L .L. Coiro et al. J. Exp. Mar. Biol. Ecol. 247 2000 243 –255
245
was expected, but the underestimation was fairly consistent and could therefore be adjusted to provide a reasonable estimate of effect which was still related to the time
component of exposure.
2. Materials and methods
2.1. Animal culture Adult marsh grass shrimp Palaemonetes vulgaris were collected from Narragansett
Bay, RI, embayments and the nearby Pettasquamscutt River. Parental stocks were maintained in 76 l flow-through aquaria supplied with 258C filtered Narragansett Bay
seawater at a salinity of 28–32‰. Individual gravid females were isolated in static, aerated 3.8 l jars of seawater that were renewed daily. Larvae were collected from the
jars within 24 h of hatching and placed directly into the test system. Adults and newly-hatched larvae were fed Artemia salina nauplii Great Salt Lake
E, Aquafauna, Hawthorne, CA daily during laboratory holding and culturing. Test larvae were fed ad
libitum rations twice daily during exposure, generally at the beginning of each D.O. phase Reference Artemia Cysts II and III, Brazilian brand; Bengtson et al., 1985.
Animals were fed at the same times in the constant D.O. treatments as in the fluctuating treatments. Excess food was removed prior to each feeding.
Juvenile grass shrimp were cultured in the same manner as larvae, except that they were maintained in the aerated jars and observed daily until transition to the first
juvenile stage. Adult Say mud crabs were collected from Narragansett Bay and Pettasquamscutt River. Maintenance of parental stocks and rearing of larvae were similar
to grass shrimp except that the larvae were removed from the jar after hatching and held in flow through mesh containers until used in the test. Post-metamorphosed summer
flounder were obtained from the University of Rhode Island Graduate School of Oceanography courtesy of D. Bengtson, Narragansett, RI. Prior to testing the fish were
maintained in 76 l flow-through aquaria and were fed Artemia salina nauplii Great Salt Lake
E, Aquafauna, Hawthorne, CA supplemented with flake fish food TetraE SM80, Tetra Werke Baensch, Melle, Germany daily during laboratory holding. During testing,
the fish were fed ad libitum rations twice daily during exposure, Reference Artemia Cysts II and III, Brazilian brand; Bengtson et al., 1985. Each feeding was supplemented
with frozen adult Artemia salina Pro Salt E, Mid Jersey Pet Supply, Carteret, NJ
2.2. Low-D.O. test system All animals were tested in a single-pass, flow-through exposure system. Specific
reduced oxygen treatments were achieved with an electronic proportioning system Miller et al., 1994 which maintained target concentrations at 60.2 mg l D.O. S.D. A
custom-designed computer program with adjustable parameters controlled both cyclic and constant treatments. Program variables included maximum and minimum con-
centration, cycle type, duration of each phase of the cycle, length of the transition period between maximum and minimum concentrations, and time of cycle initiation and end.
246 L
.L. Coiro et al. J. Exp. Mar. Biol. Ecol. 247 2000 243 –255
Treatment water was gravity-fed directly from mixing boxes to 500 ml exposure chambers at a rate of about 100 mls min. Outflows from each chamber passed through a
10 cm circular Nytex screen 220 mm mesh inserted in the lid. Each chamber was
submerged in a 7 l aquarium with a glass cover to minimize the possibility of reaeration. Low-D.O. water was produced by vacuum-degassing. CO was bubbled back into the
2
degassed water to restore and maintain a consistent pH of 7.8–8.2. Temperature was controlled electronically to 618C. Test salinity was ambient Narragansett Bay salinity,
ranging from 28 to 32 l. The light cycle was 12 h light:12 h dark.
2.3. Test design and implementation Tests usually consisted of four to six treatment concentrations and an air-saturated
control. All low-D.O. concentrations were selected to be largely sublethal, as determined by previous constant exposure testing. There were four replicates per exposure, with ten
to twenty animals per replicate. The test duration was four, seven, or eight days. Dissolved oxygen concentrations in the fluctuating exposures were continuously
monitored in the chamber with a Nester oxygen meter model 8500; BOD probes and a chart recorder Chino Corp. AH-11E Hybrid. Constant-exposure low-D.O. treatments,
included for internal comparison, were checked with a Nester meter at least daily. Meters were calibrated daily with a modified Winkler method using saturated sea water.
D.O. concentrations were checked against modified Winkler titrations at the beginning and end of each test. Salinity, temperature, and survival were also monitored daily.
This study used square-wave D.O. cycles since the results from these exposures are easier to interpret than continuously changing conditions, such as sine-wave cycles. The
cycles had equal time at low-D.O. and saturated conditions. The cycles were 6 h low:6 h high or 12 h low:12 h high, with minimal transition times Table 1. These conditions
were chosen to represent semidiurnal and diurnal tidal cycles of hypoxia. All semidiur- nal treatments started with the maximum D.O. concentration in the early morning. In this
regime, the light cycle began with the morning high-oxygen cycle. For diurnal cycles, the light cycle began 6 h after the start of the low-D.O. phase. The onset of the light
cycle in the diurnal D.O. cycles was shifted so both cycle regimes had a period of light and dark during each phase i.e. low-D.O. and saturation of the cycle.
The methods used for the tests with juvenile P . vulgaris and other species were similar
to the larval P . vulgaris tests, with modifications to the exposure chambers and test
duration Table 2. These tests only considered semidiurnal cycles and constant exposures. The exposure chambers for larval D
. sayi and juvenile P. dentatus consisted of up to twelve individual 150 ml cups attached to a common baffle for each replicate.
For P . dentatus each cup held an individual animal in order to follow the growth of that
specific individual. 2.4. Growth endpoint
After each test, the surviving animals were pooled by replicate, rinsed in deionized water, and dried at 608C for 24 h to determine their final dry weights. Growth G was
calculated for each replicate as:
L .L. Coiro et al. J. Exp. Mar. Biol. Ecol. 247 2000 243 –255
247 Table 1
a
Test parameters and growth response of newly hatched larvae P .vulgaris in five tests
Test D.O. Conc. s
D.O. Regime Test
Fractional Fractional
during and cycle
duration growth vs. sat.
difference from exposure
duration h days
control sat. control
mg l
b
1 sat.
constant 4
1 1.9,sat.
6 low 6 hi 4
0.644 0.356
1.6,sat. 6 low 6 hi
4 0.411
0.589 1.9
constant 4
0.333 0.667
1.6 constant
4 0.222
0.778 2
sat. constant
8 1
2.2,sat. 6 low 6 hi
8 0.642
0.358 1.9,sat.
6 low 6 hi 8
0.564 0.436
1.7,sat. 6 low 6 hi
8 0.438
0.562 1.4,sat.
6 low 6 hi 8
0.278 0.722
2.3 constant
8 0.541
0.459 1.9
constant 8
0.345 0.655
c
1.6 constant
8 cm
3 sat.
constant 8
1 3.0,sat.
12 low 12 hi 8
0.746 0.254
2.2,sat. 12 low 12 hi
8 0.589
0.411 1.8,sat.
12 low 12 hi 8
0.281 0.719
3.2 constant
8 0.719
0.281 2.3
constant 8
0.405 0.595
1.6 constant
8 cm
4 sat.
constant 7
1 2.8,sat.
6 low 6 hi 7
0.649 0.351
2.6 constant
7 0.494
0.506 5
sat. constant
8 1
3.2,sat. 12 low 12 hi
8 0.848
0.152 2.1,sat.
12 low 12 hi 8
0.491 0.509
1.8,sat. 12 low 12 hi
8 0.306
0.694 3.4
constant 8
0.789 0.211
2.3 constant
8 0.436
0.564 1.8
constant 8
0.252 0.748
a
Fractional growth FG calculated using Eq. 2. Difference from control calculated as 12FG. All treatments were significantly different from controls for each test Duncan’s Multiple Range Test.
b
Air saturated.
c
Complete mortality.
— —
G 5 3 dry weight animal 2 3 dry weight animal
1
final initial
where the initial dry weight was determined from a subsample taken from the test cohort at time zero. For P
. dentatus, each fish was photographed prior to test initiation to determine start length, and initial dry weight was estimated from a length:weight
regression of a subsample taken of animals taken at time zero. Growth was then
248 L
.L. Coiro et al. J. Exp. Mar. Biol. Ecol. 247 2000 243 –255 Table 2
a
Test parameters and growth responses of other life stages and species tested Species
Life stage D.O. conc.s
D.O. regime Test
Fractional Fractional
during and cycle
duration growth vs. sat.
difference from exposure
duration h days
control sat. control
mg L
b
P . vulgaris
juvenile sat.
constant 14
1 3.7,sat.
6 low 6 hi 14
1 2.5,sat.
6 low 6 hi 14
0.87 0.13
1.5,sat. 6 low 6 hi
14 0.45
0.55 3.5
constant 14
0.97 0.03
2.5 constant
14 0.87
0.13 1.5
constant 14
0.36 0.64
D . sayi
larval sat.
constant 7
1 4.5,sat.
6 low 6 hi 7
0.94 0.06
3.6,sat. 6 low 6 hi
7 0.7
0.3 2.6,sat.
6 low 6 hi 7
0.51 0.49
1.5,sat. 6 low 6 hi
7 0.11
0.89 4.2
constant 7
0.67 0.33
3.4 constant
7 0.49
0.51 2.4
constant 7
0.47 0.53
1.6 constant
7 0.1
0.9 P
. dentatus juvenile
sat. constant
10 1
1.8–4.4 6 low 6 hi
10 0.65
0.35 4.4
constant 10
0.89 0.11
1.8 constant
10 0.55
0.45 P
. dentatus juvenile
sat. constant
14 1
2.2,sat. 6 low 6 hi
14 0.82
0.18 1.8,sat.
6 low 6 hi 14
0.69 0.31
2.3 constant
14 0.67
0.33 1.8
constant 14
0.53 0.47
a
Fractional growth calculated using Eq. 2. Difference from control calculated as 12FG.
b
Air saturated.
calculated for each individual. The mean fractional growth FG Tables 1 and 2 for each treatment was calculated as:
— —
FG 5 3 growth 3 growth
2
treatment control
Time-weighted averaging was used to estimate responses to the cyclic exposures, based on responses to constant exposures. The FG
is the
time-weighted average TWA
arithmetic mean of constant exposure responses at the maximum and minimum D.O. of the cycle weighted by exposure duration using the equation:
FG 5 FG
exposure time
TWA max D.O.
max D.O.
1 FG exposure time
3
min D.O. min D.O.
L .L. Coiro et al. J. Exp. Mar. Biol. Ecol. 247 2000 243 –255
249
where FG is the fractional growth of the constant exposure which corresponds to
max D.O.
the high D.O. concentration of the fluctuation, FG is the fractional growth of the
min D.O.
constant exposure which corresponds to the low-D.O. concentration of the fluctuation, and exposure time is the portion of the whole cycle duration spent at each condition.
In this study, the cyclic exposure time was 50:50 which results in an estimate equivalent to 50 of the adverse effects observed in the continuous low-D.O exposure.
3. Results
