Lethal levels of dissolved oxygen for Haliotis diversicolor supertexta at different salinity levels.

ABSTRACT The abalone Haliotis diversicolor supertexta juveniles (shell length 3.51 [+ or -] 1.10 cm) were exposed to different concentrations of dissolved oxygen (DO) using the static renewal method at different salinity levels of 25 [per thousand] (g [kg.sup.-1]) and 35 [per thousand] at pH 8.04 and 22[degrees]C. The 72-, 84-, and 96-h L[C.sub.50] values of DO on H. diversicolor supertexta juveniles were 1.56, 1.76, 2.02 mg [L.sup.-1] at 25 [per thousand] and 1.43, 1.65, 1.83 mg [L.sup.-1] at 35 [per thousand], respectively. As the salinity decreased from 35 [per thousand] to 25 [per thousand], susceptibility to DO increased by 107%, 107%, and 110% after 72-, 84-, and 96-h exposure, respectively. The relationship between L[C.sub.50] of DO (mg [L.sup.-1]), salinity (S) and exposure time (T) in the range 72-96 h is as follows:

L[C.sub.50] = 0.541 - 0.012S + 0.018T ([R.sup.2] = 0.637)

KEY WORDS: dissolved oxygen, Haliotis diversicolor supertexta, salinity, tolerance

INTRODUCTION

Taiwan abalone (also known as small abalone) Haliotis diversicolor supertexta is commercially important in Taiwan as well as in the southeast coast of mainland China as a primary cultured species. For growth, Chen (1984) has observed that salinity in the range 30-35 [per thousand], and temperature in the range 24-30[degrees]C are the optimal levels. Culture of H. diversicolor supertexta has expanded greatly since 1986 due to successful artificial propagation and development of multiple-tier basket systems in grow-out farms (Yang and Ting 1986, Chen and Lee 1999).

Since late 2000, farmers have experienced mass mortality of abalone reared in the multiple-tier basket systems in the grow-out ponds and settlement failure of spat larvae in the nursery ponds. The bacteria Vibrio alginolyticus and V. parahaemolyticus isolated from the hemolymph of moribund abalone have been documented to cause vibriosis leading to death of abalone in warmer water temperatures (Liu et al. 2000, Lee et al. 2001). As a result, the farmed production of abalone in Taiwan declined from 2497 tons in 2001 to 1089 tons in 2003.

Yang and Ting (1989) reported that dissolved oxygen (DO) should be maintained higher than 5 mg [L.sup.-1] for the optimal growth of H. diversicolor supertexta. They also reported that the oxygen consumption of this species maintained at 32 [per thousand] and 30[degrees]C was 1.3 times that maintained at 32 [per thousand] and 25[degrees]C. Fallu (1991) reported that abalones require DO greater than about 3-4 mg [L.sup.-1]. It is very common that the pond water in multiple-tier basket systems may become hypoxic or even anoxic due to respiration of animals and decomposition of accumulated organic matter such as unconsumed food and feces. Therefore, concentrations of DO in pond water and its lethal levels on abalone are of primary concern.

Lethal levels of DO have been reported for a number species of penaeid shrimps including kuruma shrimp Marsupenaeus japonicus (Egusa 1961, Tournier 1972), caramote prawn Melicertus kerathurus (Tournier 1972), tiger shrimp Penaeus monodon (Liao & Huang 1975, Allan & Maguire 1991), southern white shrimp Litopenaeus schmitti (MacKay 1974), and northern brown shrimp Farfantepenaeus aztecus (Martinez et al. 1998). Concerning the mollusks, Miller et al. (2002) reported that the 96-h L[C.sub.90] (90% of animal killed after 96 h) of DO on the Atlantic surf-clam Spisula solidissima was 0.3 mg [L.sup.-1]. Tamai (1993) reported that the semelid small bivalve Theora fragilis survived DO as low as 1.3-1.4 mg [L.sup.-1] at least for four days. Temperature has been reported to affect the tolerance of low DO on zebra mussel Dreissena polymorpha and Asian clam Corbicula fluminea (Johnson & McMahon 1998). However, with a few minutes of literature searching, we found no information available on the lethal level of DO on marine invertebrates at different salinity levels. The purpose of the current study is to estimate the lethal level of DO on H. diversicolor supertexta at 25 [per thousand] and 35 [per thousand].

MATERIALS AND METHODS

Seawater and Test Solution

Seawater pumped from the Keelung coast adjacent to the National Taiwan Ocean University was adjusted to 35 [per thousand] (g [kg.sup.-1]) and 25 [per thousand], with municipal water dechlorinated with sodium thiosulfate, and then filtered through a gravel and sand bed by air-lifting and aerating for three days before use. The concentration of DO in water was regulated with nitrogen gas that was bubbled into the water to produce a series of DO that ranged from 6.87 to 7.46, 5.12 to 5.88, 3.19 to 3.80, 2.19 to 2.77, and 1.24 to 1.62 mg [L.sup.-1] with an average of 7.11, 5.53, 3.45, 2.47, and 1.44 mg [L.sup.-1], respectively. These served as test solutions.

Animals

H. diversicolor supertexta were obtained from a commercial abalone farm in Iilan, Taiwan, and acclimated in the laboratory with salinity of 35 [per thousand] for two weeks prior to experimentation. The abalones were divided randomly into two groups in the same salinity and then adjusted 2 [per thousand] each day to reach two different salinity levels of 25 [per thousand] and 35 [per thousand]. After the abalones reached the expected salinity levels, they were reared for one more week. The mean ([+ or -] SD) wet body weight and shell length was 4.12 [+ or -] 0.9 g and 3.51 [+ or -] 1.10 cm, respectively, with no significant difference among treatments.

Lethal Effect of Dissolved Oxygen

Short-term L[C.sub.50] (median lethal concentration) toxicity tests were carried out according to the methods described by the American Public Health Association et al. (1985). Abalones were sampled randomly from the holding tanks and transferred to test solutions. Bioassay experiments to establish tolerance limits were conducted in 20-L glass flasks containing 10-L test solutions. Each tank contained 10 test abalones. Each test solution was renewed daily, in accordance with the static renewal method for toxicity tests (Buikema et al. 1982). There were three replicates for each test solution with a total number of 30 abalones (10 per replicate for each test solution). During the experiment, abalones were fed Gracilaria tenuistipitata daily. Water temperature was maintained at 22 [+ or -] 1 [degrees]C and pH ranged from 7.87 to 8.12 with an average of 8.04.

Observations were usually made at 6-h intervals up to 168 h. Abalone that failed to respond to tactile stimulation when touched with a glass rod were defined as dead. The L[T.sub.50] (median lethal time, hours required to kill half the population) was determined. Dead abalones were removed daily. The concentration response of test organisms was determined for L[C.sub.50] (median lethal concentration) value of dissolved oxygen and their 95% confidence limits with a computer program (Trevors and Lusty 1985) based on a method described by Hubert (1980). The estimated probit line and the results of a chi-square ([chi square]) test for goodness of fit were computed.

Statistical Analysis

The L[C.sub.50]s of dissolved oxygen (DO) were subjected to one-way analysis of variance (ANOVA) followed by Duncan multiple range test (Duncan 1955). Relationship between survival, salinity, DO concentration, and exposure time (hours) and the relationship between L[C.sub.50] of DO, salinity, and exposure time were tested using the General Linear Model procedure and Regression Procedure, version 6.03, Statistical Analysis System (SAS) computer software (SAS 1998). All statistical significance tests were at the P < 0.05 level.

RESULTS

All abalones survived in the test solutions at 5.53 and 7.11 mg [L.sup.-1] DO at all salinity levels after 168 h exposure. No abalone died in 1.44 and 2.47 mg [L.sup.-1] DO after 36 h at 25%. However, all abalones exposed to 1.44 mg [L.sup.-1] DO at 25 [per thousand] died after 108 h. No abalone died in 1.44 and 2.47 mg [L.sup.-1] after 24 h and 48 h, respectively, at 35 [per thousand]. However, all abalones exposed to 1.44 mg [L.sup.-1] DO at 35 [per thousand] died after 108 h (Fig. 1). There was a significant effect of DO, salinity, and exposure time on survival. There was also a significant interaction between the effects of salinity and DO and a significant interaction between the effects of DO concentration and exposure time on survival. There was no significant interaction between the effects of salinity and exposure time on survival. In salinity levels of 25-35 [per thousand], the relationship between survival (%), DO concentration (C), salinity (S), exposure time (T), and the interactions between salinity and DO concentration (SC), DO concentration and exposure time (CT) is as follows:

Survival (%) = 123.125 + 0.340S - 1.439T - 7.340C - 0.054SC + 0.418CT ([R.sup.2] = 0.858)

[FIGURE 1 OMITTED]

For the abalones exposed to 1.44 mg [L.sup.-1] DO at 25 [per thousand] and 35 [per thousand], there was a significant effect of salinity and exposure time on survival. However, there was no significant interaction between the effects of salinity and exposure time on survival. In 1.44 mg [L.sup.-1] DO, the relationship between survival, S, and T is as follows:

Survival (%) = 116.210 + 0.138S - 0.879T ([R.sup.2] = 0.858).

For the abalones exposed to 2.47 mg [L.sup.-1] DO at 25 [per thousand] and 35 [per thousand], there was a significant effect of salinity and exposure time on survival. However, there was no significant interaction between the effects of salinity and exposure time on survival. In 2.47 mg [L.sup.-1] DO, the relationship between survival, S, and T is as follows:

Survival (%) = 97.501 + 0.460S - 0.319T ([R.sup.2] = 0.847).

In 25 [per thousand], the L[T.sub.50] (median lethal time, hours) values of H. diversicolor supertexta juveniles exposed to 2.47 and 1.44 mg [L.sup.-1] DO was 179 and 72 h, respectively, whereas in 35 [per thousand], the L[T.sub.50] of abalone exposed to 2.47 and 1.44 mg [L.sup.-1] DO was 204 and 75 h, respectively. The probit of mortality for H. diversicolor supertexta juveniles exposed to DO was linearly related to log DO at various exposure times (Table 1). A [chi square] test indicated that values of chi were less than the values in those tables, suggesting that the assumed lines were satisfactory (Trevors & Lusty, 1985).

The L[C.sub.50] of DO values (and their 95% confidence limits) at different exposure periods for H. diversicolor supertexta juveniles are shown in Figure 2. At 72, 84, and 96 h, the L[C.sub.50] values of DO were 1.56, 1.76, and 2.02 mg [L.sup.-1] at 25 [per thousand] and 1.43, 1.65, and 1.83 mg [L.sup.-1] at 35 [per thousand]. Resistance of H. diversicolor supertexta juveniles to DO was 9.0%, 6.7%, and 10.3% less at 25 [per thousand] than that at 35 [per thousand] after 72, 84, and 96 h exposure. The L[C.sub.50] of DO was inversely related with salinity level on H. diversicolor supertexta juveniles. The L[C.sub.50] values of DO at 35 [per thousand] were significantly lower than those at 25% on H. diversicolor supertexta juveniles.

[FIGURE 2 OMITTED]

Statistical analysis indicated that there was a significant effect of salinity on L[C.sub.50] and exposure time on L[C.sub.50]. However, there was no significant interaction between the effects of salinity and exposure time on L[C.sub.50]. The relationship between L[C.sub.50] of DO (mg [L.sup.-1]), S, and T is as follows:

L[C.sub.50] = 0.541 - 0.012S + 0.018T ([R.sup.2] = 0.637).

DISCUSSION

The sublethal concentrations of DO for selected marine and estuarine teleosts and invertebrates have been studied (Table 2). Reported 96-h L[C.sub.50] of DO on juvenile teleost ranged from 0.6 mg [L.sup.-1] for northern sea robin Prionotus carolinus to 1.6 mg [L.sup.-1] for summer flounder Paralichthys dentatus and striped bass Morone saxatilis (Miller et al. 2002). Reported 96-h L[C.sub.50] of DO on juvenile decapod crustaceans ranged from 0.7 mg [L.sup.-1] for daggerblade grass shrimp Palaemontes pugio to 2.9-4.4 mg [L.sup.-1] for northern brown shrimp F. aztecus (Stickle et al. 1989, Allan and Maguire 1991, Miller et al. 2002). The 96-h and 168-h L[C.sub.50] of DO on blue crab Callinectes sapidus was 2.5-4.1 mg [L.sup.-1] and 2.3 mg [L.sup.-1], respectively (Stickle et al. 1989, Das and Stickle 1993). Reported 96-h L[C.sub.50] of DO on juvenile mollusc was 0.5 mg [L.sup.-1] for Atlantic surfclam Spisula solidissima at 28-32 [per thousand] (Miller et al. 2002) and 2.02 mg [L.sup.-1] and 1.83 mg [L.sup.-1] for H. diversicolor supertexta at 25 [per thousand] and 35 [per thousand], respectively, in the current study. These facts indicated that the abalone H. diversicolor supertexta was less tolerant to low DO than P. monodon and S. solidissima but was more tolerant to low DO than F. aztecus and C. sapidus. Liao and Huang (1975) reported that the lethal level of DO was 0.2-0.3 mg [L.sup.-1] tar P. monodon. Allan and Maguire (1991) reported that all P. monodon juveniles exposed to 0.3 mg [L.sup.-1] were killed within 12 h. The current study indicated that H. diversicolor supertexta exposed to 1.44 mg [L.sup.-1] DO were killed within 108 h.

In addition to lethality, growth of H. diversicolor supertexta may be affected by low DO. Sediman and Lawrence (1985) reported that the level of DO at 1.2 mg [L.sup.-1] reduces significantly the growth, and the levels of DO higher than 2 mg [L.sup.-1] do not affect the growth of P. monodon and whiteleg shrimp Litopenaeus vannamei. Yang and Ting (1989) reported that DO should be maintained higher than 5 mg [L.sup.-1] for the optimal growth of H. diversicolor supertexta. However, we do not know the level of DO that affects the growth of abalone.

Most of the previous papers documented L[C.sub.50] values of DO for teleosts and invertebrates at one salinity level only. Abalones are generally cultured intensively in outdoor semi-static pond water or an indoor multiple-tier basket system with salinity varying from 25 [per thousand] to 35 [per thousand] (Chen and Lee 1999). Lin and Chen (2001) reported that the toxicity of ammonia to L. vannamei increased by 104% to 112% as salinity decreased from 35 [per thousand] to 25 [per thousand]. Lin and Chen (2003) reported that the toxicity of nitrite to L. vannamei juveniles increased by 161% to 197% as salinity decreased from 35 [per thousand] to 25 [per thousand]. Tsai and Chen (2002) reported that the toxicity of nitrate to P. monodon increased by 128% to 147% as salinity decreased from 35 [per thousand] to 25 [per thousand]. Martinez et al. (1998) reported that the lethality of low DO to L. setiferus increased by 160% as salinity decreased from 38 [per thousand] to 15 [per thousand]. The current study indicated that the lethality of low DO on H. diversicolor supertexta increased by 105% to 111% as salinity decreased from 35 [per thousand], to 25 [per thousand],. Therefore, abalone and shrimp farmers should note the toxicity differences of ammonia, nitrite, nitrate, and low DO at different salinity levels when marking pond management.

In ponds, DO increases during the daytime due to the photosynthesis of phytoplankton and macrophytes and decreases during the nighttime due to the respiration of both plants and animals. It is not surprising that DO depletion may occur in abalone farms, especially in multiple-tier basket systems, due to poor water circulation. Species of the bacterial Vibrio genus are ubiquitous in the marine and estuarine environments. Disease outbreaks have been reported to be associated with increases in the Vibrio population of the cultured water (Sung et al. 1999). Challenging H. diversicolor supertexta with V. parahaemolyticus at 2 x [10.sup.4] cfu [abalone.sup.-1] and placed in 35 [per thousand] water with different concentrations of DO, Cheng et al. (2004) observed that 72-h and 96-h L[C.sub.50] of DO was 4.71 and 5.17 mg [L.sup.-1], respectively. They also reported that the abalone exposed to DO at 3.57 mg [L.sup.-1] or lower showed decreases in hemocyte count, release of superoxide anion, and phagocytic activity and clearance efficiency to V. parahaemolyticus indicating depression in immune system of H. diversicolor supertexta.

The L[C.sub.50] of DO to H. diversicolor supertexta juveniles increased with exposure time. The tolerance of H. diversicolor supertexta juveniles to low DO decreased sharply by 13% and 41% after 84 and 96 h as compared with 72-h L[C.sub.50] at 25 [per thousand], and the tolerance of H. diversicolor supertexta juveniles to low DO decreased sharply by 15% and 28% after 84 and 96 h as compared with 72-h L[C.sub.50] at 35 [per thousand]. Similar phenomenon was obtained previously in penaeid shrimps. Lin and Chen (2003) reported that the tolerance of L. vannamei juveniles to nitrite decreased by 11% and 35 [per thousand] after 48 and 96 h as compared with 24-h L[C.sub.50] at 25 [per thousand], and the tolerance of L. vannamei juveniles to nitrite decreased by 19% and 38% after 48 and 96 h as compared with 24-h L[C.sub.50] at 35 [per thousand].

In conclusion, the 72 and 96 h L[C.sub.50] of DO on H. diversicolor supertexta is 1.56 and 2.02 mg [L.sup.-1] at 25 [per thousand], and 1.43 and 1.83 mg [L.sup.-1] at 35 [per thousand], respectively. It appears that in the range of 25% and 35%, the relationship between L[C.sub.50] of DO, salinity, and exposure time are L[C.sub.50] = 0.541 - 0.012S + 0.018T ([R.sup.2] = 0.637). The study has revealed that low DO concentrations might cause either lethality or depressed immune ability of abalone.

TABLE 1.
Relationship between probit of mortality (Y) and log DO as mg
[L.sup.-1] (X) at various exposure times at 25 [per thousand]
and 35 [per thousand] for H. diversicolor supertexta juveniles.

      Time                                       [X.sup.2]
       (h)               Y = a + bX        n *   ([dagger])

25 [per thousand]
       72           Y = 15.232 - 8.572X     3      0.868
       78           Y = 15.861 - 8.996X     3      0.890
       84           Y = 17.752 - 10.232X    3      0.916
       90           Y = 19.178 - 11.193X    3      0.945
       96           Y = 22.480 - 13.394X    3      0.976

35 [per thousand]
       72           Y = 13.921 - 7.729X     3      0.857
       78           Y = 14.951 - 8.424X     3      0.896
       84           Y = 16.628 - 9.559X     3      0.940
       90           Y = 18.376 - 19.736X    3      0.969
       96           Y = 19.321 - 11.339X    3      0.968

      Time          [chi square]   [chi square]
       (h)           Calculated         df        0.95

25 [per thousand]
       72              13.278           1         3.747
       78              12.840           1         3.747
       84              10.645           1         3.747
       90               7.745           1         3.747
       96               4.461           1         3.747

35 [per thousand]
       72              11.297           1         3.747
       78               9.945           1         3.747
       84               6.297           1         3.747
       90               4.033           1         3.747
       96               4.373           1         3.747

* Number of concentration for calculation.

([dagger]) Coefficient of determination.

TABLE 2.
The L[C.sub.50]s (median lethal concentrations) of dissolved oxygen
(DO) on several species of teleosts, decapod crustaceans, and mollusc
juveniles.

                                         Salinity         L[C.sub.50]
             Species                 ([per thousand])   (mg [L.sup.-1])

Teleosts
  Prionotus caralinus                     28-32          96-h 0.6
  Paralichthys dentatus                   28-32          96-h 1.6
Decapod crustaceans
  Americamysis bahia                      28-32          96-h 1.2
  Homarus americanus                      28-32          96-h 1.0
  Palaemontes vulgaris                    28-32          96-h 1.0
  Palaemontes pugio                       28-32          96-h 0.7
  Crangon septemspinosa                   28-32          96-h 1.0
  Cherax tenuimanus                     Freshwater       24-h 0.7
  Penaeus monodon                         32-35          96-h 0.9
  Farfantepenaeus aztecus                   35           96-h 2.9-4.4
  Litopenaeus setiferus                     15           72-h 1.16
  L. setiferus                              38           72-h 1.86
  Callinectes sapidus                       35           96-h 25-4.1
  C. sapidus                                35          168-h 2.3
Molluscs
  Spisula solidissima                     28-32          96-h 0.5
  Haliotis diversicolor supertexta          25           96-h 2.02
  Haliotis diversicolor supertexta          35           96-h 1.83

             Species                        Reference

Teleosts
  Prionotus caralinus                Miller et al. (2002)
  Paralichthys dentatus              Miller et al. (2002)
Decapod crustaceans
  Americamysis bahia                 Miller et al. (2002)
  Homarus americanus                 Miller et al. (2002)
  Palaemontes vulgaris               Miller et al. (2002)
  Palaemontes pugio                  Miller et al. (2002)
  Crangon septemspinosa              Miller et al. (2002)
  Cherax tenuimanus                  Morrissy et al. (1984)
  Penaeus monodon                    Allan and Maguire (1991)
  Farfantepenaeus aztecus            Stickle et al. (1989)
  Litopenaeus setiferus              Martinez et al. (1998)
  L. setiferus                       Martinez et al. (1998)
  Callinectes sapidus                Stickle et al. (1989)
  C. sapidus                         Das and Stickle (1993)
Molluscs
  Spisula solidissima                Miller et al. (2002)
  Haliotis diversicolor supertexta   Current study
  Haliotis diversicolor supertexta   Current study

ACKNOWLEDGMENTS

This paper was supported by a grant from the Council of Agriculture (Grant No. 91-No-Ke-2.5.3-Yu-F2), Taiwan, Republic of China. We appreciate Mr. Y. C. Lin for his assistance in the experiment.

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SHA-YEN CHENG, (1) YIN-HUNG CHANG (2) AND JIANN-CHU CHEN (2), *

(1) Department of Environmental Biology and Fisheries Science and (2) Department of Aquaculture, College of Life and Resource Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, Republic of China

* Corresponding author. Fax: [phone]; E-mail: jcchen@mail. ntou.edu.tw