United States        Office of Water      ERA 822-D-99-OD2
         Environmental Protection    4304         November 1999
         Agency
SERA   Draft Ambient Water Quality
         Criteria for Dissolved
         Oxygen (Saltwater): Cape
         Cod to Cape Hatteras

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 Ambient Water Quality Criteria for Dissolved Oxygen (Saltwater):

 Cape Cod to Cape Hatteras
 Introduction

       Section 304 (a)(2) of the Clean Water Act calls for information on the conditions
 necessary "to restore and maintain biological integrity of all.. . waters, for the protection
 and propagation of shellfish, fish and wildlife, to allow recreational activities in and on
 the water, and to measure  and classify water  quality." The Environmental Protection
 Agency has not previously issued saltwater criteria for dissolved oxygen (DO) because
 the available information on effects was insufficient. This document is the result of a re-
 search effort to produce the required information to support the development of saltwater
 DO criteria. The criteria presented herein represent the best estimates, based on the avail-
 able data, of DO concentrations necessary to protect aquatic life and its uses.
       The geographic scope of this document is limited to the Virginian Province of the
 Atlantic coast of the United States (i.e., southern Cape Cod, MA, to Cape Hatteras, NC).
 The document provides the information necessary for environmental planners and regu-
 lators in the Virginian Province to decide whether the DO at a given site  can protect
 coastal or estuarine aquatic life. The approach can be used to evaluate  existing localized
 DO goals (e.g., Jordan, et al., 1992) or to establish new ones. This document does not ad-
 dress direct behavioral responses (i.e., avoiding low DO) or the ecological consequences
 of behavioral responses such as changes in predation rates or in community structures.
 The document also does not address the issue of spatial extent of a DO problem. A given
 site may have DO conditions expected to cause  a significant effect  on aquatic life, how-
 ever, the environmental manager will have to judge whether the spatial extent of the low
 DO area is sufficient to warrant concern. The approach presented here for deriving crite-
 ria  is expected to  work for other regions. However, additional regionally specific data
 may be required in order to amend the database for use in other regions. Animals may
 have adapted to lower oxygen in locations where high temperatures have historically re-
 duced concentrations, or in systems with natural high demands for oxygen. In  addition,
 effects of hypoxia1 may vary latitudinally,  or site-specifically, particularly as reproduc-
 tive seasons determine risks of exposure for sensitive early life stages.
       As with the freshwater DO document (U.S.  EPA, 1986), all data and  criteria are
 expressed in terms of the actual amount of DO  available to aquatic organisms in milli-
 grams per liter (mg/L). However, unlike the freshwater document, which provides limits
 for DO in both warm and cold water,  criteria are presented for only warm saltwater  be-
 cause hypoxia in Virginian Province coastal waters is primarily restricted to the  warm
water of summer. Also, the freshwater criteria are based almost entirely on fish data even
though insects were often more sensitive than  fish. The saltwater limits, on the other
hand, use data from fish and invertebrates.
1 Hypoxia is defined in this document as the reduction of DO concentrations below air saturation.

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       The  saltwater criteria described herein are intended to maintain and support
aquatic life and their designated uses. Criteria derived using the Guidelines2 are intended
to protect aquatic communities, but they rely primarily on data generated at the organism
level, and emphasize data for the most sensitive life stage. But a population of a given
species can potentially withstand some mortality to  certain life stages without a signifi-
cant long-term effect on the population. Hence, an assessment of criteria should include
population-level considerations.  One nuance of population-level assessment is the fact
that a population's sensitivity to hypoxia may depend on which stages have been ex-
posed. For example, many populations of marine organisms may be more impacted by
mortality occurring during the juvenile and adult stages than during the larval stage(s). In
this regard, a particular individual larva is not as important to the population as a par-
ticular individual juvenile or adult. With this in mind, the saltwater criteria for DO segre-
gate effects on juveniles and adults from those on larvae. The survival data on the sensi-
tivity of the former are handled  in a traditional Guidelines manner. The cumulative ef-
fects of low DO on larval recruitment to the juvenile  life stage, on the other hand, address
survival effects on larvae. The recommended DO approach uses a mathematical model to
evaluate the effect on larvae by  tracking intensity and duration effects across the larval
recruitment season. Protection for larvae of all species is provided by using data  for a
sensitive aquatic organism (the Say mud crab Dyspanopeus sayi in this case). This model
is used to generate a DO criterion for larval survival as  a function of time.
       For the reasons listed above, the approach recommended below to derive DO cri-
teria for saltwater animals deviates from EPA's traditional approach for toxic chemicals
outlined in the Guidelines. Where practical, however, data selection and analytical proce-
dures are consistent with the Guidelines. Therefore, some of the terminology and the cal-
culation procedures are the same. Thus, knowing the  Guidelines are useful (but not es-
sential) for better understanding how  the limits were derived. Terminology from the
Guidelines used here includes Species Mean Acute Value (SMAV), Genus Mean Acute
Value (GMAV), Final Acute Value (FAV),  Genus  Mean Chronic Value (GMCV) and
Final Chronic Value (FCV). Procedures from the Guidelines include those for calculating
FAVs, Criterion Maximum Concentration3 (CMC) and Criterion Continuous Concentra-
tion (CCC).

Overview of the Problem
       The EPA's Environmental Monitoring and Assessment Program (EMAP) for the
estuaries in the Virginian Province has shown that 25% of its area is exposed to some de-
gree to DO concentrations less than 5 mg/L (Strobel  et al., 1995). EMAP has also gener-
ated field observations that correlate biological degradation in many benthic areas with
low DO in the lower water column  (Paul et al., 1997). The two reports serve to empha-
size that low DO is a major concern within the Virginian Province. Even though hypoxia
is a major concern, a strong technical basis for developing benchmarks for effects of low
DO has been lacking.
2 Guidelines for deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organ-
isms and Their Uses (Stephen, et al., 1985hereafter referred to as the Guidelines).
3 Although in the case of dissolved oxygen, CMC is more appropriately defined as the Criterion Minimum
Concentration.

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       Hypoxia in the Virginian Province is essentially a warm-water phenomenon. In
the southern portions of the Province, such as the Chesapeake Bay and its tributaries, DO
may be reduced any time between May and ^October; in the more northern coastal and
estuarine waters, any time from late June into September. Hypoxic events may be sea-
sonal or diel. Seasonal hypoxia often develops as stratified water prevents the oxygenated
surface water from mixing  downward. Low DO then appears hi the lower waters when
respiration in the water and sediment depletes oxygen faster than it can be replenished.
As summer progresses, the  areas of hypoxia expand and intensify, then disappear as the
water cools in the fall. The  cooler temperatures eliminate the stratification and allow the
surface and bottom waters  to mix. Diel cycles of hypoxia  often appear in unstratified
shallow habitats where nighttime respiration can temporarily deplete DO.

       Although the primary fauna at risk from exposure to hypoxia  in the Virginian
Province are summer inhabitants of subpycnocline4 (i.e., bottom) waters, hypoxia can
occur in other habitats as well. For example, upwelling may permit subpycnocline, oxy-
gen-poor water to intrude into shallow areas. Hypoxia also may appear in the upper water
of eutrophic water bodies on calm, cloudy days, when more  oxygen is consumed than is
produced by photosynthesis and when atmospheric reaeration is  limited. In spite of this
tendency, however, minima in DO are generally less severe above the pycnocline than
below it. Hypoxia above the pycnocline also tends to be more transient because it largely
depends on weather patterns.

       Hypoxia may persist more or less continuously over  a season (with or without a
cyclic component) or be  episodic (i.e., of irregular occurrence and indefinite duration).
Continuous hypoxia  without  a cyclic component is exemplified in the  subpycnocline
waters of western Long Island Sound and off the New Jersey coast (Armstrong, 1979).
Hypoxia in Long Island Sound may be interrupted temporarily by major storms,  but re-
turns  one or two weeks  later, when the waters again become  stratified (Welsh et al.,
1994).

       Hypoxia may oscillate with tidal,  diel or lunar  frequencies. Tidal hypoxia is
common in subpycnocline waters of the mesohaline Chesapeake  Bay main stem and the
mouth of the adjacent tributaries during summer (Sanford et al., 1990; Diaz et al., 1992).
In this case, DO concentrations oscillate as the tides alternately advect poorly oxygenated
subpycnocline water  from the mid-bay trough or tributaries and better oxygenated water
from the lower bay. Diel cycles of hypoxia are found in small eutrophic embayments and
harbors all along the coast of the Virginian Province, where oxygen is depleted overnight
by respiration and replenished by photosynthesis after dawn. The Childs River is an ex-
ample of diel hypoxia (D'Avanzo and Kremer, 1994). Lunar cycles of oxygen may occur
in various systems but have been documented most clearly at the mouths of some  Chesa-
peake Bay tributaries, where destratification  from spring tides saturates the water with
oxygen and stratification afterward depletes the  oxygen (Haas,  1977; Kuo et al., 1991;
Diaz et al., 1992).
4 The pycnocline is the region of density discontinuity in a stratified water column between surface and
bottom waters. The density difference between the two is primarily due to differences in temperature and
salinity.

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       Episodic hypoxia has been noted in shoal waters of mid-Chesapeake Bay (Breit-
burg, 1990) and in adjacent tributaries (Sanford et al., 1990). Persistent winds tilt the
pycnocline laterally and displace low DO water onto the shoals or tributaries indefinitely.
As noted above, DO may also be reduced episodically in eutrophic surface waters, par-
ticularly during calm and cloudy weather, when photosynthesis is slow and daytime re-
oxygenation is reduced.

Biological Effects of Low Dissolved Oxygen
       Oxygen is essential in aerobic organisms for the electron transport system of mi-
tochondria. Oxygen insufficiency at the mitochondria results in reduction in cellular en-
ergy and a subsequent loss of ion balance in cellular and circulatory fluids. If oxygen in-
sufficiency persists, death will ultimately occur, although some aerobic animals also pos-
sess  anaerobic  metabolic  pathways, which can  delay lethality for short time periods
(minutes to days). Anaerobiosis is well developed in some benthic animals, such as bi-
valve molluscs and polychaetes, but not in other groups, like fish and crustaceans (Ham-
men,  1976). There is no evidence that any free-living animal inhabiting coastal or estua-
rine waters can live without oxygen indefinitely.

       Many aquatic animals have adapted to short periods of hypoxia and anaerobiosis
by taking up more oxygen and transporting it more effectively to cells and mitochondria,
i.e., by ventilating its respiratory surfaces more intensely  and increasing its heart rate.  If
these responses are insufficient to maintain the blood's pH, the oxygen carrying capacity
of the respiratory pigment will decrease. An early behavioral response might be moving
faster toward better-oxygenated water. However, if the hypoxia persists, the animal may
reduce its swimming and feeding, which will reduce its need for energy and hence oxy-
gen.  Such reduce motor activity may make the animal more tolerant over the short term,
but will not solve its long-term problem. For example, even the modest reductions in lo-
comotion required by mild hypoxia may make the animal more vulnerable to predators,
and the reduced feeding may decrease its growth.

       Compensatory adaptations are well developed in marine animals that commonly
experience hypoxia, e.g., intertidal  and tide pool animals (McMahon, 1988), and bur-
rowing animals, which partly explains their reported high tolerance to low DO. In con-
trast,  compensatory adaptations are poorly developed  in  animals that inhabit  well-
oxygenated environments such as the upper water column. The animals most sensitive  to
hypoxia are among this latter group. Details on compensatory adaptations to hypoxia are
provided in reviews for marine animals (Vernberg, 1972), aquatic invertebrates (Herreid,
1980)  and fish (Holeton,  1980; Hughes,  1981;  Kramer,  1987; Rombough, 1988a, and
Heath, 1995).

Overview of the Approach
       The approach to determine the limits of DO that will protect  saltwater animals
within the Virginian Province considers both continuous (i.e., persistent), and cyclic (e.g.,
diel) exposures to low DO. The continuous situation is covered first, and deals with expo-
sures longer than 24 hr. It is followed by sections on criteria for exposures of less than
                                        10

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24 hr but that may be repeated for days. Both scenarios cover three areas of protection
(summarized here, and explained in more detail in the sections that follow):
     1. Juvenile and adult survivalA lower limit is calculated for continuous exposures
       by using Final Acute Value (FAV) calculation procedures outlined in the Guide-
       lines (Stephan et al., 1985), but with data for only juvenile or adult stages. Limits
       for cyclic exposures are derived from an appropriate time-to-death curve for ex-
       posures less than 24 hr.
     2. Growth effectsA threshold above which long-term, continuous exposures should
       not cause unacceptable effects is derived from growth data  (mostly from bioas-
       says using larvae). This  Final Chronic Value (FCV) is  calculated in the same
       manner as the FAV for juvenile and adult survival. This threshold limit as cur-
       rently presented has no tune component (it can be applied to exposures of any du-
       ration). Cyclic exposures are evaluated by comparing reductions in laboratory
       growth from cyclic and continuous exposures.
     3. Larval recruitment effectsA larval recruitment model was developed to project
       cumulative loss caused by low DO. The effects depend on the intensity and the
       duration of adverse exposures. The maximum acceptable reduction in seasonal re-
       cruitment was set at 5%, which is equivalent to the protective limit for juvenile
       and adult survival. The number of acceptable days of seasonal exposure to low
       DO decreases as the severity of the hypoxic condition increases. The severity of
       cyclic exposure is evaluated with a time-to-death model (as in the protective limit
       for juveniles and adults).


Persistent Exposure to Low Dissolved Oxygen
Juvenile and Adult Survival
       Data were used from tests with exposure ranging from 24 to 96 hr. This maxi-
mized the number of genera for the FAV calculation. Data for juveniles show that LC50
values calculated for 24 and 96 hr observations are very similar (Figure 1), therefore, all
values are applied as 24 hr data. The restriction of the data set to tests of 96 hr duration or
less was somewhat arbitrary; however, 96 hr is the duration used for most acute  tests for
traditional water quality criteria (Stephan et al., 1985). In addition, there are insufficient
test data to compare 24 hr exposures versus those longer than 96 hr. Juvenile and adult
mortality data from exposures longer than 96 hr are compared to the final criterion in the
section on Other Laboratory Bioassay Data.
       Data on the acute sensitivity of juvenile and adult saltwater animals to low DO is
available for 12 invertebrate and 11 fish species (almost all of the data are for juveniles).
The  values are summarized in Table 1 and Appendix B. Overall Genus Mean Acute Val-
ues (GMAVs) range from <0.34  mg/L for the  green crab Carcinus maenas to 1.63  mg/L
for the pipe fish Syngnathus fuscus; a factor greater than 4.8. Juvenile fish are somewhat
more sensitive than juvenile crustaceans (Table 1; Figure 2). In fact, the four most sensi-
tive  genera are all fish, and the range of values for these is 1.32 to 1.63 mg/L; a ratio of
only 1.2.
       As stated previously, the  criterion for juveniles  and adults exposed to continuous
low  DO was  calculated using  the Guidelines procedures  for  derivation of  an  FAV
                                        11

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                                     24 hr LC50 (mg/L)
         Figure 1. Relationship between 24 and 96 hr LC50 values for juvenile saltwater ani-
         mals exposed to continuous low dissolved oxygen. Each point represents a paired set of
         values calculated from the same test run. The line drawn represents a one-to-one rela-
         tionship. Data for the plot are summarized by species in Appendix A. Appendix A also
         contains data for test runs with larvae.
(Stephan, et al., 1985). However, the procedures outlined in the Guidelines were created
for toxicants. Since DO behaves in the opposite manner to toxicants (i.e., the greatest re-
sponse is associated with the lowest concentrations), DO concentration data were trans-
formed by using their inverse in the calculation. The FAV calculation is essentially a lin-
ear regression using the LC50 values for the four most sensitive genera and their respec-
tive percentile ranks. The final  FAV is the value representing the 5th percentile genus5,
which for DO is 1.64 mg/L.  This value is adjusted to a criterion of 2.27 mg DO/L by
multiplying by 1.38, the average LC5 to LC50 ratio6 for juveniles (Table 1). This value is
analogous to the CMC (Criterion Maximum Concentration) in traditional Water Quality
Criteria for toxicants.
5 Alternatively we could have modified the FAV calculation procedure to use untransformed data and es-
tablished the protective limit for the 95th percentile. However, the calculated results would be the same.
Since many researchers already have computer programs that calculate FAVs, we opted to remain consis-
tent with the Guidelines by using the inverse data.
6 The use of a ratio to adjust the FAV to a CMC is designed to estimate a negligible lethal effect concentra-
tion corresponding to the 5th percentile species. It may in fact represent an adverse effect concentration for
species more sensitive than the 5th percentile. The Guidelines use a factor of 2, however, there were suffi-
cient data available for low DO to use a factor specific to this stressor.
                                            12

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         LCSOs) against percentile rank of each value in the data set Values for each genera
         are listed in Table 1. Results from individual tests for each species are listed in Ap-
         pendix E. The value highlighted on the y-axis is the calculated Final Acute Value
         (FAV). This value is the LC50 that is higher than the values for 95% of the tested
         genera. The line drawn through the four most sensitive genera is the line of best fit
         for those four values. The LC50 values for the four most sensitive genera are the
         only values used in the FAV calculation other than the total number ("n") of values.
Growth Effects
       A threshold above which long-term, continuous exposures to low DO should not
cause unacceptable effects was calculated with growth data (mostly from bioassays using
larvae). Sub-lethal effects were evaluated with only growth data for two reasons. First,
growth is generally more sensitive to low DO than survival. There were only two excep-
tions where survival was more sensitive to low DO than growth. One test was with D.
sayi, however, growth was the more sensitive endpoint in eight other tests with this spe-
cies (Appendix C). The results from this one test were not included in Table 2. The other
exception was a 28-day early life stage test using the Atlantic silverside Menida menidia
(Appendix C). There was no effect at 4.8 mg/L DO, but there was 40% mortality and a
24% reduction in growth at a DO concentration of 3.9 mg/L. This 24% reduction in
                                           14

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growth, however, was not statistically significant. There was essentially no growth of
surviving M menidia at a DO concentration of 2.8 mg/L. Only the growth data were
summarized in Table 2.
       The second reason for restricting sub-lethal effects to growth is that results are
available from only  one saltwater test that measured reproductive effects. Data are pre-
sented in Appendix C from a 28-day life cycle test using the mysid Americamysis bahia.
Although growth was reduced 25% at 3.17 mg/L and was technically the most sensitive
endpoint in this test,  the percentage reduction in growth was essentially the same at 2.76
and 2.17 mg/L as it  was at 3.17 mg/L (20% and 27%, respectively). Reproduction was
reduced by 76% at 2.17 mg/L, the first treatment that resulted in a significant effect on
this endpoint. Although this test suggests that growth is more sensitive than reproduction,
there are insufficient data to confirm this conclusion for saltwater species. Data from two
standardized freshwater tests, however, indicate that growth is more sensitive than repro-
duction for both fathead minnows (Brungs, 1971) and Daphnia magna (Homer and Wal-
ler, 1983).  Thus, DO limits that protect against growth effects also may be protective for
reproductive effects.
       Data on the affects of hypoxia on growth are presented for four species offish and
seven species of invertebrates  from a total of 36 tests. The sensitivity of growth to low
DO has been determined in only two standard 28-day tests which meet Guidelines re-
quirements; the above life cycle test with A. bahia and the above early life stage test with
M.  menidia. Therefore, growth data from non-standard tests (i.e., not  life cycle, partial
life cycle or early life stage tests) were used to augment the chronic database. These non-
standard tests ranged from 4 to 29 days long.  Data from short duration tests were in-
cluded because effects of oxygen deprivation are assumed to be instantaneous. Oxygen is
required continuously for the  efficient production of cellular energy.  Therefore,  even
modest reductions in DO may result in the redirection of energy use from growth to  com-
pensatory mechanisms. In addition, data from larval growth of two bivalves (Morrison,
1971; Wang and Widdows, 1991) and several fish and crustaceans (Appendix C) show
that chronic values for DO do not change substantially for exposures ranging from a few
days to several weeks for most of the species tested. The Mercenaria mercenaria (Morri-
son, 1981)  andMytilis edulis (Wang and Widdows, 1991) studies show that the effect on
larval bivalve growth within the same test run is the same over a series of days (13 days
forM mercenaria and 6 to 10 days forM edulis).
       Overall Genus Mean Chronic Values (GMCVs) for effects on growth range from
> 1.97 for the sheepshead minnow Cyprinodon variegatus to 4.67 mg/L for the longnose
spider  crab Labinia  dubia; a ratio of < 2.4. Three of the most sensitive species  were
crustaceans (Figure 3; Table 2). The range of chronic values for the four most sensitive
genera is 3.97 to 4.67 mg/L; a ratio of only 1.2.  The Final Chronic Value (FCV) was cal-
culated in the same manner as the FAV (Stephan, et al., 1985). Because acutely resistant
taxa are under-represented hi the chronic database in Table 2, it could be argued that n,
the number of genera used in the calculation of the FCV, should be increased from  11 to
a higher value. We chose to increase n from 11 to 22 (the n for the FAV). This is the
same procedure that was used for the FCV in the ambient water quality.criteria for cad-
                                        16

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         Figure 3. Plot  of low dissolved oxygen effect (Genus Mean Chronic  Values  for
         growth) against  percentile rank of each value in the data set. Percentile rank was  ad-
         justed based on the total "n" from the acute data set (see text for explanation). Specific
         values for each genus included are listed in Table 2. Results from individual tests for
         each species are listed in Appendix C. The value highlighted on the y-axis is the calcu-
         lated Final Chronic Value (FCV). This value is the chronic value that is higher than the
         values for 95% of the species represented. The line drawn through the four most sensi-
         tive values is the line of best fit for those four values. The chronic values  for the four
         most sensitive genera are the only values used in the FCV calculation other than the
         total number ('n") of values.


mium7 (U.S. EPA, 1985). The final protective value for growth (the Criterion Continuous
Concentration or CCC) is  4.8 mg DO per liter, but would increase only to 5.0 mg/L if n
was kept at 11.

        As presented here, the CCC is intended as  a time-independent value. Areas where
the average minimum DO does not fall below 4.8 mg/L should have sufficient DO to
support the survival and growth of most aquatic  species in the Virginian Province. Al-
though it is generally accepted that reduced growth means reduced overall fitness, there is
7 One assumption underlying the calculation procedure for FAVs and FCVs is that the sample of values
available is representative of the population of values in the community being protected. If the dataset is
too heavily weighted with values from the sensitive end of the distribution, then this skews the interpreta-
tion of the 5th percentile value that is calculated.
                                             17

-------



1

^
^f
g
o
c
>
1
10
o
"o
10
V)
b




3.5
3.0

2.5.



2.0.



1.5.

1.0.


0.5.


0.0
D. say/-megalopa
Larvae-96 hr
D
~ n ^
A u A  D. say/
Larvae-24 hr A ,
D /
A D /
A 
A y
0 ' A

*"*o AAA
Juveniles * o 0 A A
o   o 0
* 
 5
0  o
o
o

                   0    10   20   30   40   50   60   70    80    90   100
                                          % Rank

        Figure 4. Plot of the GMAV data from Figure 2 (circles) along with 24 hr (triangles)
        and 96 hr (squares) LC50 values for larval life  stages of various saltwater animals. The
        open symbols are for invertebrates and the closed for fish. The open' square and triangle
        for D. sayi represent the mean response for all larval life stages for this species. The
        dashed line at top  represents the  LC50 for D. sayi exposed during the transition to
        megalopa. The data for the juveniles are from Table 1. The data for the larvae are listed
        in Appendix D.
little direct evidence for this in the field. In one study, Gleason and Bengtson (1996a,b)
found that for some estuarine fish bigger is not necessarily better. Bigger fish (as prey)
may be more susceptible to being  eaten by predators.  As an alternative to the growth
criterion, a criterion that addresses chronic stresses from long-term or short-term expo-
sures to low DC can be based on larval recruitment effects.

Larval Recruitment Effects
       A generic model has been developed that evaluates the cumulative effects of acute
and chronic stresses on early life stages of aquatic organisms. Early life history informa-
tion and exposure-response relationships are integrated with duration and intensity of ex-
posure to provide an ecologically relevant measure of larval recruitment. There are ex-
isting recruitment models for marine organisms (e.g., Ricker, 1954; Beverton and Holt,
1957). However, these models address other processes such as parental stock size, popu-
lation fecundity, and density dependent processes such as cannibalism and intraspecific
competition. These  existing models therefore are not appropriate for the-needs of the DO
document, which requires incorporation of abiotic stressor effects.
                                          18

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       Larvae are more acutely sensitive to low DO than juveniles (Figure 4); however,
the criteria are not being established to protect larvae and juveniles in the same manner.
A method is needed that estimates how many days a given DO concentration can be tol-
erated without causing unacceptable effects on total larval survival for the entire recruit-
ment season. This is accomplished with a generic larval recruitment8 model and applying
biological and hypoxic effects parameters for the Say mud crab (D. sayi). Parameters for
this larval crustacean are used for several reasons. Larval crustaceans are among the most
acutely and chronically sensitive larval saltwater animals, and the Say mud crab's late
larval to megalopa period is the most sensitive of the tested crustaceans (Table 2 and Fig-
ure 4). Among larvae at risk in estuaries,  considerable information is  available on Say
mud crab with respect to the biological parameters in the model. Laboratory responses of
D. sayi are indicative of a species the most at risk from hypoxia because it has a high DO
response threshold. In addition, these larvae are present in the lower water column coin-
cident with the expected hypoxia season present throughout the Virginian Province in
salinities >15 ppt, which  strengthens the  choice  of this species for  a Province-wide
model.
       The model and the major assumptions used during its development are presented
in Appendix E. The life history parameters hi the model include only those mat relate
specifically to larvae: larval  development time, larval season, attrition rate and vertical
distribution. The recruitment model assumes that the period of low DO occurs within the
larval season.  The magnitude of effects on recruitment, defined as the cumulative number
of successful transitions to megalopae, is influenced by each of the four life history pa-
rameters. For instance, larval development time establishes the number of cohorts that
entirely or partially co-occur with the interval of low DO stress. The second parameter,
the length of the larval season, is a function of the spawning period, and also influences
the relative number of cohorts which fall within the window of hypoxic stress.  The third
life history variable, natural attrition rate, gages the impact of slower growth and devel-
opment of the larvae in response to low DO by tracking the associated increase in natural
mortality (e.g., predation).  The model assumes a constant rate of attrition, so increased
residence time in the water column due to delayed development translates directly to de-
creased recruitment. Finally,  the vertical distribution of larvae in the water column de-
termines the percentage of larvae that would be exposed to reduced DO under stratified
conditions. Three exposure response curves that describe megalopa survival, zoea larval
survival, and molt delay versus DO concentration are used for estimating recruitment un-
der hypoxic conditions9.  The model makes a simplifying assumption that hypoxic days
are contiguous. The model can be applied either to establish protective conditions or to
evaluate  the severity of a given hypoxic condition.

       The dose-response data used in the model in this document are presented in Fig-
ure 5. Figure  5A is a summary response curve for exposures that included a transition
from zoea to megalopa. These tests were necessarily longer (7 to 11 days) than other lar-
val tests  to allow sufficient  time for development to megalopa. Although some of the en-
 Once the larvae are "recruited" into the juvenile life stage, the juvenile protective limit established above
is applied.
9 The model is designed to allow both biological and exposure-response data to be changed based on the
availability of appropriate data.
                                        19

-------
hanced sensitivity in these tests may be due to the longer exposures to low DO, mortality
also appeared to be associated with the molt to megalopa1 . The model assumes a con-
stant rate of reproductive output per day, and a constant rate of development during the
larval  season. Therefore,  some larvae in the plankton would be molting to this stage
daily, and it is at this point that the crab larvae may be particularly sensitive to low DO.
The model assumes that the response of the late larvae in transition to megalopae could
occur following a single day of exposure (i.e., this response is independent of exposure
prior to the day of transition). Thus, the model applies this dose response as a 24 hr expo-
sure.
       Figure 5B is a summary response curve for 24 hr exposures of zoea stage larvae.
Figure 5C shows data that suggest a delay in development time for D. sayi in going from
a stage 3 zoea to megalopa. However, the degree of developmental delay was difficult to
measure  with sufficient resolution. Further, it was difficult to distinguish it from differ-
ential survival sensitivity among individuals within a replicate. Thus, the model has been
run with and without a delayed development effect. The results of these two runs are
shown in Figure 6. Points  on the graph show which combinations of low DO concentra-
tion and  exposure duration result in a seasonal reduction of recruitment that does not ex-
ceed 5%. Until further information is  available, the output used to establish the criteria
for larval recruitment will be the one that assumes no delayed development (the solid line
in Figure 6).
       The equation for the larval line (as well as the lines in Figure 5A and 5B) was de-
rived by  an iterative process of fitting the best line through the points  generated by the
output of the recruitment model. The equation is a standard mathematical expression for'
inhibited growth (logistic functionBittinger and Morrel, 1993). This equation is:

                   P L
       P(t) =	r	    Equation 1
         U   P0+e-Lkt(L-P0)

For Figure 6, P(t) is the DO concentration at time t, PQ  is the y-intercept, and L is the up-
per DO limit L was  set as the DO concentration that allowed a 44-day exposure (the
maximum exposure period the model allowed using the current parameterssee appen-
dix page E-3 for further explanation). PO was first estimated by eye from the original plot,
and then adjusted higher or lower to minimize the residuals between the real recruitment
data and that estimated from the mathematical fit of the data. The rate constant, k, was
similarly empirically derived. For Figures 5A and 5B, the variables t and L represent DO
concentration and the upper limit for survival (100%), respectively.
10 Data for another crustacean, Cancer irroratus (rock crab), also lend some support for having separate
dose response curves for the zoea and megalopa larval life stages (Appendix F).
                                        20

-------
                          Zoea Transition to Megalopa
           V)
                                                Po = 0.10
                                                L = 100
                                                 k = 0.0208
           1
100
 90.
 80 .
 70
 60
 50
 40 -I
 30
 20.
 10.
  0
                    B.   Zoea Larvae
              0.0
                           Po = 0.01
                            L = 100
                            k = 0.0489
                  0
              1.4,

              1.2

        |  S  1.0

        S  I  0.8
        O  K_
        I  =  0.6 .
        2  S
        >  1  0.4 .

           ^  0.2.
                    C.  Delayed Development
y=-0.0785x +1.3931
     R? = 0.851
                 0      1      2      34      5      6
                          Dissolved Oxygen (mg/L)

Figure 5. Dose response curves for Say mud crab (Dyspanopeus sayi) used in
the larval recruitment model. Open symbols are the data from tests with con-
tinuous low dissolved oxygen exposures. Solid lines are the regression lines of
best fit. See text for explanation of Po, L and k.  A: dose response curve for zoea
transition to megalopa. These data are from exposures durations greater than 24
hr but are applied as 24 hr exposures in the model (see text for explanation). B:
dose response curve for zoea larvae. Data are from 24 hr exposures. C: data for
delayed development of larvae to megalopae
                                 21

-------
Application of Persistent Exposure Criteria
       The final criteria for saltwater animals in the Virginian Province (Cape Cod to
Cape Hatteras) are indicated in Figure 7 for the case of continuous (i.e., persistent) expo-
sure to low dissolved oxygen. The most uncertainty with the application of these limits
usually will be when DO conditions are between the juvenile survival and larval growth
limits. Below the juvenile survival limit,  DO conditions do not meet protective goals.
Above the growth limit, conditions are likely to be sufficient to protect most aquatic life
and  its uses. Interpretation of acceptable  hypoxic conditions when the DO values are
between the juvenile survival and larval growth limits depends on the characterization of
the duration of the hypoxia. To determine whether a given site has a low DO problem,
adequate monitoring data are required. The more frequently DO is measured the better
will be the estimate of biological effects.
       Figure 8  is a hypothetical time series for average daily dissolved oxygen minima.
The portion of the data below the CCC is all that is considered. This area of the graph is
first divided into several intervals. We recommend using no finer than 0.5 mg/L DO in-
tervals because of limitations on most monitoring programs (see Implementation section).
However, larger intervals may be necessary if monitoring data are not taken  frequently
enough. The resulting intervals in our example are (a)  below 4.8  mg/L and  above 4.3
mg/L, (b) below 4.3 and above 3.8, and so forth for intervals 'c' and 'd'. For each inter-
val, the number  of days is recorded that the DO is between the interval's limits. For ex-
ample, in interval 'a' the DO is  below 4.8 mg/L and above 4.3 mg/L from July 13th
through the 18th  and again from July 23rd through the 25th, for a total of seven  days.  This
number of days is then expressed as a fraction of the total number of days that would be
allowed for the DO rninimum for each interval. For interval 'a', the allowed number of
days is 24 (using Figure 6 at 4.3 mg/L). Table 3 lists the information for all four intervals
from this hypothetical time series. The fractions of allowed days are totaled. If the sum is
greater than one, then the DO conditions do not meet the desired protective goal for larval
survival. If the sum is less than one (as is the case in our example), then the protective
goal has been met. This procedure uses a simplifying assumption that each interval is in-
dependent. That is, there is no increased risk to recruitment due to pre-exposure to hy-
poxia. This assumption is supported by the similarity of larval survival data for 24 and 96
hr exposures in Appendix A.
       The current recruitment model is a first attempt at providing a method  that incor-
porates duration of exposure in the derivation of DO criteria. A model that could  inte-
grate gradual change in daily DO concentrations is desirable. However, the current model
may be adequate given the probable inaccuracies in assessments  of DO conditions in
coastal waters (Summers, et al., 1997).
                                        22

-------
        I
5.5
5.0
4.5
4.0-,
3.5
3.0-
2.5-
2.0-
1.5
1.0
0.5-I
0.0
                      Recruitment Model Output at 5% Protection
                 with delayed development    
without delayed development
Po=2.15
 L = 4.45
 k = 0.036
                    5      10     15     20     25    30     35     40
                                    Exposure Time (days)
                                                               45
Figure 6. Plot of model output that protects against greater than 5% cumulative im-
pairment of recruitment. Input parameters were the same for two runs of the model,
except for the inclusion of the delayed development response (Figure 5C), open sym-
bols, or the exclusion of molt delay, closed symbols, The solid line is the regression
line of best fit for the closed symbols. The area below the line represents conditions
of potential impairment. See text for explanation of Po, Land k.
55
5.0 .
4.5.
31 4.0 .
ra
S 3.5 .
g, 3.0 .
0 2.5.
a
> 2.0 .
1 1
-------
            01

            c
            ,
            >,
            
            a
            I
            o
            (0
            tn
            a
5.8 4v

5.3

4.8

4.3

3.8

3.3

2.8
               2.3
ccc
                                                             o>
                                                            <
                                             CM
                                                   <.    <.
                                                   in    o
                                                   T-    CM
             IO
             CM
O)

^
O
CO
        Figure 8. A hypothetical representative dissolved oxygen time series for one site. The
        horizontal line represents  the CCC of 4.8 mg/L.  The portion of the curve below 4.8
        mg/L is divided into four arbitrary intervals (a,b,c,d) to estimate effects on larval re-
        cruitment. The dissolved oxygen minimum, and the duration for each interval are
Table 3. Dissolved oxygen and duration data from a hypothetical persistent time series (Figure 8). The
Below and Above columns show the range of D.O. covered by each interval. Number of Days Within
Range refers to the duration that the observed D.O. is between the range given. In the last column this
duration is expressed as a fraction of the number of days allowed by the recruitment model (Figure 6) for
the D.O. minimum of the interval. These fractions are totaled to evaluate whether the larval survival
protective goal has been met.
Range (mg/L)
Interval Below Above
a 4.8
b 4.3
c 3.8
d 3.3
4.3
3.8
3.3
2.8
No. Days
Within Range
7
3
1
1
No. Days
Allowed
24
13
7
4
Fraction of
Allowed
0.29
0.23
0.14
0.25
                                                                    TOTAL
                       0.91
                                          24

-------
 Less Than 24 hr Episodic and Cyclic Exposure to Low Dissolved Oxygen
       The criteria for continuous exposure to low dissolved oxygen do not cover expo-
 sures times less than 24 hr. This section addresses this topic by describing the available
 data and how they were used to evaluate the effect of low DO on  exposure durations
 lasting less than 24 hr. These included one-time episodic events, as well as either tidal- or
 diel-influenced cycles where the DO concentrations cycle above and below the continu-
 ous CCC. The approaches described for treatment of non-constant (e.g., cyclic) condi-
 tions are intended to provide protective goals that are equivalent to those established for
 persistent conditions. The data used come from two types of experiments. The first are
 those which provide time-to-death (TTD) data and are used to derive TTD curves. The
 second are experiments in which there were treatments consisting of a constant exposure
 to a' given low DO  concentration paired with a treatment in which the DO  concentration
 cycled between that low concentration and a concentration near  saturation (or at least
 well above concentrations that should cause significant effects). The data from both of
 these experiments are discussed below.
 Cyclic Juvenile and Adult Survival
       The persistent hypoxic criterion for juveniles and adults is 2.3  mg/L. A conserva-
 tive estimate of the  safe DO concentration for exposures less than 24 hr would be to sim-
 ply use 2.3 mg/L. However, time-to-death data indicate that this would be over protec-
 tive. Data are available for two saltwater juvenile fish (Brevootia  tyrannus and Leiosto-
 mus xanthurus), one freshwater juvenile fish (Salvelinus fontinalis), and three larval salt-
 water crustaceans (D. sayi, Palaemonetes vulgaris and Homarus americanus), providing
 a total of 33 TTD curves (Appendix G). The curves represent a range of test conditions,
 including acclimation to hypoxia with S. fontinalis, and a range of lethal endpoints. Two
 general observations were made from this data. First, each curve can be modeled with the
 same mathematical expression, a logarithmic regression, of the form:

       Y=m(lnX)+b        Equation 2.

       where  X=time, Y=DO concentration, m=slope and  b=intercept where the line
       crosses the Y-axis at X=l.

 Second, the shape of the curve (i.e., the slope and intercept) was governed by the sensi-
tivity of the endpoint. This is true whether the sensitivity increase was due to interspecific
differences (including saltwater and freshwater species) or the use of different endpoint
 (e.g., LC5 is more sensitive endpoint than LC50).
       Figure 9 shows the relationship between sensitivity (i.e., 24 hr LC values) and the
slope (Figure 9A) and the intercept (Figure 9B) for all 33 TTD curves (Appendix G). The
DO value from each TTD curve at 24 hr was used as a measure of sensitivity. Plots using
other time intervals  could have been used. The value at 24 hr was chosen in order to gen-
erate a curve for juveniles that meets the constant CMC at its 24 hr value (2.3 mg/L). The
slope and intercept  for a time-to-CMC curve were calculated using Figure 9 equations
and the CMC 24 hr value of 2.3 mg/L. These were then used as the parameters in Equa-
tion 2 to generate a criterion for saltwater juvenile animals for exposures less than 24 hr
(Figure 10).
                                       25

-------
          0.6

          0.5.

          0.4 .
               y=0.191x- 0.064
                  R2 = 0.835
                         Dissolved Oxygen Concentration
                      Causing Effect Observed at 24 hr (mg /L)
          2.0 -
          1.8 .
          1.6.
          1.4.
          1.2.
          1.0 .
          0.8.
          0.6.
          0.4.
          0.2.
          0.0
B
                y = 0.392X + 0.204
                    R2 = 0.678
                           Dissolved Oxygen Concentration
                       Causing Effect Observed at 24 hr (mg /L)
Figure 9. Slope (A) and intercept (B) versus low D.O. effect values at 24 hr from
time-to-death (TTD) curves for two species of saltwater juvenile fish, one species of
juvenile freshwater fish and three species of saltwater larval crustaceans. Data used
mostly represent LT50 curves, but values for other mortality curves are included.
Species used and their associated TTD curves are presented in Appendix G. All TTD
curves were fit with a logarithmic regression.
                                  26

-------
                2.5,
                2.0 .
  1.5 J
0)

I
a
I
o
10
to
5  0.5 J
                1.0.
                0.0
                            Time-to-"CMC"
                                       = 0.370Ln(x) +1.095
                                  8      12      16
                                  Exposure Time (hr)
                                           20
24
         Figure 10. Criterion for juvenile saltwater animals exposed to low dissolved oxygen
         for 24 hr or less. The line represents the same protective limit as the CMC for juve-
         niles for continuous exposure. The line is a logarithmic expression with a slope and
         intercept calculated from the regressions in Figure 9 at the dissolved oxygen con-
         centration of 2.3 mg/L (the CMC).
Cyclic Growth Effects
       The CCC for continuous exposure was derived based on growth effects data (Ta-
ble 2). The simplest way to determine effects from cyclic exposure to low DO is to com-
pare growth of organisms under cyclic conditions to those for the same species under
continuous conditions. Growth data are available from cyclic exposures to low DO for
three species of saltwater animals, D. sayi, P. vulgaris and Paralichthys dentatus (Coiro,
et al, 1999). These data are listed in Appendix H and summarized in Figure 11. Data are
from experiments in which a low DO treatment was paired with a treatment cycling be-
tween the same low DO concentration and one that was above the continuous CCC (usu-
ally saturation). All cyclic treatments had 12 hr of low DO within any one 24 hr period.
Most of the cycles consisted of 6 hr at the low concentration followed by 6 hr at the high
concentration. Only two  tests (both with P.  vulgaris) were conducted using a 12hr:12hr
cycle. There were a total  of 20 paired treatments spread among the three species.
     .  As expected, at the end of each test, cyclic exposures generally resulted in more
growth than constant exposures to the minimum DO of the cycle (Figure 11). However, if
the effects of DO on growth were instantaneous (i.e., growth reduction begins as soon as
the DO concentration drops and growth rate returns to normal as soon as DO returns to
above CCC concentrations), then the cyclic exposures in the above experiments would
have been expected to cause one half of the growth reduction observed in the constant
treatment of each pair. (As noted above, the DO cycles had a total of 12 hr of low DO per
day.) If this were true, then the  slope of the line in Figure 11 would be 0.5. However, the
slope of the line for the data (forced through the origin) is 0.778, a factor of 1.56 greater.
                                        27

-------
                     Percentage Reduction in Growth Relative to Control
                                                       y = 0.778x
                                                          = 0.815
                 0
                                                                       y = 0.5x
10    20     30    40     50    60     70    80     90

               Constant Exposure
         Figure 11. Plot of test results from growth experiments pairing constant low dis-
         solved oxygen exposure with exposures to various cycles of low dissolved oxygen
         and concentrations above the CCC. The dark line is a linear regression of the data
         with the line forced through the origin. The lighter weight line is the "expected" rela-
         tionship from a slope of 0.5 (see text for explanation). Species used and the experi-
         mental conditions are listed in Appendix H.

Thus greater growth impairment occurs from  cyclic exposures than expected. One hy-
pothesis for this discrepancy is that recovery from the low DO portion of the cycle is not
instantaneous, and the actual low DO effect period is then greater than 12 hr within each
day (by a factor of 1.56)11.
        Figure 12 shows a dose-response for growth of larval Say mud crab (D. sayi) over
a range of constant  DO concentrations12. The data are from ten tests (see Appendices C
and H) with durations ranging from 4 to 11  days. The  percentage growth reduction is
relative to a control response.  Growth reduction  effects are considered instantaneous,
therefore the % reduction can be applied to any time period. Data for this  mud crab are
emphasized, because it was the only sensitive species tested in cyclic exposures. In addi-
11 The data used to establish the relationship between cyclic and constant exposures (Figure 11) came from
experiments with a total low DO exposure of 12 hr per 24 hr period. We assume that as the total time of
exposure per 24 hr decreases the discrepancy between expected and observed should also decrease. Thus
the 12-hr data can be considered a worst case for any daily cycle of 12 hr or less exposure to low DO.
There is insufficient information for cycles with greater than  12 hr exposure periods per day. We recom-
mend assuming constant exposure conditions for these latter situations.
12 The relative sensitivity of Say mud crab growth to low DO versus other species tested is shown in Ap-
pendix I.
                                            28

-------


c:
g
"o
T3
&
.c
1
0
SS



100 .. .
90 .
80 .

70 -

60 -
50.
40 -
30 -
20 .
10 -
0
 Constant Exposure
* y = -19.4x + 116.2
**
  t
jf 9 
"*'., 
 v
 
^
* ***.
v--.


                   0.0   0.5  1.0
1.5  2.0  2.5  3.0  3.5
 Dissolvsd Oxygen (mg/L)
4.0  4.5  5.0
         Figure 12. Plot of dose-response data for growth reduction in Say mud crab (Dys-
         panopeus sayi) exposed to various continuous low dissolved oxygen concentrations.
         Percentage growth reduction is relative to a control. The dashed line is a linear re-
         gression through the data points.

tion, this species is used to represent larval crustaceans in the recruitment model for con-
stant exposures.
       To evaluate a cycle for chronic growth effects, the above relationship between cy-
clic and constant exposure is needed as well as monitoring data from a representative,  or
worst case, cycle of low DO for a given site.  Figure 13 provides a hypothetical DO time
series. To estimate the expected growth reduction during this cycle the curve is divided
into three DO intervals13 for that  portion of the cycle that falls  below 4.8  mg/L (the
CCC). The DO mean, and the total duration that the cycle is within the interval's range  of
DO, are determined for each interval. Data from this example are presented in Table  4.
Interval  'c' lasts a total of five hours. Interval 'b' lasts a total of three hours  (bl before
plus b2 after interval 'c'). Similarly, interval 'a' lasts for a total of four and a half hours.
Each of these time intervals is multiplied by 1.56 to adjust for the cyclic effect.
       A DO mean concentration for each interval is used with the equation from Figure
12 to estimate a daily growth reduction that is expected for larval crustaceans during con-
stant exposure to hypoxia. This value is then normalized for the interval's cyclic adjusted
duration. The normalized reductions for all intervals are added (growth effects are cu-
mulative) for an  estimated growth reduction for the cycle. This reduction is compared  to
the reduction estimated to occur at the  CCC for constant  exposures (23%, using the
13Any number of intervals can be chosen, even one. For simplicity, different DO ranges can be selected for
each interval so that each interval has approximately the same total time below the CCC. Alternatively, the
cycle can be divided by selecting a constant DO range (e.g., 0.5 mg/L), giving each interval a different time
value. Monitoring data, however, must be frequent enough to justify the chosen interval size.
                                          29

-------
equation from Figure 12 at 4.8 mg/L DO). The percentage reduction in our example is
34%. This reduction is greater than the maximum allowed by the CCC, thus our hypo-
thetical cyclic hypoxic event does not meet the protective goal for growth.



"oi
^,
c
D)
o
o

0
to
CO
Q




6.5
6.0 .
5.5.
5.0.
4.5.

4.0 .


3.5.

3.0.

2.5.
2.0



.


















>
a1













t

__

b1










4

^
*.[""

b2
c








^
,

a2






.**.

m ccc











                  12:00 15:00 18:00  21:00  0:00   3:00   6:00  9:00  12:00 15:00  18:00
                                        Time (hr)


           Figure 13. A hypothetical representative dissolved oxygen time series for one cycle.
           The horizontal line represents the CCC of 4.8 mg/L. The portion of the curve below
           4.8 mg/L is divided into three arbitrary intervals (a,b,c) to estimate effects on growtii.
           The range of dissolved oxygen, the mean dissolved oxygen and the duration for each
           interval are listed in Table 4.
     Table 4.  Dissolved oxygen and duration data from a hypothetical cyclic time series (Figure 13).
     These data are used to estimate the growth reduction occurring for the recruitment modeled species
     during the cycle. Percentage reductions in growth for constant exposure are calculated with the
     equation in Figure 12. These in turn are normalized for the cyclic adjusted duration.
Interval
al-a2
bl-b2
c
D.O. Range
(mg/L)
4.8-4.0
4.0-3.5
3.2-3.5
D.O. Mean
(mg/L)
4.40
3.75
3.35
% Daily
Reduction
in Growth
31
43
51
Actual
Duration (hr)
4.5
3
5
Cyclic Ad-
justed Dura-
tion (hr)
7.0
4.7
7.8
% Reduction
for Duration
9
8
17
                                                 Total % Reduction for Cycle
34
                                             30

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 Cyclic Larval Recruitment Effects
       In order to evaluate cyclic" exposures for their potential impact on larval recruit-
 ment to the juvenile life stage two pieces of information are needed. First, a set of larval
 crustacean time-to-death curves to estimate the expected daily mortality for a given low
 DO cyclic exposure. Second, a way to translate that predicted daily larval mortality into
 allowable  days for the given low DO cycle using the constant exposure recruitment
 model output. Creation of the larval TTD curves is straightforward using the sensitivity
 information (dose response curve) for the Say mud crab late larval to megalopa transition
 period in Figure 5A14 and the sensitivity dependent relationships for TTD slopes and in-
 tercepts in Figure 9. Creation of a series of larval TTD curves followed the same proce-
 dure used to create the time-to-CMC curve for juveniles (Figure 10). Figure 14 shows the
 results for nine calculated curves for mortalities ranging from 5 to 95%.
       Estimating the daily mortality expected to occur with the model species also is
 straightforward, and as with cyclic growth protection, requires representative or worst
 case DO monitoring data. Figure 15 is a hypothetical monitoring data set for a single cy-
 cle. As with growth, the portion of the cycle below the CCC is first divided into several
 intervals. The DO minimum is determined for each interval. It should not matter how the
 intervals are selected. All that is needed is a set of paired time and DO values. Table 5
 lists the data for the intervals in this example. These data were plotted among the family
 of larval TTD curves (Figure 16). In the example, the greatest effect datum lies  closest to
 the 10% mortality curve. Therefore, the hypothetical cycle of DO is  expected to cause
 10% daily mortality to  the modeled larval crustacean. We are only concerned with the
 greatest effect datum because survival effects are not cumulative (i.e., an individual can
 only die once).
       Now all that is needed is to translate the expected 10% mortality into the number
 of allowable days for this hypothetical cycle to occur. This is accomplished using the fit-
 ted curves  in Figures 5A and 6. Figure 5A is the dose response  curve for the Say mud
 crab late larval transition to megalopa period used in the recruitment  model. The infor-
 mation in the figure is for percentage survival, but it can be converted easily into percent-
 age mortality. Thus the information shows the  expected cohort mortality to occur for a
 given DO concentration. For the example, 10% mortality occurs at a DO concentration of
 4.4 mg/L. From the equation used to fit the data in Figure 6, the  4.4 mg/L is allowed to
 occur for up to  26 days without significant impairment to seasonal recruitment.  Thus, the
 cycle that resulted in an estimated 10% daily mortality to larval crustaceans can be re-
peated for up to 26 consecutive days without exceeding a 5% reduction in seasonal larval
recruitment to the megalopa life stage. All of the above can be simplified by merging the
information from Figures 5A and 6 into one cyclic translator figure using the DO axis
that is common between Figures 5A and 6. This  is shown in Figure 17.
 4T"he late larval to megalopa dose-response curve was selected because it is the most sensitive curve used
in the recruitment model.
                                        31

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               5.0 1


               4.5.


               4.0 .


               3.5.


               3.0.


               2.5.


               2.0 .


               1.5.


               1.0 .


               0.5.


               0.0
                                                            5

                                                            10
                                                            15
                                                            25
                      2   4
                                   i   10  12   14   16  18  20  22

                                    Time to Death (hr)
                                                                    24
Figure 14. Time-to-death (TTD) curves generated for the recruitment model species.
Data to generate the curves were taken from Figures 5A, 9A and 9B. The numbers ad-
jacent to each TTD curve are the percentage mortality that each curve represents. The
dashed lines represent curves created with slopes and intercepts outside the range of the
original data used in Figure 9.
       I
       I
       s
       in
       5
6.5,


6.0 .

5.5 .




4.5 .


4.0.

3.5 .


3.0 .

2.5.
          2.0 .
            12:00
                                                                ccc
                  15:00  18:00  21:00   0:00   3:00   6:00   9:00   12:00  15:00  18:00
Figure 15. The same hypothetical dissolved oxygen time series as  Figure 13. This time
the portion of the curve below 4.8 mg/L is divided into several arbitrary intervals to es-
timate effects on mortality. The dissolved oxygen minimum and  its duration for each
interval are listed in Table 5.
                                   32

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Table 5. Dissolved oxygen and duration data from the intervals selected from the hypothetical
cyclic time series in Figure 15. These data are plotted in Figure  16 to  estimate the expected
mortality occurring for recruitment modeled species during the cycle.
Interval
a
b
c
d
e
D.O. Minimum for Inter-
val (mg/L)
4.2
3.9
3.6
3.4
3.2
Duration of Interval
Off)
12
10
8
6
4
                1
5.0


4.5


4.0


3.5


3.0 .
                f
                1  2.0
                1
                U)
                5  1.5


                   1.0


                   0.5


                   0.0
                                                                         5

                                                                         10
                                                                         15
                                                                         25
                      0    24   6   8   10   12  14  16  18  20   22

                                        Time to Death (hr)
                                                                       24
     Figure 16. The dissolved oxygen minima and the durations listed in Table 5 superim-
     posed on Figure 14 (solid circles). The expected mortality from the cyclic exposure is
     determined by the data point falling closest to a TTD curve of greatest effect, in this
     case 10% mortality.
                                           33

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                                   Cyclic Translator
          70

          65

          60

          55
     c
     >    50
     O
                                       I   !      I
                                     UNACCEPTABLE
                5  10 15 20 25  30 35 40 45 50 55 60 65 70  75 80 85 90 95
                              Daily Cohort Mortality (%)
Figure 17. A plot that combines the information from Figures 5A and 6 into a single
cyclic translator to convert expected daily mortality from cyclic exposures into allow-
able number of days of those cycles.
                                  34

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Other Laboratory Bioassay Data
       Additional available data on lethal and sublethal effects of hypoxia on saltwater
animals (Appendix J) do not indicate significantly greater sensitivity than indicated pre-
viously. The other data are divided into effects  on juveniles and adults, and effects on
larvae. Figure 18 shows all of the juvenile mortality data from Appendix J plotted against
the criteria for juvenile and adult survival (limits for both persistent and cyclic exposures
are included). Most of the  other survival data are well below the criteria. There are three
notable exceptions. The first is a single datum (LC50 of 1.9 mg/L) for the Atlantic men-
haden Brevoortia tyrannus at 6 hr (Voyer and Hennekey, 1972). However, several other
LC50 values (Burton et al., 1980) for Atlantic menhaden with durations ranging from 2 to
72 hr were much less (0.70 to 0.96 mg/L). The second is a single datum for the Atlantic
silverside M. menidia at 6  hr (also Voyer and Hennekey, 1972).  There are no other data
for juvenile Atlantic silversides, but the unusually high sensitivities reported by Voyer
and Hennekey for the other species suggest that their exposure system might be a con-1
founding factor. In addition, the authors provided no information on control response for
either the Atlantic menhaden or the Atlantic silversides.
          "S>
          d
          d
          o
          o
          s
          .
          
             2.5
             2.0 .
1.5 .
1.0 .
             0.5 .
             0.0
                              M. menidia

                          B. tyrannus   ^ 0
                  E. affinis
                                  o  o
                                           o w o
               0.1
                                           10            100
                                     Time to Hfect (hr)
                                                                 O O  O
                                                         1000
        Figure 18. A plot of the other juvenile/adult mortality data from Appendix J (open
        symbols) along with the proposed dissolved oxygen criteria for juvenile/adult survival
        (solid line).
                                         35

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       The third set of data above the criteria is a series of values at 0.5 hr for the cope-
pod Eurytemora affinis. Some are below the criteria, but many are above it (Vargo and
Sastry, 1978). However, the authors did not give any details on their experimental meth-
ods, including the number of replicates, the number of animals in each replicate, or on the
response in the control. Thus, it is difficult to adequately access the significance of these
results. However, in the absence of data to the contrary, it is worth noting the DO limit
for juveniles and adults may not be protective of copepods. Alternatively, one could con-
sider that short-lived species with high reproductive outputs (such as copepods) may be
more appropriately protected in a manner similar to larval recruitment.  In this case all of
the E.  affinis LC50 values would fall below the criterion provided by the larval recruit-
ment (see explanation for Figure 19A below).
       Figures 19A and  19B  present all of the lethality data  from Appendix J for tests
using larval life stages. All of these data are from tests for effects on individuals, and the
criterion for larval survival  acknowledges that some larval mortality is acceptable. Most
of the data for larvae are LC50 values for exposure durations other than 24 or 96 hr (these
two durations are used elsewhere in the document). The LC50 data are plotted in  Figure
19A. The most appropriate protective limit to compare these values with is the time-to-
death (TTD) curve for 50% mortality for the Say mud crab (from Figure 14), because the
larval survival protective limit is based on data for this species. There are two series of
data points for LC50 values for larval rock crab Cancer irroratus for exposure durations
of two and four hours; each has some values above the 50% TTD curve (Vargo and Sas-
try, 1977). The more sensitive values in these sets are for tests run at 25C, thus the ani-
mals were likely exposed to multiple stressors (temperature and low DO).
       The rest of the other lethality data for larvae are plotted in Figure 19B. These data
are separated into three categories, LC5 toLC35, LC40 to LC65, and LC90 to LC100. As
with the  LC50 values in Figure  19B, these values are plotted along with time-to-death
curves (10, 50 and 90% mortality) for late larval Say mud crab (From Figure 14). All of
the LC5 to LC35 values are at or below the 10% TTD curve. All of the LC40 to LC65
values are well below the 50% TTD curve. Finally,  all but one of the LC90 to LCI00
values are below the 90% TTD curve. This one value is for 100% mortality of stripped
bass larvae, M. saxatilis that occurred after a 2  hr exposure to 1.90 mg/L DO. However,
there are two other stripped bass tests were 100% mortality of the larvae did not occur
until 24 hr of exposure to similar low DO.
       The are fewer  other data on sublethal effects than for lethality effects (Appendix
J). The sublethal effects included reduced feeding, growth, locomotion, and bivalve set-
tlement, as well as delays in hatching and molting. However,  none of these values indi-
cate that the CCC would not be protective against these effects.
                                       36

-------
    6.0 ,
    5.0.
    4.0 .
 en
 E
 O
 Q,
 6

 J
 13
 I
3.0 .
2.0 .
    1.0 .
    0.0
      0.01
                 0.10            1.00
                         Time to Effect (days)
                                                   10.00
                                                                 100.00
    6.0
    5.0.
~  4.0.
_j
^>
E

El.  3.0 .
o
O
c
I
2.0.
    1.0
    0.0.
           B
          C. bosguianus
           5% mortality
         M. saxitalis
        100% mortality
                      F. heteroclitus
                       10% embryo
                         mortality
      0.010
                                                      10%TTD
                                                       50% TTD
                                                  90%TTD
                    0.100          1.000          10.000

                               Time to Effect (days)
                                                                100.000
     Figure 19. A plot of the other larval survival data from Appendix J.
     Figure  19A presents the available LC50 data (open circles) along
     with the 50% time-to-death (TTD) curve for the Say mud crab. Fig-
     ure 19B presents mortality data for other than 50%. Open circles
     represent 5 to 35% mortality, open squares 40 to 65% mortality, and
     closed circles 90 to 100% mortality. Figure 19B also  includes the
     10, 50 and 90% TTD curves for the Say mud crab.
                                        37

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 Laboratory Observed Behavioral Effects of Hypoxia
       A number of laboratory studies report behavioral alterations following exposure
 to hypoxia. The effects include low DO avoidance, changes in locomotion, burrowing
 and feeding activity, and altered predator-prey behaviors. Most of the effects observed
 occurred <2.3 mg/L, hence would be protected by even the 24 hr acute limit CMC. The
 most hypoxia-sensitive behavioral effect occurs in red hake (Urophycis  chuss). In red
 hake, age 0+ fish leave their preferred bottom habitat and begin to swim continuously as
 DO concentrations fall below 4.2 mg/L (Bejda et al., 1987). Food search time is also re-
 duced as a consequence. Below 1.0 mg/L, most locomotor and other behavioral activity
 ceases, and at 0.4 mg/L there is loss of equilibrium. Older red hake, (age 1+ and 2-3+),
 did not exhibit these responses with low DO, except for loss of equilibrium at 0.6 mg/L.
       The following effects are reported at less than the 2.3 mg/L protective limit. In the
 red morph of green crabs (Carcinus maenas) the low DO avoidance EC25 was <2.3 mg/L
 and the EC50 was 1.8 (Reid and Aldrich, 1989). The green morph was less sensitive. In
 naked goby (Gobiosoma bosc) larvae, avoidance at 2.0 mg/L occurred  with > one hr ex-
 posure (Breitburg, 1994). No avoidance was observed at 3.0 mg/L. This same author re-
 ported 100% avoidance in larval bay anchovy (Anchoa mitchilli) at 0.75 mg/L following
 a one hr exposure. Reduced locomotor activity occurred in daggerblade grass shrimp (P.
pugid) at 1.8 mg/L (Hutcheson et al., 1985). Burrowing in the northern quahog (M. mer-
 cenarid) was reduced 1.4 to 2 fold when exposed to 1.8 to 0.8 mg/L and slowed 4 fold in
 Atlantic surfclam (Spisula solidissimd) at 1.4 mg/L (Savage, 1976). The polychaete,
 Nereis virens, EC25 for emergence from the sediment was 0.9 mg/L  (Vismann, 1990).
 The shelter guarding and nest guarding behavior by adult male naked goby (G. bosc) was
 not altered at 0.7 mg/L, but they abandoned shelters at 0.38 mg/L and nests at 0.3 mg/L.
 Death occurred in these animals at 0.26-0.24 mg/L (Breitburg,  1992).
       The following low DO effects on feeding are reported in a bivalve and four poly-
 chaetes. In eastern  oyster (Crassostrea  virginicd) early post-settlement stage (436 um
 mean  shell height), exposure to 1.9 mg/L for 6 hr resulted in 54 to  61% reduction in
 feeding rate; at <0.4 mg/L for the same period, 86 to 99 % reduction occurred (Baker and
 Mann, 1994b). In older post-settlement animals (651 um mean shell height), feeding rate
 was not altered with 1.9 mg/L exposure for 6 hr, but at < 0.4 mg/L it was reduced 97 to
 99%. In the polychaetes, feeding stopped in Nereis diversicolor at 1.2 mg/L and in N. vi-
 rens at 0.9 mg/L (Vismann, 1990). In adult Loimia medusa, feeding stopped at 1.0 mg/L
 during <20 hr exposure, then resumed in 42 to 113 hr in 42% of the animals (Llanso and
 Diaz, 1994). At 0.5  mg/L, there was no resumption of feeding after initially ceasing  dur-
 ing the same initial  exposure period. Following exposure in Streblospio benedicti adults,
 the initial response to 1.0 mg/L was cessation of feeding, but it resumed in 3.5 days; with
 0.5 mg/L exposure,  the initial response was the same,  with feeding resuming in 4.5 days
 (Llanso, 1991).
       Changes were observed in predator-prey activities in two fishes in low DO In na-
 ked goby (G. bosc)  larvae, avoidance of the sea nettle (Chyrsaora quinquecirrhd) preda-
 tor was reduced 60% following 3 hr exposure to 2.0 mg/L. In striped bass (M. saxatilis)
juveniles, predation on naked goby larvae was reduced 50% following  1-hr 35 min expo-
 sure to 2.0 mg/L (Breitburg et al., 1994).
                                       38

-------
Observed Field Effects
       Field reports of the biological consequences of hypoxia could be used to derive
DO criteria if they include information to describe the exposure conditions. Yet sufficient
data are rarely available. In most cases, DO conditions prior to observed effects are un-
known, making it difficult to predict an exposure threshold for the observed effect.  A
field report of hypoxic effects must, at a minimum, provide a description of the concur-
rent DO exposure conditions if it is to be useful  in deriving criteria. Ten studies in the
Virginian Province have provided concurrent DO measurements.  The DO observations
often are only point measurements, not continuous records, and they rarely provide in-
formation on DO  conditions prior to the observed effects. The biological effects reported
include alterations in the following: presence of fish and crustaceans, diel vertical migra-
tion of copepods,  recruitment and population density of an oyster reef fish (naked goby),
recruitment and growth of eastern oyster spat, and macrobenthic community parameters.
Effects were usually not observed above 2 mg/L.  Exceptions are the Long Island Sound
trawl studies, where effects were reported in the 2.0 to 3.7 mg/L range.
       The relationship between low DO and presence of fish and shellfish in Long Is-
land Sound was examined in two trawl studies.  Howell and Simpson (1994) reported
marked declines in abundance and diversity in 15 of 18 study species when  DO was be-
low 2  mg/L. When DO was between 2 and 3 mg/L,  there  were significantly reduced
abundances of three species: winter flounder, windowpane flounder and butterfish. In a
subsequent three year study, the aggregate data for 23 species of demersal finfish showed
a decline for two  community indices, total biomass and species richness, with declining
DO (Simpson et al., 1995). The DO concentration that corresponded with a 5% decline
below a response asymptote was 3.7 mg/L for total biomass and 3.5 mg/L for species
richness. Dissolved oxygen declines below these concentrations resulted in further exclu-
sion of these animals, which has implications for the secondary productivity  of these wa-
ters.  Reduced  species number implies reduction of community resilience, should this
condition persist. The consequences of habitat crowding on animals occurring in adjacent
waters is unknown.
       Hypoxia-induced changes in the distribution of fish and crustaceans have also
been reported in the lower York River, located in the  Virginian portion of Chesapeake
Bay (Pihl et al., 1991). Subpycnocline DO <2 mg/L developed during neap  tide periods
and the study species (spot, croaker, hogchoker, blue crab, and mantis shrimp) migrated
to shallower and better oxygenated habitats. The degree and order of vertical movement
was believed to be a function of the water column DO concentration and species sensi-
tivity to hypoxia, i.e. croaker > spot = blue crab > hogchoker   mantis shrimp. Water
column destratification and reaeration occurred with spring tide or strong winds and all
species except the burrowing mantis shrimp returned to the deeper strata,  indicating a
preference for the deeper habitats.
       Diel vertical migration of copepods Acartia tonsa and Oifhona colcarva is dis-
rupted by hypoxia (Roman et al., 1993). In mid-Chesapeake Bay during the summer,
these copepods typically occur near the bottom during the day and  migrate to the surface
waters at night. However, when DO concentrations fell below 1 mg/L in subpycnocline
waters, the copepods were displaced to the pycnocline, where the highest numbers were
                                       39

-------
 found both day and night. When mixing occurred during the summer, the bottom waters
 were reaerated, and the copepods once again were found at depth during the day. Vertical
 migration is believed adaptive in that it places the copepods in the chlorophyll maximum
 at night to maximize food intake, yet it provides day-time avoidance of the surface wa-
 ters, protecting the copepods from visual feeding bay anchovy.
       The consequences of hypoxia on recruitment were examined for two species at a
 mid-Chesapeake Bay site: the naked goby G. bosc, a benthic oyster reef fish, (Breitburg,
 1992), and Eastern oyster C. virginica (Osman and Abbe, 1994). In the naked goby study,
 low DO episodes were short-lived, but extreme (<0.5 mg/L), the result of movement of
 deep, oxygen-depleted bottom water into the near shore reef habitat. Following each se-
 vere intrusion, the naked goby population density fell dramatically at the deeper stations,
 which experienced the lowest DO (0.4 mg/L). Small, newly recruited, juveniles were ab-
 sent, presumably due to extremely high mortality. There is evidence, based on observed
 densities, that older juveniles and adults  survived these events by temporarily moving to
 inshore portions of the reef where DO was not as low, then return during the weeks fol-
 lowing  the event. Embryonic development was also affected. Males  abandoned egg-
 containing tubes placed at deeper sites, and the majority to all of the embryos were dead.
 In addition, the youngest embryos collected from the shallower, less hypoxia-stressed site
 developed abnormalities following laboratory incubation. The severe intrusions occurred
 during peak periods of recruitment, with the lowest DO occurring on portions of the reef
 where recruitment was expected to be highest. These adverse effects were not observed at
 sites experiencing low DO >0.7 mg/L.
       In the study with the eastern oyster C. virginica (Osman and Abbe, 1994), mor-
 tality was observed in newly-set (2 to 4 days old) animals during periods of prolonged
 intrusions of low DO water (<1 mg/L 40% of the time  in bottom water during the first
 two weeks of two experiments).  Mortality was proportional with depth, which corre-
 sponded to severity of hypoxia. Growth rate of surviving spat decreased after 1, 2, and 4
 weeks following deployment, with a greater effect also occurring at the deeper stations.
 Survival and growth of juvenile oysters were unaffected following simultaneous deploy-
 ment at the same stations, indicating greater tolerance of the older animals. The authors
 concluded hypoxia to be a plausible causative  factor, acting directly or indirectly,  al-
 though other causative factors also are possible.
       Responses of the macrobenthic community to DO < 2 mg/L are reported for  the
 lower Chesapeake Bay and tributaries (Dauer and Ranasinghe,1992; Diaz et al., 1992;
 Llanso,1992; Pihl, et al.,1991, 1992). Two community effects are reduced species num-
 ber and abundance, with these effects increasing spatially and temporally with increasing
 severity and duration of hypoxia. There also is a shift with hypoxia from dominance of
 longer-lived, deeper burrowing  species of a mature community to short-lived, shallow
burrowing opportunistic species. The response of benthic species, and their  subsequent
recoveries following hypoxia, depends on species tolerance, the timing of the hypoxic
 event relative to larval availability and settlement, and life history strategy. Some infau-
nal organisms migrate towards the sediment surface with hypoxia, beginning around 2
mg/L (Diaz et al.,1992). Animals that migrate to the surface are exposed to predation by
hypoxia-tolerant fish and crustaceans (Pihl et al., 1992). Defaunation may  only occur
                                       40

-------
below 1 mg/L. These studies support 2 mg/L as the hypoxic effect threshold for the mac-
robenthos, which is consistent with the global literature (Diaz and Rosenberg, 1995).
       To summarize, demersal finfish community biomass has been observed to dimin-
ish at DO <3.7 mg/L, and species richness to diminish at <3.5. These effects become in-
creasingly pronounced with further DO decline. Below 2.0 mg/L, migration of the infau-
nal species to the sediment surface and movement of epifaunal species to better aerated
water were observed. All effects reported at <1 mg/L DO concern hypoxia-tolerant spe-
cies and life stages (i.e. disruption of die! vertical migration in copepods, reduced growth
and survival of newly settled oysters, and lethality in larval goby) as demonstrated in par-
allel laboratory studies (Breitburg, 1992, Roman et al., 1993) or by other workers (Baker
and Mann, 1992 and 1994a).


Data not used
       Data from a variety of published literature were not used. The literature on effects
of anoxia was not used, as it provides negligible information on threshold requirements of
aerobic animals. Information on anoxic effects may be found in a recent symposium (Ty-
son and Pearson, 1991) and a review (Diaz and Rosenberg, 1995)  of this subject. Results
of hypoxia effects studies were not cited for species which do not commonly occur in
coastal and estuarine waters between southern Cape Cod, MA and Cape Hatteras, NC
during the spring to autumn period which brackets the occurrence of hypoxia. Reports for
occasional visitor species that occur in these waters during  a favorably warm or cold
summer were excluded.
       Data were not cited if the test temperature was outside the temperature range of
Virginian  Province waters during the hypoxic season, e.g. American lobsters tested at 5
C (McLeese, 1956). Data were not used if they are probably not reliable. Examples in-
clude indications that  the test animals may have been stressed,  e.g. American lobster
tested  at 25 C which were not fed during a 8-10 week  acclimation period (McLeese,
1956); excessive control mortality (> 10% for juveniles or adults and > 20% for early life
stages); the DO  exposure concentration was uncertain, whether due to questionable DO
measurements or failure to  directly  measure test chamber DO  conditions (e.g. Reish,
1966); or if test animals were removed and handled during the test to make other meas-
urements,  e.g. for an energetics study (Das and Stickle, 1993). Literature on physiological
responses of animals to hypoxia was reviewed, but was not found useful to determine low
DO effect thresholds. See Herreid (1980) for a discussion of difficulties in using oxygen
consumption results to describe DO requirements of invertebrates. Rombough (1988b)
has developed an approach to identify the DO requirements for fish embryos and larvae,
but this approach has not been  employed with species applicable to Virginian Province
saltwaters.
       Some data are not used for juvenile blue crabs, C. sapidus (Stickle, 1988; Stickle
et al.,  1989). Effect concentrations for this  species from this laboratory are an order of
magnitude higher than values from an earlier study using adult  C. sapidus (Carpenter and
Cargo, 1957). In addition, these effect concentrations for juvenile blue crabs are almost
all higher than values for larvae of all tested species. Another study (DeFur et al., 1990)
showed that adult C. sapidus make respiratory adjustments that allow them to tolerate
                                       41

-------
long-term (25 days at 22 C) exposure to 2.6 to 2.8 mg DO/L. These data for juvenile
blue crabs are considered outliers until further testing shows otherwise.
       Just prior to final completion of this document, a paper appeared (Secor and Gun-
derson, 1998) describing the effects of hypoxia and temperature on juvenile Atlantic
sturgeon, Acipenser oxyrinchus. There was 22% mortality at 19 C and an average within
tank DO concentration of 2.7 mg/L (within tank data provided by author). This sensitivity
is not that different from that of stripped bass. However, a combination of low DO (ca.
3.5 mg/L)  and high temperature (26 C) resulted in 100%  mortality of A. oxyrinchus
within approximately 24 hr. Because the greatest sensitivity was associated with the high
temperature the data were not included in this document. In addition, the salinity during
the experiments only ranged between one and three ppt, therefore it is likely that this data
is more appropriately associated with  freshwater  criteria  which are much higher than
those for saltwater (see Implementation section).


National Criteria

       The national criteria for ambient dissolved oxygen for the protection of saltwater
aquatic life from Cape Cod to Cape Hatteras  are summarized in Table 6 and presented
graphically on Figure 20 (for persistent exposure) and Figure 21 (for episodic and cyclic
exposure). These criteria are briefly described below:

(1) Protection of Juvenile and Adult Survival from Persistent Exposure
This limit is derived following the Guidelines procedures and is analogous the criterion
maximum concentration (CMC), except that a protective DO concentration limit is ex-
pressed as a minimum as opposed to a maximum, as would be the case for a toxicant.
This limit represents the floor below which dissolved oxygen conditions (for periods of >
24 hours) must not occur.  Shorter  durations  of acceptable exposure to  conditions less
than the CMC have been derived from laboratory studies,  as described in  (4) below.
Please refer to Table 1 for a detailed explanation of the derivation of this limit.

(2) Protection of Growth Effects from Persistent Exposure
This limit is derived following the Guidelines procedures and is analogous to the criterion
continuous concentration (CCC) for a toxicant. This  limit represents the ceiling above
which  dissolved oxygen conditions should support both the survival and growth of most
aquatic species from Cape Cod to Cape Hatteras. Please refer to Table 2 for a detailed
explanation of the derivation of this limit. This limit may be replaced with a limit derived
in (3) as described below, when exposure data are adequate to derive an allowable num-
ber of days of persistent exposure.

(3) Protection of Larval Recruitment Effects from Persistent Exposure
This limit is derived from a generic larval recruitment model using data for the Say mud
crab, a sensitive species native to the waters from Cape Cod to Cape Hatteras. It provides
a degree of protection equivalent to the CCC described above in (2). The limit represents
allowable dissolved oxygen conditions below the CCC, provided the exposure duration
does not exceed a corresponding allowable number of days that assure adequate recruit-
ment during the recruitment season. The cumulative effects of all exposure interval dura-
                                       42

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tions at a given DO below the CCC can be accounted for by totaling the fractions of the
actual (or projected) exposure duration (in days) divided by the allowable exposure dura-
tion for each interval of a specific DO concentration. Please refer to Table 3 and Figure 6
of this document for a detailed explanation of the derivation of this limit.

(4) Protection of Juvenile and Adult Survival from Episodic or Cyclic Exposure
This time dependent limit was derived to represent the responses of the most sensitive ju-
veniles  tested in the laboratory.  It provides an equivalent degree of protection as the
CMC, but for shorter exposure durations than a day. It is assumed that adults are no more
sensitive than juveniles. This limit represents the minimum dissolved oxygen conditions
that must be maintained on an hourly basis (e.g., one-hour minimum, two-hour minimum,
etc.). The limit applies to conditions occurring on a single given day; even if this limit is
met, recurring exposure patterns  still must be checked for agreement with the larval re-
cruitment limit described in  (6) below. Please refer to Figure 10 of this document for a
detailed explanation of the derivation of this limit.

(5) Protection of Growth Effects from Episodic or Cyclic Exposure
This limit is derived from the dose-response relationship for DO vs. growth reduction for
the Say mud crab, and comparisons of the effects of cyclic exposure versus  constant ex-
posure on growth for a variety of species. It  provides an equivalent degree of protection
as the CCC, but for shorter exposure durations than a day. The limit represents the DO
conditions that maintains a daily percent growth reduction in Say mud crab not greater
than the level provided at the CCC for whole day exposures (23%). The cumulative ef-
fects of all exposure interval durations at a given DO below the CCC are accounted for
by summing the percent reductions for time intervals at representative D.O. concentra-
tions. An adjustment factor  of 1.56 was derived to estimate time-variable effects from
intermittent exposure tests that indicated residual, or delayed recovery effects from vari-
ous growth-inhibiting conditions. The limit applies to  conditions that may occur as a re-
curring  pattern throughout the year without adverse growth effects at the CCC level of
protection. However, a recurring pattern of exposure may be limited for a certain number
of days  based on the larval recruitment limit (6). Recurring patterns of DO conditions that
do not meet the growth limit may be allowed for a limited number of days  in a recruit-
ment season, provided the larval recruitment limit is met according to (6). Please refer to
Table 4 and Figure 12 of this document for a detailed explanation of the derivation of this
growth  limit. The  larval recruitment limit can be substituted in whole for the growth
limit.

(6) Protection of Larval Recruitment Effects from Episodic or Cyclic Exposure
This limit is derived from the modeled relationships  between daily cohort mortality for
the Say mud crab and the allowable number of days  at a given maximum  daily cohort
mortality that protects against greater than 5% cumulative  impairment of recruitment
over a season. It provides an  equivalent degree of protection as the limits described in (3)
above, but for recurring patterns of low DO as opposed to continuous low DO conditions.
Figure 16 of this document illustrates how to determine the maximum daily cohort mor-
tality from duration intervals of DO minima. Figure 17 of this  document illustrates how
to determine the allowable number of days of cyclic exposure for a given maximum daily
                                       43

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       cohort mortality. This limit provides additional information that should be used in con-
       junction with the limits described in (4) and (5) above. The limit determines the number
       of days that recurring episodic or cyclic conditions may occur, including whether the
       pattern may occur for an unlimited number of days. For example, a cyclic pattern that in-
       cludes a DO minimum of 3.6 mg/L for 8 hours results in  a daily cohort mortality of 10%
       (see Figure 16). Assuming this represents the maximum daily cohort mortality for the cy-
       clic pattern, the allowable number of days for the cyclic  exposure is 26 (see Figure 17).
       Please refer to pages 31-34 of this document for a detailed explanation of the derivation
       of this limit.

       In summary, limits (1) and (4) establish one day and hourly minimum conditions that
       should be maintained for persistent and cyclic exposures, respectively; limits (3) and (6)
       establish conditions that may occur for a limited number of days for persistent and cyclic
       exposures, respectively; and limits (2) and (5) establish long term conditions that should
       be maintained for the remaining number of days for persistent and cyclic exposures, re-
       spectively.


Implementation
       Dissolved oxygen criteria should be implemented differently from those of toxicants, but
not for reasons associated with biological effects or exposure. Uncertainties associated with
aquatic effects of DO, such as behavior, synergistic relationships with temperature, salinity, or
toxics, apply to toxics as well. Dissolved oxygen also does not differ from toxics for reasons as-
sociated with exposure. Dissolved oxygen can vary greatly in the environment, but so can toxics.
Effluents and their receiving waters can vary daily, even hourly, in their toxicity to aquatic life.
Toxicity of saltwater receiving waters also can vary with the tide and the depth of water. It may
be mistakenly perceived that DO  varies more in concentration simply because it can be measured
easily and nearly continuously.
       From the standpoint of environmental management, DO differs from toxic compounds
primary because it is not regulated directly. Hypoxia  is a symptom of a problem; not a direct
problem. Dissolved oxygen is regulated primarily by controlling discharges of nutrients (in the
marine environment, most commonly nitrogen). Dissolved oxygen also differs from most toxic
compounds because hypoxia can have a large natural component. Therefore,  criteria for hypoxia
should not automatically be applied in the same way as limits for toxicants are.
       This document provides the information necessary for environmental planners and regu-
lators in the Virginian Province to address the question of whether DO at a given site is sufficient
to protect coastal or estuarine aquatic life. The document does not address how compensatory
mechanisms such as avoidance can influence the response of local populations to seemingly ad-
verse DO conditions. The document also does not address the issue of spatial extent of a DO
problem. In other words, even if the DO at a site is low enough to significantly affect aquatic
life, the environmental manager will have to judge whether the hypoxia is widespread enough for
concern.
                                           44

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Table 6. Summary of Saltwater Dissolved Oxygen Criteria.
                                Persistent Exposure
                       (24 hour or greater continuous low DO
                                   conditions)
                                              Episodic and Cyclic Exposure
                                          (less that 24 hour duration of low DO
                                                      conditions)
  Juvenile and Adult
       Survival
      (minimum
      allowable
      conditions)
(1) a limit for continuous exposure
DO = 2.3 (mg/L)

(criterion minimum concentration,
CMC)
                                       (4) a limit based on the hourly duration of
                                       exposure.
                                       DO = 0.37*ln(t) +1.095

                                       -where:
                                       DO = allowable concentration (mg/L)
                                       t = exposure duration (hours)
    Growth Effects
      (maximum
 conditions required)
(2) a limit for continuous exposure
DO = 4.8 (mg/L)

(criterion continuous concentration,
CCC)
                                       (5) a limit based on the intensity and hourly
                                       duration of exposure.
                                       Cumulative cyclic adjusted percent daily
                                       reduction hi growth must not exceed 23%.
                                                                   ti*\56*Gredi
                                                                        24
                                                           < 23%
                                                             and
                                                             Gredi = -19.4*DOi + 116.2

                                                             -where:
                                                             Gred; = growth reduction (%)
                                                             DO; = allowable concentration (mg/L)
                                                             t; = exposure interval duration (hours)
                                                             i = exposure interval
  Larval Recruitment
       Effects1
  (specific allowable
     conditions)
(3) a limit based on the number of days
a continuous exposure can occur
Cumulative fraction of allowable days
above a given daily mean DO must not
exceed 1.0
    n  .(actual)
                             Z*.
                            7
                           i  */
       .(allowed)
                      and
                         DO, =
                                      9.57
                                (2.15  + 2.3eai6ti)
                      where:
                      DO; = allowable concentration (mg/L)
                      t; = exposure interval duration (days)
                      i = exposure interval
                                       (6) a limit based on the number of days an
                                       intensity and hourly duration pattern of
                                       exposure can occur.
                                       Maximum daily cohort mortality for any
                                       hourly duration interval of a DO minimum
                                       must not exceed a corresponding allowable
                                       days of occurrence.

                                       where:
                                       Allowable number of days is a function of
                                       maximum daily cohort mortality (%).

                                       Maximum daily cohort mortality (%) is a
                                       function of DO minimum for any exposure
                                       interval (mg/L) and the duration of the
                                       interval (hours).
1 model integrating growth and survival effects to maintain a minimally impaired Say mud crab larval population
                                                  45

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              2 .
                >**> !>>
                                                                   .  _  . Larval Recruitment
                                                                   Juvenile Survival
                        10        20        30        40

                           Exposure Time (days)
Figure 20. Summary of Criteria for Persistent Exposure. The larval recruitment line represents the minimum DO
concentration that may persist for a given exposure interval duration (number of days). The cumulative effect of
multiple intervals during a season must be accounted for as described in (3) above and in the equation provided on
Table 6.
                                                                          Growth
                                                                      _  .Larval Recruitment
                                                                      +   Juvenile Survival
                       4        8       12      16      20       24

                          Exposure Time (hours)
Figure 21. Summary of Criteria for Episodic and Cyclic Exposure. The growth line represents the minimum DO
concentration that may persist for a given exposure interval duration (i.e., the exposure duration/DO concentration
that results in a 23% daily growth reduction). The cumulative effect of multiple intervals during the course of a day
must be accounted for as described in (5) above and in the equation provided on Table 6. The larval recruitment
line represents the hourly exposure duration/DO concentration intervals that may recur for an unlimited number of
days (corresponds to the 8% daily cohort mortality as shown on Figure 17).
                                                   46

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Finally, as with all criteria, this document does not address changes in sensitivity to low
DO that accompany other stresses such as high temperature, extremes of salinity, or toxi-
cants. Chief among these concerns would be high temperature because high temperature
and low DO often appear together. Low DO will be more lethal at water temperatures
approaching the upper thermal limit for species. This effect has been seen for freshwater
species (U.S. EPA, 1986; Secor and Gunderson, 1998), and saltwater species (e.g., C. ir-
roratus and E. affinis). The limits provided here should be sufficient under most condi-
tions where aquatic organisms are not otherwise unduly stressed.
       Many programs that monitor coastal DO with electronic equipment cannot meas-
ure DO to better than 0.5 mg/L due to limitations of instrument accuracy and resolution
(e.g., Strobel, et al., 1995;  Strobel and Heltshe, 1999) or  sampling design (Summers, et
al., 1997). Attempts to refine the limits presented here or  to apply these limits in assess-
ing field DO conditions should take  this  into account. Criteria for DO can be used in a
risk assessment framework. The approach outlined in this document can be easily used to
compare among areas the DO conditions that are adequate to support aquatic life. Envi-
ronmental managers can determine which sites need the most attention, and evaluate the
spatial and temporal extent of hypoxic problems from one year to the next for sites  of
major concern.
       Environmental managers who wish to use the protective approach presented here
will have to decide several questions  about how the limits  will be used, four of which are
described  below.
       1.  Accuracy of monitoring dataThe most important decision  is to determine
          how accurate the monitoring  data arethe better that hypoxia is character-
          ized, the more reliably it  can be  decided whether it meets  the criteria. Data
          from  existing monitoring programs may not always be accurate enough  to
          take full advantage of the approach provided here. For example, a recent as-
          sessment of conventional sampling procedures along the Atlantic and Gulf
          coasts has suggested that hypoxia in estuarine waters is substantially more
          widespread that previously believed (Summers, et al.,  1997). Deciding what
          data can adequately characterize hypoxia is a matter of risk management. Cy-
          clic conditions may require measurements every 30 min  for several days,
          whereas persistent hypoxia may need only several measurement a week. Deci-
          sions  also have to be made about the number and locations of sampling sites
          to properly represent a given area.
       2.  Biological effectsPotential biological effects are most difficult to predict
          when DO lies between the limits for juvenile and adult survival and larval
          growth. Concentrations below the juvenile and  adult limit do not protect; con-
          centrations above the limit for growth probably protect most  aquatic life and
          its uses15. Deciding whether concentrations between the limits are acceptable
          will depend on the duration of hypoxia and on the acceptable impairment  of
          larval recruitment. The acceptable impairment  can be a risk-management de-
15 The larval growth protection limit is based on statistically significant differences that result in chronic
values similar to EC25s for growth of many organisms. EC25 values are listed as a part of Appendix C for
four species of crustaceans and two species offish. The geometric mean of these values (by species) corre-
lates with the geometric mean of the chronic values.
                                        47

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    cision. The 5% impairment level was selected to be consistent with the pro-
    tection provided to juvenile and adult life stages. In addition, a model that in-
    tegrates  gradual change in daily DO conditions may more accurately predict
    recruitment effects than the current simplified model and its application.
3.  Spatial extentAfter environmental managers have  found a hypoxic area,
    they must decide whether it is small enough relative to nearby unaffected .ar-
    eas to allow the coastal region as a whole to meet the criteria.
4.  Freshwater versus saltwaterIt  is not trivial to decide whether the DO in
    certain parts of estuaries should be judged by freshwater criteria or saltwater
    criteria, particularly where the tides vary the salinity between near fresh and a
    few parts per thousand.  This decision  is important because the criteria for
    freshwater can be up to twice as great as the saltwater limits developed here,
    depending on water temperature and the life stage being protected (U.S. EPA,
    1986). A reasonable way to start is by considering an  estuary's biological
    communities. If they are more like freshwater organisms, freshwater criteria
    should be applied. If they are more like saltwater, then saltwater criteria apply.
5.  Threatened or endangered speciesIn cases where a threatened or endan-
    gered species occurs at a site, and sufficient data exists  to suggest that it is
    more sensitive at  concentrations below the  criteria, it is appropriate to con-
    sider development of a site-specific criterion.
                                 48

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                                      55

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 Appendix A. Comparison of 24 hr and 96 hr acute sensitivity to low dissolved oxygen for saltwater animals.
 Each pair is from the same test run.
 bpecies
 Common name
24 hr LC50   96 hr LC50  Reference
 Americamysis bahia
 Americamysis bahia
 Apeltes quadracus
 Brevoortia tyrannus
 Brevoortia tyrannus
 Crangon septemspinosa
 Leiostomus xanthurus
 Morone saxatilis
 Morone saxatilis
 Palaemonetes pugio
 Palaemonetes vulgaris
 Paralichthys dentatus
 Pleuronectes americanus
 Pleuronectes americanus
 Prionotus carolinus
 Tautoga onitis
 Tautoga onitis
                   Juveniles
 mysid shrimp                1.22         1.29
 mysid shrimp                1.20         1.25
 fourspine stickleback          0.92         0.91
 Atlantic menhaden            1.14         1.21
 Atlantic menhaden            0.88         1.04
 sand shrimp                  0.77         0.97
 spot                        0.67         0.70
 striped bass                  1.50         1.53
 striped bass                  1.62         1.63
 daggerblade grass shrimp      O.55         0.72
 marsh grass shrimp            0.84         1.02
 summer flounder              1.10         1.10
 winter flounder               1.44         1.45
 winter flounder               1.28-         1.30
 northern sea robin         .    0.55          0.55
 tautog                       0.82          0.82
 tautog                       0.80          0.82

                    Larvae
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Burton, etal. 1980
                         Poucher and Coiro, 1997
                         Burton, etal. 1980
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
                         Poucher and Coiro, 1997
Cancer irroratus
Cancer irroratus
Cancer irroratus
Cancer irroratus
Cancer irroratus
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Eurypanopeus depressus
Homarus americanus
Homarus americanus
Homarus americanus
Homarus americanus
Homarus americanus
Homarus americanus
Homarus americanus
Libinia dubia
Menidia beryllina
Morone saxatilis
rock crab
rock crab
rock crab
rock crab
rock crab
Say mud crab
Say mud crab
Say mud crab
Say mud crab
Say mud crab
Say mud crab
Say mud crab
flat mud crab
American lobster
American lobster
American lobster
American lobster
American lobster
American lobster
American lobster
longnose spider crab
inland silverside
striped bass
  2.20         3.09     Poucher and Coiro, 1997
  2.14         2.80     Poucher and Coiro, 1997
  <1.72         2.17     Poucher and Coiro, 1997
  <1.75         2.22     Poucher and Coiro, 1997
  1.85         2.20     Poucher and Coiro, 1997
  1.66         2.50     Poucher and Coiro, 1997
  <1.18         1.73     Poucher and Coiro, 1997
  1.61          1.73     Poucher and Coiro, 1997
  1.88         2.13     Poucher and Coiro, 1997
  1 -95         1.97     Poucher and Coiro, 1997
  <1.55         1.57     Poucher and Coiro, 1997
  <1.83         2.40     Poucher and Coiro, 1997
  2.09          2.10     Poucher and Coiro, 1997
  3.31          3.43     Poucher and Coiro, 1997
  2.66          3.21     Poucher and Coiro, 1997
  2.46          2.82     Poucher and Coiro, 1997
  2.27          2.27     Poucher and Coiro, 1997
  2.14          3.08     Poucher and Coiro, 1997
  2.44          2.83     Poucher and Coiro, 1997
  <2.32         3.19     Poucher and Coiro, 1997
  1.83          2.71 '   Poucher and Coiro, 1997
  1-43          1.44     Poucher and Coiro, 1997
  1.96          1.96     Poucher and Coiro, 1997

-------
                                           Appendix A. Continued
Palaemonetes pugio
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
daggerblade grass shrimp       1.24          1.58
marsh grass shrimp            0.84          1.02
marsh grass shrimp            1.50          2.18
marsh grass shrimp           <2.05         2.16
marsh grass shrimp           O.48         0.98
marsh grass shrimp           <1.56        >1.92
marsh grass shrimp           <1.59         2.05
marsh grass shrimp            1.77          1.87
marsh grass shrimp        .    1.70          1.72
marsh grass shrimp            1.66          2.15
marsh grass shrimp            1.95          2.10
marsh grass shrimp           <1.79        <1.79
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Poucher and Coiro, 1997
Appendix A: page A-2

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-------
 Appendix E. Explanation of the larval recruitment model and how it is used.


       The  model is available as a Microsoft Excel file (APPEND-E.XLS).  The input
 parameter fields and the model output are on the sheet labeled "I-O". A sample  of the I-O
 sheet is shown in Figure E-l. The input parameters are divided into three categories: biology,
 bioassay, and exposure. The model is run by inputting the necessary biological and bioassay
 parameters, selecting the dissolved oxygen (D.O.) concentration (interval) to model, and then
 iteratively assessing various exposure days until the "% Recruitment Impairment" is at or
 near 5% (Figure E-l). Alternately, one can assess the expected impairment for a given site by
 inputting the D.O. interval that represents the  minimum for  than site, and inputting the
 number of days that the  site is experiencing D.O.  concentrations  less than the  CCC (4.8
 mg/L). The  second sheet in the Excel file is labeled "Model" and contains the calculation
 fields for recruitment -without hypoxia and with hypoxia (sample outputs are shown in Tables
 E-l and E-2, respectively).

 Model Input Parameters

       The recruitment model is a discrete time,  density-independent model. The input
 parameters for the model include: the length of the recruitment season (R), the duration of
 larval development (L), the cohort initial abundance (N0), percentage of the initial cohort
 exposed to low dissolved oxygen (p), the attrition rate without hypoxia (a), the duration of
 the hypoxic event in days (E), and the minimum D.O. experienced during that event (DOmin).
 Three exposure response models describe the following: late larval to megalopa survival vs.
 D.O. concentration, larval survival vs. D.O. concentration, and delayed development vs. D.O.
 concentration.  These exposure  response models are used for estimating recruitment under
 hypoxic conditions. The general model presented for assessment of ecological risk, while
 developed for  use with hypoxia, is applicable to any type of time-variable environmental
 stress. In addition, life history parameters of the model can be redefined to reflect site-
 specific qualities or to describe another species of concern.
      The cumulative impact of low D.O. on recruitment is expressed as a proportion of the
potential annual recruitment for the species of concern (Say mud crab, Dyspanopeus sayi, in
the current run of the model). The recruitment season (R) takes into account information hi
the literature from various Virginian Province locations. Consideration was given to capture
the period of predominant recruitment, rather than observance of the first and last dates for
zoeal presence in the water column. Peak larval abundance between June and September is
typical of brachyurana crustaceans in the Virginian Province (Hillman, 1964; Sandifer, 1973;
Dittel and Epifanio, 1982; Johnson, 1985; Jones and Epifanio, 1995). Settlement of D. sayi in
the megalopal  stage is relatively continuous, and unrelated to lunar periods (van Montfrans,
et al., 1990). The larval season, or period of presence in the water column, chosen for  the
running of the model for D. sayi is 66 days. This  value is derived from a representative
hatching  season of 45 days and a larval development time of 21  days. The development time
                                                                  Appendix E: page E-l

-------
 of 21 days was estimated from field  data (Hillman, 1964), as  well  as  from laboratory
 observations made during EPA's D.O. testing with D. sayi.

       Only one data set (Hillman, 1964) was available to represent natural attrition. It was
 generated  from a full  season of weekly collections  in Narragansett Bay, Rhode Island.
 Mortality per day was estimated by applying the assumption that the observed densities of
 each zoeal life stage represented the relative survivorship  of each stage, and that the total
 number of zoeal development days was 21. The rate of attrition, 7.8%  loss per day, is the
 exponential loss constant based on the best fit to these data.

       The model assumes that only 75% of the available mud crabs are exposed to low D.O.
 on any given day (i.e., the other 25% remain above the pycnocline). This assumption is based
 on observations  of water column position  of  these  larvae and  the  recognition of the
 importance of observed vertical migration for estuarine retention of these larvae (Hillman,
 1964; Sandifer 1973,1975). The choice to apply the 75% lower water column distribution to
 all stages  is a conservative  assumption,  which  particularly emphasizes risk  hi the more
 sensitive later stages. A general assumption regarding vertical (and horizontal) distribution is
 that zoea do not successfully avoid hypoxia.

       For each individual run of the model, the exposure input parameters are limited to one
 exposure duration and one D.O. concentration. The conservative approach to deriving the
 exposure parameters  used by the model is to treat each D.O. time series  as a number of
 intervals of D.O. less than the CCC (see  main text for a more detailed explanation). This
 approach defines the duration as  the total number of sequential days of hypoxia for each
 interval. The D.O. value for the model is the minimum D.O. concentration that occurs during
 that duration of time.

       Bioassay input parameters are presented and discussed in the main body of the D.O.
 document,  however,  specific values  used in the model are presented in Table E-3. The final
 protective  limit for  larval survival  that is presented  in the D.O.  document was derived
 assuming the there was no delayed development (i.e., a value of 1.0 was  used for each D.O.
 exposure interval).

 Model Assumptions

       The creation of any  model necessitates  the use of simplifying assumptions that
introduce some limitations to the application of the  model. A complete understanding of the
utility of the model output for a given set of circumstances requires an understanding of these
underlying assumptions. The model divides the recruitment period into 24 hr time periods.
The model assumes that a new cohort of larvae (those released within a 24 hr period) are
available each day of the recruitment period. This is a reasonable assumption for larval mud
crabs. However, if the model is adopted for use with species for which daily cohorts are not
available, then the model may be overprotective1.  Under these conditions  the  model may
'The model applies a daily effect on each cohort that is included in the sum of effects for the total number of
days of exposure. If on a given day there is no new cohort, then there is no effect registered for that day (i.e., if


Appendix E: page E-2

-------
need to be modified for a different time period (e.g., weekly or lunar cycle).  The model
assumes that there is no change in sensitivity for an individual zoeal larva (the "early life
stage" in the model) exposed to low D.O. for multiple days. In other words, the  same 24 hr
dose response relationship is used for each day of exposure without any consideration as to
whether or not an available individual was exposed on the previous day. The  model also
assumes that once a zoeal larva has made the development transition to megalopa, then there
is no further low D.O. effect  (the model only applies the late larval to megalopa dose-
response curve for one 24 hr tune period).

       The recruitment model has two assumptions with respect to duration of exposure to
hypoxia. First, the model assumes that exposure to low D.O. will not occur over the entire
recruitment season (R).  The maximum number of days that low D.O. can exist in the model
is R-(L+1), or 44 days in the current run. Any exposure longer than 44 days gives the same
output as 44 days.  This is not a serious limitation  for the current D.O. protective limit for
larval survival because the protective limit essentially reaches an asymptote at around 30
days (Figure 7, main text). Second, the model was developed with the maximum number of
exposure days equal to the length of the development period  of the modeled species (21 days
in the current run). This only affects the zoeal life stage portion of the model. If exposure
exceeds 21 days, then the model behaves as if there were delayed development and the output
is not as accurate and is slightly over protective. The inaccuracy associated with exposures
longer than 21 days does not show up under conditions that allow for such long exposures to
low D.O. (i.e., which keep the percentage impairment at or below 5%). This is demonstrated
in Figure E-2.

       An  implicit assumption that the model makes  is that the various over- and  under-
protection  issues  more or less cancel each other out. The  assumptions were  necessary  to
construct a reasonably simple and tractable model.

Model Equations

Recruitment under non-hypoxic conditions

       In  the non-hypoxic example, the number  of recruits from  each  daily cohort  is
expressed by the following equation:
                                        Eq.l
NR represents the number of surviving recruits from the initial cohort, all other parameters are
as described in the above section on Model Input Parameters.  The total number of recruits
for non-hypoxic conditions is then determined by summing NR for all daily cohorts.
there are no recruits, then there is nothing to have an effect on for that day). The model cannot apply a "zero':
effect to a cohort that does not exist.
                                                                   Appendix E: page E-3

-------
 Recruitment under hypoxic conditions

        In the hypoxia example, the above equation is modified to account for D.O. effects on
 megalopae, larvae, duration of larval period, and percentage of larvae  exposed.   These
 modifications are performed using several intermediate calculations, but the overall equation
 is:

                                        Eq.2
The variable Sy represents the number of unexposed individuals from a cohort that survive to
recruitment during the hypoxic event. Note that this equation is Eq. 1 (the recruitment model
for non-exposed cohorts) multiplied by the proportion of the population that  does not
experience hypoxic conditions.
                                        Eq. 3

                           Su = (1 - p 1 100) * N0 (1 - a 1 100)L

The variable SH represents the number individuals from a cohort exposed to low D.O. that
survive to recruitment during the hypoxic event.


                                        Eq.4

                                             N0]*(l-a/10Q-)L'}
SDQ represents the total number of individuals from a daily cohort that survive the hypoxic
event. The equation describing SDO will follow below. L' represents the duration of larval
development modified by the developmental delay due  to D.O.  exposure.  The equation
describing L' will follow below. All other variables have been described previously.

                                        Eq.5
The variable ER surv represents the proportion of the cohort surviving at the selected D.O.
concentration using the laboratory exposure-response data (Table E2). Two possible exposure
response models can be used in the calculation of the survival of low D.O. conditions and
their selection is based on whether the individuals  of a particular cohort experience  the
hypoxic  condition during the larval stage or the megalopa stage (Table El).   All other
variables have been described previously.
                                       Eq. 6
       The variable ER delay represents the molt delay observed at the selected D.O.
concentration using the laboratory exposure-response data (Table E3). All other variables
used in this equation have been described previously.
Appendix E: page E-4

-------
       As described for the non-hypoxic condition, the total number of recruits for hypoxic
conditions is determined by summing NR's for all daily cohorts. The percent recruitment
impairment due to hypoxic conditions is calculated as follows:
                                        Eq. 7

                          O/T         i   1  JVn(hypoxic) 4.1nr.
                          %lmpairment =		 * 100
                                          JY^J(non - hypoxic)
                                                                    Appendix E: page E-5

-------
Table E-l. Calculation field from the recruitment model showing
summed to estimate the seasonal recruitment -without hypoxia. Only
(Table E-3).
Cohort
(W
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Days Exposed
(E)
0
0
1
2
3
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Molt During E?
N
N
Y
Y
Y
Y
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N.
N
the results for
the biological
each cohort that are then
input parameters are used
No. Surviving D.O. Realized L No. Surviving 
Exposure (days) Attrition
100
100
100
 100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17.
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
"18.17
18.17
18.17
Appendix E: page E-6

-------
Table E-2. Calculation field from the recruitment model showing the results for each cohort that are then
summed to estimate the seasonal recruitment -with hypoxia. The input parameters for this run are shown in
Table E-3.
Cohort
W
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Days Exposed
(E)
0
0
1
2
3
4
5
5
5
5
5
5
.5
5
5
5
5
5
5
5
5
5
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Molt During E?
N
N
Y
Y,
Y
Y
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
No. Surviving D.O.
Exposure
100
100
50.425
50.425
50.425
50.425
50.425
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7
99.7-
99.7
99.7
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Realized L
(days)
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
No. Surviving
Attrition
18.17
18.17
9.16
9.16
9.16
9.16 '
9.16
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12
18.12 
18.12
18.12
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
18.17
Appendix E: page E-7

-------
 Table E-3. Bioassay input parameters used in  the current run of the larval recruitment model. The final
 protective limits for larval survival assumed no delayed development (i.e., Delayed Development set to 1.0 for
 each dissolved oxygen interval).
    D.O. Cone.
      Interval
D.O. Minimum
    (mg/L)
SurvivalLarvae1
Survival
Megalopae1
Delayed
Development
(fraction of
control)1
         1
         2
         3
         4
         5
         6
         7
         8
     2.0
     2.1
     2.2
     2.3
     2.4
     2.6
     2.8
     3.0
   Data Set I

      0.639
      0.745
      0.825
      0.885
      0.926
      0.971
      0.989
      0.996

  Data Set H
      0.060
      0.073
      0.089
      0.107
      0.128
      0.183
      0.253
      0.339
       1.24
       1.23
       1.22
       1.21
       1.20
       1.19
       1.17
       1.16
1
2
3
4
5
6
7
8
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
0.998
0.999
1.000
1.000
1.000
1.000
1.000
1.000
0.438
0.541
0.641
0.731
0.804
0.862
0.904
0.935
1.14
1.13
1.11
1.09
1.08
1.06
1.05
1.03
'From regression equations for Figure 5.
Appendix E: page E-8

-------






























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-------
                  80
                  70
                  60.
              C?  50.
               I  40 J
               
-------
Appendix F. Justification for treating transition to megalopa as a more
sensitive early life period than zoea life stages. Data are summarized for
      both the Say mud crab Dyspanopeus sayi and the rock crab
                         Cancer irroratus.
                                                     Appendix F: page F-l

-------
                              Say mud crab (Dyspanopeus say/)
           10Ch
            80-
            60-
        CO
            40-I
            20-
                            zoea
                                              zoea 3-megalopa
0
0.0
1.0       2.0       3.0       4.0
          Dissolved Oxygen (mg/L)
                                                                      5.0
       Figure F-l.  Comparison of effects of low dissolved oxygen on zoea and megalopa of the Say mud
       crab Dyspanopeus sayi. Zoea data are from five tests with a four day duration. Megalopa data are from
       2 tests with durations of 8 and 10 days. The test began with stage 3 zoea and the longer exposure times
       were required for test animals to molt to megalopae. Observations made during the tests suggested that
       most of the mortality occurred during the transition to megalopa. For the zoea data, the point is the
       mean, the box the standard deviation and the line the range. Data are from this study.
Appendix F: page F-2

-------
                   Atlantic rock crab (Cancer irroratus)
   100
    80-
    60H
 ca
    40-
    20-
                        zoea
zoea 5-megalopa
      0
      O.O       1.0       2.0        3.0       4.0        5.0       6.
                             Dissolved Oxygen (mg/L)
Figure F-2. Comparison of effects of low dissolved oxygen on zoea and megalopa of the rock crab
Cancer irroratus. Data are -from four zoea tests each with a four day duration, one zoea 5 to megalopa
test of four day duration, and one megalopa test of seven day duration. The longer durations were
necessary to allow sufficient time for individuals to molt to megalopae. Observations made during the
tests suggested that most of the mortality occurred during the transition to megalopa For the zoea
tests, the point is the mean and the line is the range. Data are from this study.
                                                                  Appendix F: page F-3

-------
This page intentionally left blank.

-------
Appendix G. Time-to-death curves used to generate the regressions in
                       Figures 9A and 9B.
                                                       Appendix G: page G-l

-------
                                       Dyspanopeus sayi
                2.5.
                2.0.
a
f  1.5.
            I
            o
            to
            CO
            s
                1.0.
                0.5.
                0.0
                                         LT10: y = 0.511 Ln(x) + 0.892
                                                  r2=0.98
                                                     LT25: y = 0.493Ln(x) + 0.672
                                                              r2=0.99
                                    750: y= 0.291 Ln(x)+0.568
                    LT90: y=0.378Ln(x)+0.329
                             r2=0.79
                                              10

                                           Time (hr)
                                                15
20
        Figure G-l. Time-to-death curves for LT10, LT25, LT50 and LT90 for larvae of the Say mud crab
        Dyspanopeus sayi exposed to  low dissolved  oxygen.  Data are from  this study.  Solid lines are
        logarithmic regressions of the four data sets. Regressions were calculated using Microsoft Excel 5.0.
Appendix G: page G-2

-------
    -J
    "S
     o
     D)


    I

    13


     >
     O
     CD
     CO
        2.5
        2.0
        1.5.
1.0.
        0.5.
        0.0
                               Palaemonetes vulgaris
                                    LT10: y = 0.449Ln(x) + 0.856

                                             r2=0.94
                                                      LT25: y = 0.299Ln(x)+0.881

                                                      r2=0.87
                       LT50: y = 0.287Ln(x) + 0.819

LT90: y = 0.268Ln(x) + 0.750        r2=0.89

         r2=0.97
                                     10
                                         15
                                   20
                                                                           25
                                        Time (hr)
Figure G-2. Time-to-death curves for LT10, LT25, LT50 and LT90 for larvae of the marsh grass

shrimp Palaemonetes vulgaris exposed to low dissolved oxygen. Data are this study. Solid lines are

logarithmic regressions of the four data sets. Regressions were calculated using Microsoft Excel 5.0.
                                                                        Appendix G: page G-3

-------
                                    Homarus americanus
    4.0


    3.5


~  3.0.


1  2.5.
g
O!


O
|  U
o
w
S  1.0.


    0.5.


    0.0
                                     LT10: y = 0.487Ln(x) +1.827
                                              r2=0.71
                                                             LT50: y = 0.363Ln(x) + 1.413
                                                                              r2=0.91
                                       LT90: y = 0.255Ln(x) +1.340
                                               r2=0.83
                                          10          15

                                            Time (hr)
                                                     20
25
        Figure G-3. Time-to-death curves for LT10, LT25, LT50 and LT90 for larvae of the American lobster
        Homarus americanus exposed to low  dissolved oxygen. Data are from this study. Solid lines are
        logarithmic regressions of the four data sets. Regressions were calculated using Microsoft Excel 5.0.
Appendix G: page G-4

-------
    1.6

    1.4

J  1.2.
0)
f  1.0J
0)
I  0.8 J
O     1
|  0.6.
"5
i8  0,
5
    0.2.

    0.0
    O)

    e
    CD
    O>
           0
    0.9.

    0.8.

    0.7.

    0.6.

    0.5

    0.4.

   0.3.

1   0.2.]
Q
    0.1 J

    0.0
              B
                             BreVoortia tyrannus

                       LT5: y=0.156Ln(x)+0.804
                       R2=0.91
                                        LT50:  y=0.084Ln(x)+0.618
                                                R2 = 0.96
                                            LT95: y=l).043Ln(x)+0.469
                                                    R2 = 0.90
                     20
                                 40
                                            60
                                                    80
                                                                  100
                                   Time (hr)
                           Leiostomus xanthurus
                                 LT5: y=0.058Ln(x)+0.542
                                         R2 = 0.96
                          LC95: y = 0.043Ln(x) + 0.421
                                  R2 = 0.95
                      20         40         60          80         100
                                   Time (hr)
Figure G-4. Time-to-death curves for LT5, LT50 and LT90 for juveniles of the saltwater fish Atlantic
menhaden Brevoortia tyrannus (A) and spot Leiostomus xanthurus (B) exposed to  low dissolved
oxygen. Data are from Burton et al., 1980. Solid lines are logarithmic regressions of the four data sets.
Regressions were calculated using Microsoft Excel 5.0.
                                                                        Appendix G: page G-5

-------
                              Salvelinus fontinalis -small fingerlings
            
            B>

            I
            D
            >
            O
            CO
            CO
            5
1.6-

1.4.

1.2.

1.0 .

0.8.

0.6.

0.4.

0.2.

0.0
                               y = 0.204Ln(x) + 0.97
                                    R2 = 0.97
                                     y= 0.21 OLn(x)+0.844
                                             = 0.91

                                             1   "~A
                                        y= 0.150Ln(x)+0.884
                                             R2 = 0.97
                                                               x
                   y = 0.111Ln(x)+0.907
                         R2 = 0.91
y = 0.083Ln(x) + 0.880
      R2 = 0.85
                                        10
                                   15

                               Time (hr)
                                    20
25
30
                                      Salvelinus fontinalis - fry

                                y = 0.235Ln(x) +1.177
                                            R2 = 0.95
                                          y=0.207Ln(x)-H.013
                                                R2 = 0.87
        Figure G-5. Time-to-death curves for LT50s of small fingerlings (A) and fry (B) of the freshwater
        brook trout Salvelinus fontinalis acclimated to different concentrations of low dissolved oxygen and
        then exposed to different concentrations of low D.O. Data are from  Shepard, 1955. Solid lines are
        logarithmic regressions of the four data sets. Regressions were calculated using Microsoft Excel 5.0.
Appendix G: page G-6

-------
                     Salvelinus fontinalis - large fingerlings
        o>
        
        c
        o
        O)
        I
        "5
 2.0
 1.8
 1.6
 1.4.
 1.2.
 1.0.
 0.8.
 0.6.
 0.4.
 0.2.
 0.0
                            y = 0.345Ln(x) + 0.773
                            R2 = 0.990       O
                                       y = 0.260Ln(x) + 0.709
                                            R2 = 0.945
                                        y = 0.200Ln(x) + 0.645
                                       /    R2 =0.984
                                            y = 0.180Ln(x) + 0.606
                                                 R2 = 0.963
                                 y = 0.165Ln(x) +0.616
                                      1^ = 0.913
                                  10       15        20
                                        Time (hr)
                                                    25
                                                       30
       I
       o
       o
       o
       "5
       
-------
This page intentionally left blank.

-------
Appendix H. Growth data for constant versus cyclic exposure to low dissolved oxygen (Coiro, et al., 1999).
Species
Dyspanopeus sayi
Dyspanopeits sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Dyspanopeus sayi
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Palaemonetes vulgaris
Paralichthys dentatus
Paralichthys dentatus
Paralichthys dentatus
Paralichthys dentatus
Paralichthys dentatus
Paralichthys dentatus
Lifestage
larva!
larval
larval
larval
larval
larval
larval
larval
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
newly hatched
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
juvenile
Cycle (mg/L)
4.5-sat.
3.6-sat.
2.6-sat.
1.5-sat
4.2
3.4
2.4
1.6
1.9-sat.
1.6-sat.
1.9
1.6
2.2-sat.
1.7-sat.
2.3
1.9
3.0-sat.
2.2-sat.
3.2
2.3
2.8-sat
2.6
3.2-sat.
2.1-sat.
1.8-sat.
3.4
2.3
1.8
3.7-sat.
2.5-sat.
1.5-sat.
3.5
2.5
1-5
1.8-4.4
1.8
2.2-7.2
1.8-7.2
2.3
1.8
Cycle Duration Test Duration
(hr) (days)
6 low/6 hi
6 low/6 hi
6 low/6 hi
6 low/6 hi
constant
constant
constant
constant
6 low/6 hi
6 low/6 hi
constant
constant
6 low/6 hi
6 Iow/6 hi
constant
constant
12 low/12 hi
12 low/12 hi
constant
constant
6 low/6 hi
constant
12 low/12 hi
12 low/12 hi
12 low/12 hi
constant
constant
constant
6 low/6 hi
6 low/6 hi
6 low/6 hi
constant
constant
constant
6 low/6 hi
constant
6 low/6 hi
6 low/6 hi
constant
constant
7
7
7
7
7
7
7
7
4
4'
4
4
8
8
8
8
8
8
8
8
7
7
8
8
8
8
8
8
14
14
14
14
14
14
10
10
14
14
14
14
D.O.
Minimum
(mg/L)
4.5
3.6
2.6
1.5
42
3.4
2.4
1.6
1.9
1.6
1.9
1.6
2.3
1.7
2.3
1.9
3
2.2
3.2
2.3
2.8
2.6
3.3
2.2
1.8
3.4
2.3
1.8
3.7
2.5
1.5
3.5
2.5
1.5
1.8
1.8
2.2
1.8
2.3
1.8
% Reduction
in Growth
6
30
49
89
33
51
53
90
36
59
67
78
36
56
46
66
25
41
28
60
35
51
15
5l'
69
21
56
75
0
13
55
3
13
64
35
45
18
31
33
47
                                                                         Appendix H: page H-l

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Appendix I. Comparison of Say mud crab growth effects with other saltwater
                    species. Data are from this study.
                                                        Appendix I: page 1-1

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                                      Dissolved Oxygen (mg/L.)
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     Figure 1-1. Plot of growth  (percentage impairment realtive  to  control) for several species of
     saltwater animals. The  Say mud crab (Dyspanopeus sayibold solid  line)  is among  the  most
     sensitive tested. Experimental conditions are listed in Table 1-1.
Appendix I: page 1-2

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