Combined effects of dissolved oxygen and salinity on growth and body composition of juvenile green-lipped mussel Perna viridis.
Subject: Book publishing (Growth)
Aquaculture industry (Growth)
Authors: Wang, Youji
Hu, Menghong
Wong, W.H.
Cheung, S.G.
Shin, P.K.S.
Pub Date: 12/01/2011
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2011 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2011 Source Volume: 30 Source Issue: 3
Topic: Computer Subject: Company growth
Product: Product Code: 2731000 Book Publishing NAICS Code: 51113 Book Publishers SIC Code: 2731 Book publishing; 0273 Animal aquaculture
Organization: Company Name: Cambridge University Press
Geographic: Geographic Scope: United Kingdom; China Geographic Code: 9CHIN China
Accession Number: 278595628
Full Text: ABSTRACT The green-lipped mussel Perna viridis is distributed widely in estuarine and coastal areas of the Indo-Pacific region and is regarded as a cultured mussel or by-product in aquaculture. However, in estuarine and coastal waters where salinity varies with freshwater input and rainfall during the wet season, hypoxia frequently occurs, especially when waters are highly eutrophic. The current study aimed to evaluate the effects of two key environmental factors in estuarine and coastal waters-- dissolved oxygen (DO) and salinity--on growth and body composition of juvenile green-lipped mussels P. viridis. Parameters studied included shell length (SL), tissue dry weight (TDW), condition index (CI), specific growth rate (shell length, [SGR.sub.L]; tissue dry weight, [SGR.sub.w]), moisture content (MC), crude protein (CP), crude fat (CF), crude carbohydrate (CC), crude ash (CA), and energy content (EC). Mussels were cultured for 6 wk at 4 salinities (15, 20, 25, and 30) and 3 dissolved oxygen concentrations (1.5 [+ or -] 0.3, 3.0 [+ or -] 0.3, and 6.0 [+ or -] 0.3 mg [O.sub.2]/L) in a 4 x 3 factorial design. All growth parameters (SL, TDW, CI, [SGR.sub.L], and [SGR.sub.w]) decreased under reduced DO and salinities, but interactive effects between these 2 factors were statistically indistinguishable except for [SGR.sub.w]. Higher percentages of CF and CP, and lower percentages of CC were obtained at reduced salinities and DO. When changes in biochemical content (weight per individual) were compared, both CP and CC content decreased significantly as salinity or DO decreased, whereas no pattern was observed for CF. EC (calories per gram) was not significantly different among DO treatments, but varied significantly with salinity. Total energy content (calories per individual), however, increased significantly with both DO and salinity, but the interaction between salinity and DO was statistically indistinguishable.

KEY WORDS: Perna viridis, green-lipped mussel, growth, body composition, hypoxia, salinity


Hypoxia has been recognized as a major problem on a global scale in many estuarine and coastal regions during the past several decades (for reviews see Diaz (2001), Rabalais et al. (2007), and Kidwell et al. (2009)). Even though hypoxic and anoxic environments have existed throughout geological time, anthropogenic nutrient input to coastal waters has been increasing during recent years, leading to a greater frequency, severity, and duration of hypoxia in various water masses worldwide (Diaz & Rosenberg 1995, Chen et al. 2007, Diaz & Rosenberg 2008). Affected areas include shallow seas that provide essential nursery habitats for many marine molluscs with ecological and economic importance. Hypoxia can have acute effects on fish populations such as fish kills; however, coastal molluscs are more commonly exposed to sublethal levels of hypoxia, with growth being disturbed in the range of 1-4 mg [O.sub.2]/L (Kidwell et al. 2009). Although hypoxia is known to affect individual growth of many organisms (Gray et al. 2002, Wu 2002), relatively few studies have investigated the effect of hypoxia on growth and biochemical composition in bivalves, especially in ecosystems where salinity may be a confounding factor.

Salinity is an important abiotic factor that affects growth and survival of marine organisms, especially in shallow waters where sessile invertebrates such as mussels live (Brown & Hartwick 1988a, Brown & Hartwick 1988b). In estuarine and coastal systems, salinity can change dramatically across spatial and temporal scales (Kirkpatrick & Jones 1985). Marine

invertebrates inhabiting estuarine and coastal areas are exposed to short-term (tidal) and long-term (rainy seasons) changes in salinity. Low salinity undoubtedly has significant effects on physiological processes in marine invertebrates, including osmoregulation, active intracellular transport, feeding rate or nutrient absorption, respiration, and excretion (Kinne 1971, Schmidt-Nielsen 1997). The regulation of intracellular osmotic effectors affects amino acid metabolism and protein composition under osmotic stress (Bayne et al. 1976a, Bayne et al. 1976b). Extracellular osmoregulation, on the other hand, is associated with energy expenditure for active ion transport, involving the degradation of energy-rich compounds (Bayne et al. 1976a, Bayne et al. 1976b). These mechanisms could result in biochemical changes in response to salinity variation. Reduced salinity is known to influence metabolic and physiological parameters in mussels, including heart rate (Bakhmet et al. 2005, Braby & Somero 2006), respiration (Stickle & Sabourin 1979), energy acquisition (Gardner & Thompson 2001), and growth (Westerbom et al. 2002). However, biochemical changes under hyposalinity in bivalves have received little attention in contrast to crustaceans (Anger et al. 1998, Lemos et al. 2001, Torres et al. 2002, Torres et al. 2008).

Environmental stresses rarely occur in isolation. Their effects on marine invertebrates, however, are frequently studied in isolation, regardless of potential interaction among the stresses. Estuaries and coastal areas with intensive aquaculture activities are highly stressful environments, with fluctuating salinity and common occurrence of hypoxia resulting from the supply of inorganic and organic nutrients from aquaculture activities and neighboring rivers.

The green-lipped mussel Perna viridis is widely distributed in the Indo-Pacific region and is extensively cultured in Asia (Rajagopal et al. 1998, Kripa & Mohamed 2008, Laxmilatha et al. 2011) as a cheap protein source because of its fast growth and natural abundance. In Hong Kong, P. viridis is distributed widely from oceanic waters to estuarine waters (Huang et al. 1985), and is a dominant species of epibenthic marine communities in sheltered harbors, where seawater experiences extensive hypoxic (Chan et al. 2008) and salinity changes during the summer (Thiyagarajan & Qian 2003). The current study investigated the combined effects of hypoxia and hyposalinity on the growth and body composition in juvenile P. viridis. The results may help assess the impact of these stresses on the culture of this species and provide information on the selection of potential aquaculture sites.


Experimental Animals

Juvenile mussels (shell length (SL), 26.05 [+ or -] 1.55 mm: dry weight, 70.0 [+ or -] 5.0 mg) were collected from a sheltered bay at Lok Wo Sha, Hong Kong. On return to the laboratory, they were maintained in a fiberglass tank (500 L) equipped with a filtering system and air supply, and fed the brown alga Thalassiosira pseudonana daily (concentration, 5.0 x [10.sup.5] cells/mL). The seawater was maintained at 21[degrees]C and salinity at 30. Individuals of P. viridis were allowed to acclimate to laboratory conditions for 1 wk prior to experimentation.

Experimental Design

To examine the combined effect of hypoxia and salinity, the experimental ranges of dissolved oxygen (DO) and salinity were selected to cover the natural ranges that P. viridis experience in Hong Kong waters. The experiment was set up in a factorial design with 12 treatments, using 3 levels of DO (1.5 [+ or -] 0.3 mg/L as severely hypoxic, 3.0 [+ or -] 0.3 mg/L as moderately hypoxic, and 6.0 [+ or -] 0.3 mg/L as normoxic) and 4 levels of salinity (15, 20, 25, and 30). Each treatment consisted of 3 replicates with 25 mussels per replicate (aquaria, 3 L). The system by Chan et al. (2008) was adopted, and comprised an experimental tank, a digital DO controller (model no. 01972-00; Cole-Parmer, Vernon Hills, IL), a cylinder of compressed N, and an air pump. The DO level in each experimental tank was monitored automatically by the oxygen probe of the controller. When the desired DO level deviated from the preset value, the DO controller would send a signal to the valves connecting to the N gas tank or air pump to restore the desired DO level by delivering either N or air into the experimental tank. Different salinities were obtained by diluting 0.22-[micro]m filtered seawater (30) with double-distilled water.

Growth and Biochemical Composition

At the start of the experiment, 30 mussels were killed and dried for initial SL and body composition analysis. All parameters were evaluated again after the mussels were cultured in the previously noted system for 6 wk. SL was measured with vernier calipers to the nearest 0.01 mm, and weight was measured with an electronic balance to the lowest 0.1 mg. Mussels were dried at 80[degrees]C to determine the tissue dry weight (TDW) and moisture content (MC). Condition index (CI) was calculated according to the formula (Orban et al. 2004) CI = (TDW/SDW) x 1,000, where TDW is tissue dry weight in grams and shell dry weight (SDW) in grams. The specific growth rates (SGRs) of mussels were determined as [SGR.sub.L] - ([ln([L.sub.t]) ln([L.sub.0])]/t) x 100%, and [SGR.sub.w] - ([ln([W.sub.t]) - ln([W.sub.0])]/t) x 100%, where [SGR.sub.L] and [SGR.sub.w] are specific growth rate of SL and TDW, respectively: [L.sub.0] and [L.sub.t] were initial and final SL, respectively; [W.sub.o] and [W.sub.t] were initial and final TDW, respectively; and t was the time elapsed in days (Wang et al. 2009). Then CHNS/O analyzer (PerkinElmer 2400 Series II; PerkinElmer, Waltham, MA, USA) was used to analyze the N content, and crude protein (CP) was calculated from the N content by multiplying by 6.25 (Chen et al. 2009). Crude fat (CF) was determined by ether extraction whereas crude ash (CA) was determined by combusting samples at 550[degrees]C for 6 h (Chen et al. 2009). Crude carbohydrate was estimated by subtracting the sum of percentages of protein, fat, and ash from 100% (Vasconcelos et al. 2009), and energy content (EC) of tissue was determined using an oxygenic bomb calorimeter (model 1261 Isoperibol Calorimeter; Parr Instrument Company, Moline, IL, USA). At the end of the experiment, all mussels in each replicate were killed and pooled for body composition analysis. The body composition was calculated based on TDW.

Statistical Analyses

Growth parameters obtained from all experimental mussels in each replicate aquarium were averaged, and 3 replicates were prepared for each treatment. Prior to the analysis, normality of the data was evaluated by using the Shapiro-Wilk W test, and homogeneity of variance was checked by Levene's test using the statistical software SPSS 16.0 (SPSS, Inc., Chicago, IL). Percentage data were arcsine transformed. The effects of DO, salinity, and their interactions were analyzed using 2-way analysis of variance (ANOVA) with DO and salinity as fixed factors. When a significant difference was detected, Tukey's HSD post hoc multiple comparisons were performed to determine the differences among treatments for each factor (i.e., DO and salinity).


During the experiment, only 5 mussels died (1 at 3.0 mg [O.sub.2]/L x salinity 25, 2 at 1.5 nag [O.sub.2]/L x salinity 20, and 2 at 6.0 mg 02/L x salinity 15). Growth (i.e., SL and TDW) increased after a 6-wk culture in all treatment groups. The results of 2-way ANOVA on growth parameters and biochemical composition are summarized in Table 1.

SL was significantly lower at reduced DO and salinities (P < 0.001; Table 1, Fig. IA). Values obtained at different DO levels were significantly different from each other, with the highest value at 6.0 mg [O.sub.2]/L and lowest at 1.5 mg [O.sub.2]/L. For the salinity effect, highest SL was obtained at 25 and 30, which were not significantly different from each other, but was higher than at 20 and 15. The values at 20 and 15 were similar. No interactive effect between DO and salinity was found (P > 0.05, Table 1).

TDW was significantly lower (P < 0.001) at reduced DO and salinities (Table 1, Fig. 1B). Values obtained at different DO levels were significantly different from each other. TDW at different salinities were also significantly different from each other, with the lowest value at 15 and the highest at 30. No significant interaction between DO and salinity was observed (P > 0.05, Table 1).

CI was significantly lower (P < 0.001) at reduced DO and salinities (Table 1, Fig. 1C), but there was no significant interaction between DO and salinity (P > 0.05, Table 1). Values obtained at salinities of 15 and 20 were similar, but significantly lower than at 25 and 30. Values at 25 were also significantly lower than those at 30. For the effect of DO, highest values were obtained at 6.0 mg [O.sub.2]/L, which were significantly higher than those at 3.0 mg [O.sub.2]/L and 1.5 mg [O.sub.2]/L. Values obtained at 3.0 mg [O.sub.2]/L and 1.5 mg [O.sub.2]/L, however, were not significantly different.

[SGR.sub.L] was significantly lower (P < 0.001) at reduced DO and salinities (Table 1, Fig. 1D), but the interactive effect between DO and salinity was not significant (P > 0.05, Table 1). [SGR.sub.L] at salinities 25 and 30 was similar, but was significantly higher than at 20 and 15. Values at 20 were also significantly higher than at 15. For the DO effect, values obtained at different DO levels were significantly different from each other. [SGR.sub.w] was lower at reduced DO and salinities (Fig. 1E), with a significant interactive effect between DO and salinity (Table 1).

Moisture content was not influenced by DO, salinity, and the interaction between DO and salinity (P > 0.05, Table 1). The tissue biochemical compositions--CP, CF, and CC--varied significantly with DO, salinity, and the interaction between DO and salinity (Table 1). In general, the lower the salinity and DO, the higher the CP and CF (Fig. 2 A, B). CC showed an approximate reverse pattern to the CP and CF (Fig. 2C). The disadvantage of expressing the results as a percentage is that changes in one biochemical component are reflected by reciprocal changes in all the other components (Gabbott 1976). When changes in biochemical content (weight per individual) were compared, both CP and CC content decreased significantly as salinity or DO decreased, whereas no pattern was observed for CF.

CA (Fig. 2D) and EC (Fig. 2E) were not significantly different among DO treatments (P > 0.05), but were significantly different among salinity treatments (P < 0.001). No interactive effect of DO and salinity on these 2 parameters was found (Table 1). For CA, higher values were obtained at higher salinities, with values obtained at different salinities being significantly different from each other. A reverse pattern was observed for EC, except the values obtained at 25 and 30 were statistically nonsignificant. Total energy content, expressed as calories per individual, showed a reverse pattern, with energy content increased significantly with both DO and salinity, but the interaction between salinity and DO was statistically indistinguishable.


Both DO and salinity significantly affected growth performance (measured as SL, TDW, CI, [SGR.sub.L], and [SGR.sub.w]) of P. viridis, with a lower growth at low DO levels and salinities. Because there was no interactive effect of DO and salinity on growth (except for [SGR.sub.w]), this indicated that DO and salinity affected growth independently, with their effects being additive.

The current study has demonstrated that P. viridis has high tolerance to hypoxia. Similar observations were found for other bivalves, and this was a result, in part, of a reduction in metabolic activity and energy utilization (Widdows 1987, Storey & Storey 2004). Oxygen consumption of the green-lipped mussel P. viridis and the brown mussel Perna indica exposed to declining oxygen tension decreased (Hawkins et al. 1987). For example, P. viridis started to reduce oxygen consumption at 80% air saturation, and a more abrupt decrease in oxygen consumption occurred when air saturation was reduced to 20% (Davenport 1983). Oxygen consumption of 3 species of lamellibranch molluscs--the black clam Arctica islandica, the Norway cockle Laevicardium crassum, and the blue mussel Mytilus edulis also declined under low oxygen tension (Bayne 1971a). In the blue mussel M. edulis, physiological responses for reduced oxygen tension included a decline in heart rate, a reduction in the ventilation-to-perfusion ratio, an increase in the extraction efficiency for oxygen (Bayne 1971b, Bayne 1975), a reduction in oxygen uptake (Bayne & Livingstone 1977), and an accumulation of end products of anaerobic metabolism (such as glutamate, alanine, malate, and succinate) in the posterior adductor muscle of the mussels (Livingstone & Bayne 1977). Similar responses were observed in the California mussel Mytilus californianus during aerial exposure (Bayne et al. 1977). General metabolic depressions at low DO eventually lead to growth reduction in marine invertebrates (Diaz & Rosenberg 1995, Gray et al. 2002). This adaptive response may allow P. viridis to dominate sheltered and eutrophic harbors in Hong Kong (Huang et al. 1985), where growth was much reduced compared with that in clean waters (Cheung 1993). Some bivalves are also capable of adapting to short-term anaerobic metabolism under hypoxia (De Zwaan 1977, Carroll & Wells 1995). This results in a metabolic deficit during each period of low tide, which, coupled with the reduced time available for feeding, imposes a physiological stress on mussels distributed on the shore (Bayne et al. 1977). The Eastern oyster Crassostrea virginica is able to lower oxygen consumption, when exposed to declining oxygen tensions and low salinities (Shumway & Koehn 1982). Le Moullac et al. (2007) further demonstrated that the oxygen level is important in the regulation of the energy metabolism of the Pacific oyster Crassostrea gigas, and metabolic depression occurred quickly when oxygen availability was reduced. Recent studies showed that low DO could increase reproductive success and distribution in the freshwater clam Sphaerium sp. (Joyner-Matos et al. 2007, Joyner-Matos et al. 2011).



Although P. viridis could survive at a salinity of 15, this was achieved at the expense of growth. Lee (1986) and Cheung (1991) have reported slower growth of P. viridis in the western waters of Hong Kong (salinity, 19-31) compared with mussels in the east (salinity, 24-34). Reduction in growth at reduced salinities was also reported in other invertebrates (Torres et al. 2002). Unfavorably low salinity may cause a reduction in the average rate of feeding or growth efficiency, presumably as a result of metabolic maladjustments induced by osmotic stress. Such effects should be stronger in osmoconformers like P. viridis (Parulekar et al. 1982, Nicholson 2002) or weak osmoregulators compared with strong osmoregulators. Salinity influence on respiratory metabolism is usually thought to be an energy consumption adjustment caused by an osmotic pressure difference between the environment and body fluid. In the blue mussel M. edulis, physiological compensation for reduced salinity involves increased rates of excretion of ammonia, loss of certain amino acids, and a decline in the concentration of free amino acids in the mantle tissue consistent with previous studies of volume regulation (Bayne 1975). These energy consumptive processes eventually reduce energy for growth. However, an alternative theory suggested that reduced growth in high- or low-salinity conditions could be attributed to reduced food assimilation or consumption. Brown and Hartwick (1988a, 1988b) reported that growth and survival of the Pacific oyster C. gigas was inhibited under conditions of low salinity because of reduction of the assimilated ration and possible limitations on the supply of minerals essential to shell formation. Our previous experiments (Wang et al. 2011) have indicated that feeding efficiency in P. viridis was also reduced when the salinity was outside the range between 20 and 30. Considering that P. viridis is an osmoconformer (Parulekar et al. 1982), poor growth at low salinities, therefore, was also likely a result of a reduction in food assimilation.

DO, salinity, as well as the interaction between DO and salinity had significant effects on the body composition in P. viridis, with lower carbohydrate content being obtained in mussels exposed to hypoxic and/or salinity stress. Glycogen is the major energy storage in mussels because it can yield energy by breakdown to lactic acid under hypoxia or even anoxia (Schmidt-Nielsen 1997), and its accumulation in the tissue is dependent on its relative rates of production and utilization. Under hypoxia, carbohydrate in the form of glycogen is used to produce energy for metabolism. A lower metabolic rate, associated with a lower rate of energy acquisition, hence may impede the rate of glycogen accumulation. Salinity stress has a similar effect on glycogen accumulation. Additional energy is expended on isosmotic intracellular ion regulation under salinity stress for both osmoconformers and osmoregulators (for reviews see Schoffeniels and Gilles (1970) and Pequeux (1995)). Together with a lower food assimilation or consumption as salinity was reduced, the rate of carbohydrate accumulation was lower. An increase in body weight (i.e., growth) was observed for all treatment groups, including those exposed to hypoxic and/or salinity stress, and the time course for utilization of body reserves for energy metabolism in marine mussels is first carbohydrate, then lipid. Protein is the last substrate to be used when other substrates are exhausted (Gabbott 1976). A lower percentage in protein and lipid in the control, therefore, could be a result of faster accumulation of glycogen instead of utilization of protein and lipid for energy metabolism. This can also be supported by the fact that protein content (measured as milligrams per individual) was lower under hypoxia and/or salinity stress, although a reverse pattern was observed when protein content was expressed in a percentage. Energy content was also higher under salinity and/or hypoxia stress in terms of calories per gram, but was lower when it was expressed in calories per individual--a result of tissue growth.

The current study has demonstrated that P. viridis is tolerant to hyposalinity and hypoxic stress with a high survival rate. This may help explain its territorywide distribution as well as its dominance in sheltered eutrophic harbors (Huang et al. 1985, Cheung 1993) in Hong Kong. In some local waters with salinities more than 20 (e.g., Yim Tin Tsai West in Tolo Harbour and Kau Sai Bay in Port Shelter on the east coast ofHong Kong), P. viridis may experience hypoxia around 1.5 mg/L (Wu & Lam 1997; Gao et al. 2008). With such a high tolerance to DO, these areas are possible ideal sites for mussel culture. The current results hence provide useful information in site selection and culturing practice for mass culture of this mussel species that is cultured widely in Asian-Pacific (Wong & Cheung 2001) sheltered harbors using hanging ropes on floating rafts (Gosling 2003). Poor flushing and accumulation of wastes in aquaculture sites may lead to localized eutrophication and hypoxia, especially at high temperatures during the summer (Wu et al. 1999), which is also the major growing season of P. viridis (Wong & Cheung 2001, Wong & Cheung 2003). Improving aeration/water circulation during the summer and locating aquaculture sites away from areas with major freshwater inputs may help in enhancing the production of the species.


The work described in this article was fully supported by a grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (AoE/P-04/04) from the City University of Hong Kong.


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YOUJI WANG, (1) MENGHONG HU, (1) W. H. WONG, (2) S. G. CHEUNG (1,3) AND P. K. S. SHIN (1,3) *

(1) Department of Biology and Chemistry, City University of Hong Kong, Tat Chee A venue, Kowloon, Hong Kong; (2) University of Nevada at Las Vegas, (4505) Maryland Parkway, Box (453064), Las Vegas, N V (89154) ; (3) State Key Laboratory of Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

* Corresponding author. E-mail:

DOI: 10.2983/035.030.0326
Summary of 2-way ANOVA results on effects of dissolved oxygen (DO) and
salinity (S) on shell length (SL), tissue dry weight (TDW), condition
index (CI), specific growth rate (shell length, [SGR.sub.L]; tissue dry
weight, [SGR.sub.W]), moisture content (MC), crude protein (CP), crude
fat (CF), crude carbohydrate (CC), crude ash (CA), and energy content

                          SL                         TDW

Source     df    MS        F         P     MS         F        P

DO          2   14.97   21.07   <0.001   0.002      73.64   <0.001
S           3   30.03   42.27   <0.001   0.00 5    145.79   <0.001
DO x S      6    0.41    0.53     0.74   7.54E-5     2.39     0.06

                         CI                     [SGR.sub.L]

Source     df    MS       F         P      MS        F        P

DO          2   567.44   5.64     0.01   0.088    20.47   <0.001
S           3   965.34   9.60   <0.001   0.184    42.62   <0.001
DO x S      6    76.80   0.76     0.61   0.003     0.59     0.74

                        [SGR.sub.W]              MC

Source     df   MS          F       P     MS      F      P

DO          2   1.10     87.81   <0.001    0   2.77   0.08
S           3   2.20    176.60   <0.001    0   0.95   0.44
DO x S      6   0.044     3.56    0.012    0   1.43   0.25

                            CP                        CF

Source     df     MS         F        P     MS        F        P

DO          2    0.001     4.69     0.02   0.001    15.19   <0.001
S           3    0.008    51.07   <0.001   0.004    40.33   <0.001
DO x S      6    0.001     6.09    0.001   0.001     7.83   <0.001

                          CC                           CA

Source     df    MS         F         P        MS        F        P

DO          2    0.003    10.69   <0.001   3.88E-5     0.44     0.65
S           3    0.001     4.44    0.013      0.01   119.12   <0.001
DO x S      6    0.001     4.33    0.004      0        1.76     0.15


Source     df      MS         F         P

DO          2     8,054.59     1.20     0.32
S           3   471,646.35    70.53   <0.001
DO x S      6     9,623.77     1.44     0.24

Dissolved oxygen, 1.5, 3.0, and 6.0 mg [O.sub.2]/L; salinity, 15, 20,
25, and 30.
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