
Morphometrics—Growth of JZ. marina, as measured by shoot length and
number of leaves, was clearly Inhibited at 100 ppb atrazine over 21 days. It
267
-------
z
t-4
U]
OS
a
tn
§
til
U
z
t-l
N
2
H
•<
>4
a
CM
os
u
£
o
w
o
5
s**
s
>••
CX
Q
••"^
1
O
>
h-l
&
u
a.
£
^«
•3
CO
ua >-*
O v£>
M n
H z
o •**
w
J — >
o
3 H
Z Z
H £
Z 1-4
l-l OL
Z W
ID CL.
Q X
< W
.
«•
in
r*«
bl
CD
2
J3
«^
a
s_^
(U
c
• )^
N
CO
IM
4J
<
CO
C
• p*
Q
Z
3
•3
•H
O
•«t
O
o
-
«^
^H
o
Contr
c^
m m m \o o
CM CM
-H +4, +4 -H -H
U
H ji o Xt co
*-H o to QO in
(N -^ — . ^J
* <••» o o o
-4 CM •
44. 44 44 -H -H
01 O o XI c«
o •* o m oo
CM ON in >£> »n
CM _ ^ . ,
o
-H m P-. eo
44 41 44 44 44 |
-O -O U Xl XI
i^> »»4 r*^ >A <^
•H <«^ -H ^ •
O
CM C^l >* \O
44 44 41 44 44
U
CJ Xt U XI XI
O vO 00 -* r^
vO O oo in -*
-. _ -4 ^
ON v£t O >O "*
— I CM
r- «
-vl O
m ^ (n -H •
••-< V
41 44 41 41 4)
co n co co eg
in \£ v£> r** o*
p^i in oo r^ in
— « CM
a, a, a.
H^*s ^* t^ C_l
fcj AI r^ '•••'
< «3 < < w
m
O
A
OL,
^»*
>
^4
4-1
C
a
u
• H
1*1
• H
00
• H
W
^t
C1J
I**
• -«
T)
4J
0
c
o
T>
^•x
10
u
c
01
u
co
4
u
CJ
CO
Vi
CU
a.
3
CO
W
CU
4J
4J
01
CD
8u
en O
u
x: M
u cu
•p*
» T3
^j
ca *
to
-- CM
268
-------
Q
_H
CM
OS
U)
H
o
w
a
ij
/-N
E-l
3
O.
Q ~»
-3-
— II
1 Z
CJ ^
M ^
3 H
Or Z
W M
O4 1-4
H a:
a.
u x
n w
z
en 1-1
Q [T]
1-4 as
H 3
O W
U O
J 0<
O X
z
u z
t— 4 Cs]
uj as
Q H
.
CN
in
i —
W
03
(-1
^
a
a
^/
01
c
N
CO
kt
4j
<
l~4
C
• l-t
2j
at
X-
CO
k>
co
o
o
o
_^
"^
^^
o
c
o
m o oo r* o
44 44 44 44 44
^ c7* in oo
r*^ \4O oo m ^
CN
^^
in ^0 ^ ao o^ •—< in
— . en
CU O> Oi
H Q Z H U
< -2 3 < u
1^
01
14-4
<4-l
•^
T3
4J
O
c
o
/— s
CD
4J
c
Oj
,5
CO
01
^t
iJ
c
01
3
u
01
^x
4J
a.
• ^4
U
u
ca
OI
&
3
en
•
^4 *^
01 in
4J O
4-1
01
01 04
C <**^ •
H 14
U> >^ O
— • 1-
J= «J u
u C 01
•H CO
S u ^
•F4 kl
W «-" CO
oi ••* •«
3 e c
—i 60 eo
CO •-* 4J
> en en
-< CM
269
-------
Q
••*
CM
at
Id
H
CO
•3 E"1
cr z
U4 U
2
Pu h«4
H OS
< td
a.
u x
3- w
z
Cfl i-l
U)
ss
H 3
O "3
id o
-j a.
0 X
3 U
z
U]
u z
Z i-l
l-l N
z •<
u oc
Q H
< <
.
m
.
f^
fo-]
( 1
BQ
^*
H
a
a
>>— t
OJ
c
*p4
N
Q
^4
u
**
CO
c
• (^
e
o
z
Ol
^^
r>
CO
-H
tl
CO
>
o
o
p— 4
o
^
f>^
*
1— 1
o
c
o
u
00
44
XI
CM
_H
CM
^
CO
0
CO
XI
co
vO
o
^
^1
XI
CO
_H
^^
•H
CM
44
_*
CO
00
a.
<
co -* -< o
44 44 44 44
XI X) U CO
CO P* i"^ \O
vO ^T CO vD
CM
O
— i CM CO
co xi X) CO
ON in »3" t£
CM •
CM
in in CM o
•^ '
XI
CO co XI CO
CM CM C7\ \C
m ^^ &* \c
** •
^^
O
CM »-i r»
V
44 44 44 44
CO CO CO XI
CO J- r^ -H
*^ CO 00 f**
•-* •
^H
co -H m o
•
44 44 44 44
CO Cd eg
eg
01
3
CO
^
-H
•
^•^
in
O
/\
a,
IH
>> o
4J V4
C Ol
CO
o -a
*
-------
i
an
Id
§
u
Id
Q ^
vO
.-. II
I Z
CM
44
XI
CM
O
44
U
CM
44
U
< M
Ou
O X
3. W
H 3
O w
id O
J Ou
U X
(d
•O
U
00
B) CO
—I CM
271
-------
I
8
m
1
8
i
8
i
o
o
PJ
1
o
g
1
o
C « >-"
6 V" T3
a> *i 4J
ex 1-1 co
x -a
4) i-l
at
•a
ca
o 01
c u
CO CO
U O
i-! T> W
—• u
o. i-i ca
Ol O) .O
*-l 4-1
4-1 U
01 o
o
0)
1-1
u
c
01
c
1-1
c
01
•o
<
w
ca ca
4J CO •
CO 10
Q « 4J
C
01
m -C 4-1
4J 4J CO
c ^ 01
I*5
M 01 00
01 3 C
o,^ o
X * I
ai > co
vO
CM
4)
(-1
00
Ajp 6 /unba
S3ail0310nN 3NIN30V
272
,/•*
-------
TABLE 7.55. SPEARMAN CORRELATION COEFFICIENTS OF 21 DAY ADENINE
NUCLEOTIDES AND EC WITH NOMINAL ATRAZINE CONCENTRATION IN
LONG-TERM ATRAZINE EXPERIMENTS. MEANS FROM REPLICATE
EXPERIMENTS ARE POOLED (N=20)
Variable Correlation Coefficient
ATP .3956
ADP .4844*
AMP .1901
AT .3679
EC -.0400
* P < .05
273
-------
appears Chat 10 ppb was also inhibitory, but to a lesser extent. Apparent
stimulation of £. marina growth at 1 ppb may have been an indirect result of
selective atrazine toxicity toward epiphytic algae, since epiphytes can
inhibit "L. marina photosynthesis by interfering with carbon uptake and by
reducing light intensity (Sand-Jensen, 1977). Other studies have demonstrated
inhibitory effects of atrazine on algal photosynthesis (Plumley and Davis,
1980) and growth (Veber et al., 1981), although at higher concentrations.
Section VI reports 21 day EC 50 values (equivalent to 150. the
concentration inhibiting growth 50%) of 410 and 60 ppb atrazine for shoot
length and number of leaves, respectively, with "L. marina in laboratory
bioassays. Forney and Davis (1981) have calculated 3-6 week 150 values of
80-1104 ppb atrazine, based on leaf length measurements with other submerged |
aquatic macrophyte species under various laboratory conditions. These results I
are in general agreement with those of the present study. I
Mortality—Conservative estimates of the 21 day LC 1 and LC 50
(concentrations lethal to 1 and 50% of the test organisms, respectiively) for
"L." mar^na are 1 ar>d 100 ppb atrazine, respectively. Forney and Davis (1981)
have calculated LC 1 and LC 50 values of 11 and 53 ppb atrazine, respectively,
for Potamogeton perfoliatus (redheadgrass pondweed).
I
|
Tolerance of plants toward triazine herbicides may have been influenced
by temperature (Ebert and Dumford, 1976). Incomplete mortality at 1000 ppb
atrazine, over 21 days in replicate Experiment 1, may be related to cooler
prevailing temperatures. Atrazine toxicity appears to increase with warmer
temperature, perhaps due to accelerated rates of uptake and translocation.
Adenine nucleotides — Inability to remove AMP from the adenylate pool f
contributed to a reduction in "L. marina EC at 0.1, 1.0, and 10 ppb atrazine I
over 21 days. At 100 ppb, corresponding to the estimated LC 50, ATP and EC
unexpectedly rebounded before plant death resulted. Apparently, severe stress
(100 ppb) elicits an adaptation response. For example, increased rates of
respiration and associated oxidative phosphorylation may have supplied ATP in
sufficient amounts to maintain metabolic homeostasis. Continued stress at 100
ppb atrazine, however, became lethal.
ATP and AT respor.se patterns at 100 ppb atrazine appear to follow the
triphasic general adaptation syndrome, outlined by Selye (1976). Over the
short-term (6 hrs), ATP and AT were reduced (alarm reaction). Over the
long-term (21 days), ATP and AT increased beyond control levels (stage of
resistance) until death resulted (stage of exhaustion). Giesy et al. (1981)
have re^irted a similar response pattern for ATP, AT. as well as EC, in
Palaeomonetes paludosis (glass shrimp) with 30 g 1~* cadmium exposure.
Morphometric and mortality data facilitate interpretation of adenylate
response. EC indicated stress as low as 0.1 ppb atrazine, but failed to
reflect visually apparent stress at 100 ppb. It appears, then, that EC is a
sensitive monitor of long-term, sublethal herbicide stress. When £. marina
was confronted with more severe stress, however, physiological adaptation
increased EC before death resulted. The utility of EC as an index of
long-term herbicide stress in JJ. marina may, therefore, be limited.
274
-------
Conclusions—
Adenylate and EC response in £. marina to selected environmental
variables are useful measures of metabolic state under cer lin conditions.
The response is integrative, representing the interaction of genetic
disposition with the environmental matrix, both stressful and beneficial.
This may be advantageous in an ecological context, but can pose difficulties
when attempting to evaluate effects of a single variable. Adenylate and EC
response may also change in accordance with physiological adaptation over
time. Chronic and severe herbicide stress was observed to elicit this
adaptive response in Z_. marina.
ATP or AT response may be more appropriate than EC in certain cases, as a
monitor of environmental stress. ATP and AT decreased in "L_, marina with
short-term herbicide stress, but EC remained constant. In contrast, EC was
reduced with long-term, sublethal herbicide stress. Limitations of adenylare
and EC utility must be recognized in order to allow sound interpretation of
results. It is suggested that more conventional quantitative analyses
accompany adenine nucleotide measurements in any effort to evaluate
physiological response to environmental variation.
275
-------
REFERENCES
Atkinson, D. E. 1977. Cellular energy metabolism and its regulation.
Academic Press, N. Y. 293 p.
Chapman, A. G. and D. E. Atkinson. 1973. Stabilization of adenylate energy
charge by the adenylate dearainase reaction. J. Biol. Chem.
248:8309-8312.
Correll, D. L., J. W. Pierce and T. L. Wu. 1978. Herbicides and submerged
plants in Chesapeake Bay. Pp. 858-877 In: Proc. Symp. on Technical,
E •/ironmental , Socioeconomic, and Regulatory Aspects of Coastal Zone
Management, ASCE, San Francisco.
Davis, D. E. 1968. Atrazine effects on mitochondrial respiration. Proc. So.
Weed Conf. 21:346.
Decleire, M. and W. Decat. 1981. Modification des teneurs en ATP, glucose,
fructose, et saccharose dans des plantules de coneombre (Cucumis sativus)
sous 1'action de divers herbicides. Weed Res. 21:93-97. "
den Hartog, C. l^O. The seagrasses of the world. North Holland Pub. Co.,
Amsterdau. 275 p.
Ebert, E. and S. W. Dumford. 1976. Effects of triazine herbicides on the
physiology of plants. Residue Rev. 65:1-103.
Forney, D. R. and D. E. Davis. 1981. Effects of low concentrations of
herbicides on submersed aquatic plants. Weed Sci. 29:677-685.
Gardner, G. 1981. Azidoatrazine photoaffinity label for the site of triazine
herbicide action in chloroplasts. Science 211:937-940.
Giesy, J. P., S. R. Denzer, C. S. Duke, and G. W. Dickson. 1981.
Phosphoadenylate concentrations and energy charge in two freshwater
crustaceans: Responses to physical and chemical stressors. Vehr.
Internal. Verein. Linnol. 21:205-220.
Gruenhagen, R. D. and D. E. Moreland. 1971. Effects of herbicides on ATP
levels in excised soybean hypocotyls. Weed Sci. 19:319-323.
Hull, C. H. and N. H. Nie (eds.). 1981. SPSS Update 7-9. McGraw-Hill, N. Y.
402 p.
276
-------
X
Ivanovici, A. M, 1980. Application of adenylate energy charge to problems of
environmental impact assessment in aquatic organisms. Helg. Meets.
33(l-4):556-565.
Litchfield, J. T. and F. Wilcoxon. 1949. A simplified method of evaluating
dose effect experiments. J. Pharmacol. Exp. Ther, 96:99-113.
McRoy, C. P. and C. Kelfferich (eds.). 1977. Seagrass ecot.-/stems: A
scientific perspective. Maicel Dekkar, Inc., N. Y. 314 p.
McRoy, C. P. and C. McMillan. 1977. Production and physiology of seagrasses.
Pp. 53-87 In: Seagrass ecosystems: A scientific perspective (C. P.
McRoy and C. Helfferich, eds.), Marcel Dekkar, Inc., N. Y.
Mendelssohn, I. A. and K. L. McKee. 1981. Determination of adenine
nucleotide levels and adenylate energy charge ratio in two Spartina
species. Aq. Hot. 11:37-55.
Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H. Bent. 1975.
Statistical Package for the Social Sciences (SPSS), 2nd Ed. McGraw-Hill,
N. Y. 675 p.
Orth, R. J., K. A. Moore, M. H. Roberts, and G. M. Silberhorn. 1981. The
biology and propagation of eelgrass, Zostera marina, in the Chesapeake
Bay, Virginia. Final Report, US EPA, Chesapeake Bay Program, Grant No.
R805953, VIMS.
Phillips, R. C. and C. P. McRoy (eds.). 1980. Handbook of seagrass biology:
An ecosystem perspective. Garland STPM Press, N. Y. 353 p.
Plumley, F. G. and D. E. Davis. 1980. The effects of a photosynthesis
inhibitor atrazine, on salt marsh edaphic algae, in culture,
microecosystems, and in the field. Estuaries 3:271-277.
Romano, J. C. and R. Daunias. 1981. Adenosine nucleotide "energy charge"
ratios as an ecophysiological index for microplankton communities. Mar.
Bioi. 62:281-296.
Sand-Jensen, K. 1977. Effect of epiphytes on eelgrass photosynthesis. Aq.
Bot. 3:55-63.
Selye, H. 1976. Stress in health and disease. Butterworth, Inc., Boston.
334 p.
Shimabukuro, R. H., D. S. Frear, H. R. Swanson, and W. C. Walsh. 1971.
Gluthathione conjugation: An enzymatic basis for atrazine resistance in
corn. Plant Physiol. 47:10-14.
Sprague, J. B. 1973. The ABC's of pollutant bioassay using fish. Pp. 6-30
In: Biological methods for the assessment of water quality (J, Cairns,
Jr. and K. L. Dickson, eds.), Amer. Soc. Test. Mat. STP 528,
Philadelphia.
277
-------
Stevenson, J. C. and N. M. Confer. 1978. Sumnary of available information on
Chesapeake Bay submerged vegetation. USFWS/OBS-78/66. 335 p.
Thompson, 0. C. B., B. Truelove, and D. E. Davis. 1974. Effects of triazines
on energy relations of mitochondria and chloroplasts. Weed Sci.
22:164-166.
Veber, K., J. Zahradnik, I. Breyl, and F. Kredl. 1981. Toxic effect and
accumulation of atrazine in algae. Bull. Environ. Contarn. Toxicol.
27:872-876.
Wetzel, R., K. Webb, P. Penhale, R. Orth, J. van Montfrans, R. Diaz, J.
Merriner, and G. Boehlert. 1981. Functional ecology of eelgrass. Final
Report, US EPA, Chesapeake Bay Program, Grant No. R805974, VIMS.
WSSA. 1974. Herbicide Handbook of the Weed Science Soc. of America, 3rd Ed.
Champaign, III. 430 p.
Wu, T. L. 1981. Atrazine residues in estuarine water and the aerial
deposition of atrazine into Rhode River, Maryland. Water, Air, and Soil
Pollut. 15(2):173-184.
Wu, T. L., L. Lambert, D. Hastings, and D. Banning. 1980. Enrichment of the
agricultural herbicide atrazine in the microsurface water of an estuary.
Bull. Environ. Contain. Toxicol. 24:411-414.
Zar, J. H. 1974. Biostatiscical analysis. Prentice-Hall, Inc., Englewood
Cliffs, N. J. 620 p
278
-------
-------
Removal of epiphytes from Z_. marina leaf blades was essential in order to
quantify adenine nucleotides in eelgrass tissue alone. Epiphytes can be a
significant proportioi. of an aboveground tissue sample. For example,
epiphytes comprise an .iverage of 242 of Che total eelgrass leaf plus epiphyte
biomass (dry wt) in a North Carolina estuary (Penhale, 1977). Low epiphyte
adenylate levels, relative tc levels in £. mar:na leaf tissue, may be
attributed to the inclusion of small amounts of sediment, as well as siliceous
diatom frustules, in epiphyte preparations.
Storage techniques are aimed at halting enzyme activity, which can alter
adenine nucleotide composition. Enzyme activity nay be minimized by either
dehydration or freezing. Frozen-lyophilized-ground-desiccated tissue (Wilson,
1978) and frozen extract (Holm-Hansen, 1973) consifitute two forms of storage.
In this study, frozen-lyophilized-ground tissue was stored desiccated-dark for
periods up to 5 days.
Extraction—Extraction of adenylates at in situ levels requires rapid
nucleotide release and enzyme deactivation by either heating or lowering pH.
Destruction of the semipermeable characteristics of cell membranes with
boiling extractants causes all soluble constituents (e.g. adenylates) to
rapidly diffuse out of the cells, ultimately resulting in a uniform
concentration of each constituent throughout the entire suspension
(Holm-Hansen, 1973). Hydrolases are released upon disruption of cellular
integrity (DeGreef et al., 1979). Deactivation of these enzymes relies on the
effectiveness of heat conduction or acid permeation through the tissue. The
resultant thermal or [H+] gradients (Karl et al., 1978) are dependent on
tissue chemical and physical properties (e.g. surface to volume ratio,
density, chemical composition). Thermal gradients are minimized by
homogenization of tissue and by using a low tissue to extractant ratio (
------- |