Comparative Biochemistry and Physiology Part C 125 (2000) 333 – 343 www.elsevier.com/locate/cbpc Ammonia toxicity as a criterion for the evaluation of larval quality in the prawn Macrobrachium rosenbergii Ronaldo O Cavalli *, Els Vanden Berghe, Patrick Lavens, Nguyen T.T Thuy, Mathieu Wille, Patrick Sorgeloos Laboratory of Aquaculture and Artemia Reference Center, Uni6ersity of Gent, Rozier 44, 9000 Ghent, Belgium Received 17 August 1999; received in revised form 12 November 1999; accepted 18 November 1999 Abstract The feasibility of a short-term ammonia toxicity test as an evaluation criterion for larval quality was assessed in three trials In each one, Macrobrachium rosenbergii larvae originating from the same spawn were nutritionally differentiated in two groups by feeding them either a nutrient-rich (Artemia nauplii enriched for 24 h with n-3 highly unsaturated fatty acids (HUFA) and ascorbic acid (AA)) or a nutrient-poor diet (Artemia nauplii starved for 24 h) Throughout their development, larvae from both treatments were exposed during 24 h to six concentrations of total ammonia (NH+ + NH3) and a control (no ammonia added) Based on mortality rates, the median lethal concentration for 50% of the population (LC50) was estimated As expected from earlier work, larvae fed the optimal diet presented higher n-3 HUFA and AA contents as well as higher growth and metamorphosis rates From the moment the effect of diet quality was analytically detectable in the tissues of the larvae, the ammonia test was able to distinguish both groups of larvae Differences in ammonia tolerance were observed as early as larval stage and remained evident throughout larval development The short-term ammonia toxicity test proved to be a valuable, sensitive and reproducible criterion for the establishment of larval quality © 2000 Elsevier Science Inc All rights reserved Keywords: Ammonia toxicity; Larval quality; Macrobrachium rosenbergii; Prawn; Stress test; Aquaculture; Highly unsaturated fatty acids; Ascorbic acid Introduction World aquaculture production has increased tremendously over the last two decades It is estimated that only from 1987 to 1996 the volume of production rose by 250% (FAO, 1998) To support this expansion, the technology for the mass production of fry was established for several species of economic importance Nowadays, commercial aquaculture operations rely mainly on the * Corresponding author Tel.: + 32-9-264-3754; fax: +329-264-4193 E-mail address: ronaldo.cavalli@rug.ac.be (R.O Cavalli) production of hatchery-reared fry For the most successful species current hatchery practices enable the supply of sufficient numbers of fry and, hence, larval quality is becoming of major concern Unfortunately, up to the present limited information is available on standardised methods for the detection of larval and postlarval quality Probably one of the first developments of a specific test for the evaluation of fry quality took place in Japan when Watanabe et al (1983) measured the survival of red sea bream (Pagrus major) larvae 24 h after exposure to the air for s Since then, several methods have been proposed These usually involve the exposure of the animals 0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc All rights reserved PII: S - ( 9 ) 0 1 - 334 R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 to a short but extreme environmental stress (salinity, temperature, pH, or formalin) at which their physiological condition will determine their ability to survive (Tackaert et al., 1989; Briggs, 1992; Dhert et al., 1992; Fegan, 1992; Ako et al., 1994; Samocha et al., 1998) Other methods use more subjective observations, e.g body pigmentation, colour and activity, morphological (muscle to gut ratio) indicators (Bauman and Jamandre, 1990), biochemical (fatty acid) composition (Arellano, 1990) and even a score comprising several parameters like chromatophore development, body deformity, fouling, and muscle opaqueness (Fegan, 1992) Most often these criteria are considered complementary and therefore used together to evaluate larval quality However, most of them were conceived to evaluate the quality of fry in later developmental stages, i.e at the end of the hatchery cycle and prior to their release in grow-out facilities For the early larval stages, other than the usual estimates of spawn size, egg size and hatching rate, time to hatching and larval survival, very few evaluation methods have been devised For penaeid shrimps, Bray et al (1990) suggested the use of protozoea I length, while Browdy (1992) indicated that larval quality could be evaluated according to the phototactic response of the nauplius stage A similar principle was used to separate ‘healthy’ and ‘weak’ larvae of the freshwater prawn Macrobrachium rosenbergii (Singh and Philip, 1995) Although all these criteria are useful, none of them is considered sufficiently sensitive or standardised to provide a conclusive and reliable assessment of the physiological condition of larvae Ammonia is the principal excretory product in aquatic animals (Kinne, 1976) and its mechanisms of toxicity and lethal concentrations to various fishes and crustaceans of commercial importance are relatively well documented (Tomasso, 1994) In water, ammonia is found primarily as the ammonium ion (NH+ ) and the non-ionised molecule (NH3), which co-exist in an equilibrium reaction governed mainly by pH (Emerson et al., 1975) Higher pH levels increase the concentration of NH3 in relation to NH+ Non-ionised ammonia freely diffuses across cell membranes in the direction favoured by its pressure gradient (Fromm and Gillette, 1968) Therefore, if ammonia levels increase in water, ammonia excretion diminishes, and levels of ammonia in blood and other tissues increase This might result in an elevation of blood pH and adverse effects on membrane stability and enzyme-catalysed reactions (Tomasso, 1994), which may eventually lead to death Since acute static bioassays are a standard practice in aquatic toxicology studies, a similar procedure could also prove valuable to ascertain the physiological condition of larval stages of aquatic animals Accordingly, this study aimed to assess the feasibility of a short-term ammonia toxicity test as a larval quality evaluation criterion The freshwater prawn M rosenbergii was chosen as a model species since it easily reproduces under captive conditions and its larvae can be cultured following standardised hatchery practices Materials and methods 2.1 Origin of animals Prawn larvae were obtained from broodstock imported from Thailand and reared indoors in a standardised maturation system (Cavalli et al., 1999a,b) The broodstock animals were fed to satiation twice a day with a shrimp broodstock feed (INVE Technologies N.V., Baasrode, Belgium) 2.2 Experimental procedures Three independent trials were repeated in time In each trial, newly hatched larvae originating from the same spawn were divided in two groups and reared under standard conditions in an experimental hatchery system In order to create two different quality groups, the larvae were fed either a nutrient-rich or a nutrient-poor diet Dietary supplementation of ascorbic acid (AA) and n-3 highly unsaturated fatty acids (HUFA) are known to increase the survival and stress tolerance of M rosenbergii postlarvae (Devresse et al., 1990; Merchie et al., 1995; Romdhane et al., 1995) Accordingly, the nutrient-rich diet consisted of Artemia franciscana (Great Salt Lake, USA) enriched with a n-3 HUFA rich emulsion (ICES enrichment emulsion 50/0.6/C, containing 50% n-3 HUFA) to which 20% ascorbyl palmitate was added The enrichment of Artemia was performed according to Merchie et al (1995) Freshly hatched nauplii were enriched for 24 h Two doses of 0.3 g emulsion l − were added at 12-h inter- R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 vals The density of nauplii for enrichment was around 200 ml − After 24 h, the enriched metanauplii were harvested and rinsed with tap water over a 120-mm sieve to remove any remaining emulsion The nutrient-poor diet consisted of freshly hatched Artemia nauplii starved for 24 h under the same conditions as the enriched Artemia group (stocking density of 200 nauplii ml − 1, temperature 2891°C, and natural seawater) 2.3 Lar6iculture system and feeding For each trial, the two groups of larvae were reared to postlarvae in separate recirculation units at an initial density of 100 l − Each unit consisted of a 100-l black cylindro-conical PVC tank connected to an 80-l submerged biological filter and a 20-l overhead tank Water was continuously pumped from the filter to the overhead tank at a rate of 2.5 l − and then forced back through the bottom of the rearing tank by gravity An outlet screen (150 mm) at the top of the rearing tank led the water back to the biological filter and at the same time retained the larvae and Artemia within the rearing tank Two shortcuts, at the pump outlet and the rearing tank inlet, enabled the adjustment of the water flow rate The filter screen was cleaned twice a day to prevent overflow A 300-W thermostatic heater placed in the filter maintained the water temperature at 2891°C Water salinity at 1291 g l − was obtained through the mixture of deionised water and natural seawater Deionised water was added to the system to compensate for losses due to evaporation The photoperiod was automatically controlled at 12 h light and 12 h dark Aeration in the rearing tanks and filters assured oxygen levels above mg O2 l − Ammonium, nitrite and ni−1 , 0.03 mg NO2 trate were below 0.2 mg NH+ l −1 −1 l and 50 mg NO3 l , respectively, while pH varied from 7.8 to 8.2 Prawn larvae were fed solely on Artemia metanauplii, which were maintained in the rearing tanks at a density of 10– 15 nauplii ml − Feeding started on the second day after hatching and was carried out twice a day (10:00 and 18:00 h) The enriched and starved groups were prepared daily and stored at 4°C (Merchie, 1996) for the evening feeding Every morning, Artemia remaining from the previous day were filtered out of the larvicul- 335 ture tanks to maintain a constant quality of the prey organisms This was accomplished by increasing the water flow rate (to around 19 l − 1) and the filter screen mesh sizes (from 150 mm for normal functioning to 350 mm, for the first days, and 500 mm from day onwards) At least 30 larvae from both treatments were periodically sampled and conserved in 4% formalin for the determination of total length (from the tip of rostrum to the tip of the telson; TL) and larval staging (Uno and Kwon, 1969) The larval stage index (LSI) was then estimated according to Maddox and Manzi (1976): LSI =SSi /N where Si is the stage of the larvae (i= – 12) and N is number of larvae examined 2.4 Ammonia toxicity test set-up The general procedures for the ammonia toxicity tests were based on the methodology presented by the Standard Methods for the Examination of Water and Wastewater (Greenberg et al., 1992) The tests were performed in duplicate in a series of 1-l glass cones immersed in a water bath at 289 1°C Starting from newly hatched larvae (day 0) up to complete metamorphosis to postlarvae, groups of 30 animals from both treatments were exposed during 24 h to six increasing concentrations of total ammonia (TAN; NH+ + NH3) and a control (no ammonia added) TAN concentrations were based on preliminary tests to identify suitable ranges for each LSI, and varied according to the age of the larvae The ammonia stock solution was prepared with reagent grade NH4Cl and was added to the glass cones immediately before stocking the test animals Measurements of pH were carried out at the beginning (after adding the NH4Cl solution) and at the end of the tests Initial values ranged from 7.8 to 8.3 Ammonium (NH+ ) concentrations were also measured at the beginning and at the end of trial with a selective ammonium electrode (model 6833; Consort, Turnhout, Belgium) As in the larval rearing tanks, salinity and temperature were kept at 12 91 g l − and 289 1°C, respectively The animals were not fed and water was not renewed during the exposure The concentrations of non-ionised ammonia (NH3) were estimated according to the general formula for bases (Albert, 1973) for the mean 336 R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 values of pH, salinity and temperature as presented by Armstrong et al (1978): [NH3] =[TAN]/1 +10[pK − pH] where pK= 9.31 at a temperature of 28°C and salinity of 12 g l − 1; pH is the mean value measured at the beginning and the end of test After 24 h of exposure, larvae presenting no movement of appendages and not responding to mechanical stimuli were considered dead Based on the mortality rates, the mean lethal concentrations for 50% of the population (24-h LC50) were estimated with the Trimmed Spearman Karber Method (Hamilton et al., 1977) 2.5 Salinity stress test Postlarvae from trial and larvae (LSI =8.8– 9.0) from trial were also subjected to salinity stress tests (Tackaert et al., 1989) The tests were run in five replicates, with groups of 10 larvae or postlarvae being transferred to 1-l plastic beakers containing water at 28 91°C and a salinity of 65 or 45 g l − 1, respectively The test medium was a mixture of natural seawater (33 g l − 1) and artificial salts (Instant Ocean, Aquarium System, Sarrebourg, France) The mortality was monitored at 3-min intervals during h The animals presenting no movement of pleopods and giving no reaction to prodding with a pipette were considered dead The sensitivity to the salinity stress was expressed by the cumulative stress index (CSI), which was calculated as the sum of cumulative mortality observed Table Biochemical composition (mean S.D.) of enriched and starved Artemia meta-nauplii fed to M rosenbergii larvaea Enriched Total lipids (% DW) Selected fatty acids (mg g−1 DW) 20:5n-3 22:6n-3 Sn-3 ( ± 20:3n-3) Sn-6 ( ± 18:2n-6) Total FAME Ascorbic acid (mg g−1 DW) Starved 22.5 6.3b 14.1 3.2c 40.3 7.5b 15.8 2.9b 61.7 10.9b 15.9 2.8b 230.79 31.7b 3.8 0.5c 0.3 0.1c 5.3 0.8c 7.5 2.8c 104.2 40.6c 3911.8 36.7b 600.7 35.8c a Within rows, superscripts express significant differences between treatments (PB0.05) over the test period Higher sensitivity to osmotic stress resulted in earlier and/or higher mortalities and thus a higher value of CSI (Dhert et al., 1992) 2.6 Biochemical analyses Enriched and starved Artemia meta-nauplii, prawn larvae and postlarvae were periodically sampled for the determination of total lipids (TL), fatty acid methyl esters (FAME) and AA levels Larvae and postlarvae were food deprived for at least h prior to sampling All samples were rinsed with tap water over a sieve and frozen until analysis Samples for TL and FAME were maintained at − 20°C, while those for AA were kept at − 80°C TL were determined by Folch et al (1957), modified by Ways and Hanahan (1964), while FAME analysis followed the methodology of Coutteau and Sorgeloos (1995) AA levels were analytically determined according to Nelis et al (1997) 2.7 Statistical analysis Data were subjected to one-way analysis of variance-ANOVA (PB0.05) and, where appropriate, Tukey’s Honest Significant Difference (HSD) Student’s t-test was used to establish differences between nominal and measured ammonia concentrations Differences on the LC50 values were graphically determined through the comparison of polynomial regressions Results In all ammonia tests performed in trial 1, the nominal concentrations were significantly related to the mean ammonium values measured (P B 0.05) Therefore, no measurements were performed in trials and Only low pH variations within single tests and among subsequent tests were observed, hence these were considered negligible The effects of enrichment and starvation on the biochemical composition of Artemia are summarised in Table The content of TL, n-3 HUFA, 20:5n-3, 22:6n-3 and AA were significantly higher in the enriched Artemia than in the starved ones (P B0.05) The differences in the biochemical composition of Artemia were R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 337 (P B0.05) in morphological development (LSI) between larvae of the same age but from different treatments started to be detected and days after hatching in trials and 2, respectively (Fig 2) The evolution of the LC50 values according to the morphological development of the larvae (LSI) from both treatments is shown in Figs and LC50 values for both groups were differentiated after a certain larval stage, usually around LSI This trend of differentiation was even more apparent as larval development proceeded For both total and non-ionised ammonia, differences in LC50 values between treatments were clearer in trial than in trial A significantly higher tolerance to the salinity stress was observed for the postlarvae fed enriched Artemia compared to those fed starved Artemia (Fig 5) On the other hand, no difference to the response to the salinity Fig Total length (mm) as a function of age (days after hatching) of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials and *Significant differences between means; PL indicates postlarvae reflected in the tissue composition of larvae fed the different diets (Table 2) Animals fed enriched Artemia presented comparatively higher levels of TL, n-3 HUFA and AA than those fed starved Artemia The biochemical composition of day larvae differed between trials, i.e larvae from trial had an average AA content of 149 mg g − DW while those from trial had mean AA levels of 265 mg g − DW Higher growth (Fig 1) and metamorphosis rates (Fig 2) were observed for larvae fed enriched Artemia, as expected (Merchie et al., 1995; Romdhane et al., 1995) Significant differences Fig Larval stage index (LSI) as a function of age (days after hatching) of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials and *Start of significant differences between treatments 338 Trial 1; days after hatching Stage Artemia Total lipids (% DW) Fatty acids (mg g−1 DW) 20:5n-3 22:6n-3 Sn-3 ( ± 20:3n-3) Sn-6 ( ± 18:2n-6) Total FAME Ascorbic acid (mg g−1 DW) a n.a., not available Lv – n.a n.a n.a n.a n.a n.a n.a 148.8 14 Lv rich 14 Lv stv Trial 2; days after hatching 22 Lv rich 22 Lv stv 26 PL rich 26 Lv stv 29 Lv stv 12.0 1.8 11.4 6.2 12.6 10.2 21.9 12.6 5.0 20.5 6.6 94.1 7.5 1.7 10.5 5.1 49.9 18.2 6.5 28.3 7.4 113.5 7.6 0.7 9.6 5.4 55.5 17.4 7.0 26.9 6.4 105.9 13.6 5.3 20.9 5.2 83.8 12.4 9.7 23.2 31.8 184.4 910.7 451.6 652.0 579.6 487.2 n.a n.a 35 Lv stv n.a 6.7 0.5 7.9 4.2 39.1 n.a 35 PL stv Lv – Lv rich Lv stv 14 Lv rich 14 Lv stv 21 PL rich 21 Lv stv 40 PL stv 7.6 24.1 21.8 12.2 15.4 6.7 11.5 5.5 3.6 7.0 1.0 9.3 6.0 62.3 24.6 13.8 41.3 24.1 279.0 26.9 20.9 50.5 67.9 388.8 7.9 2.4 11.4 6.0 66.0 16.2 6.0 25.4 7.5 113.4 7.9 1.0 9.9 5.0 55.0 11.7 4.6 17.9 4.8 76.3 8.5 0.9 10.2 4.9 49.7 6.7 0.1 7.7 8.1 110.8 193.6 265.1 844.4 384.9 835.7 447.7 484.9 442.5 191.9 R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 Table Biochemical composition of M rosenbergii larvae (Lv) and postlarvae (PL) fed enriched (rich) or starved (stv) Artemia in trials and 2a R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 339 −1 Fig Polynomial regressions of the estimated 24-h LC50 values for total ammonia (mg NH+ ) according to the larval stage + NH3 l index of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials and shock was detected between larvae fed enriched or starved Artemia (Fig 5) Discussion As expected, the biochemical composition of Artemia nauplii was influenced by the enrichment and starvation procedures, which in turn affected the composition of the larvae that fed upon them Higher TL, n-3 HUFA and AA contents were found in the tissues of larvae fed enriched Artemia versus those fed starved Artemia Larvae fed enriched Artemia also presented higher growth and metamorphosis rates than those fed starved Artemia These results confirm the positive effects of these nutrients in the enhancement of larval development and correspond well with previous findings (Devresse et al., 1990; Merchie et al., 1995; Romdhane et al., 1995) Hence, our goal to 340 R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 produce two groups of larvae with distinct quality properties as study material for the ammonia testing was considered achieved Comparison of the ammonia tolerance (LC50) of larvae at the same stage of morphological development (LSI) indicated pronounced differences between treatments The estimated LC50 values for both total and non-ionised ammonia were consistently higher for larvae fed enriched Artemia Similarly, postlarvae fed enriched Artemia presented a higher tolerance to the salin- ity stress All this indicates the superior ability of the animals fed enriched Artemia to cope with changing environmental conditions, i.e a better physiological condition Furthermore, these observations confirm the possibility of differentiating larval quality in terms of ammonia tolerance and, more importantly, demonstrate the potential of the short-term ammonia toxicity test as a criterion for the establishment of larval quality It is also worth noting that the ammonia test was sensitive enough to detect differences in the Fig Polynomial regressions of the estimated 24-h LC50 values for non-ionised ammonia (mg NH3 l − 1) according to the larval stage index of M rosenbergii larvae fed enriched or starved Artemia meta-nauplii in trials and R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 Fig Mean ( S.D.) cumulative stress index (CSI) of M rosenbergii postlarvae and larvae (LSI of 8.8 – 9.0) fed enriched or starved Artemia meta-nauplii in trials and 3, respectively early stages of larval development From the moment the effect of diet quality was analytically detectable in the larval tissues, the ammonia test was able to distinguish both groups of larvae, i.e as early as larval stage Furthermore, this pattern of differentiation remained evident throughout the experimental period Another fundamental aspect in the determination of the feasibility of the ammonia test as a quality criterion is its reproducibility over time All trials had overall results with similar patterns, demonstrating that the test is indeed reproducible However, a within-trial comparison also revealed differences in the LC50 values of larvae with similar LSI This indicates that some variation may be found when working with different batches of larvae Although the reasons for these differences are not clear, this may be due to differences in the nutritional background of the broodstock, as suggested by the differences in AA content in the batches of newly hatched larvae Other sources of variation could be the genetic differences among 341 broodstock, the inherent variation in brood quality, and minor differences in management and/or environmental conditions during the distinct larval rearing periods The salinity stress test is probably the most used method to estimate fry quality It is based on the principle that a short-term exposure to a salinity shock will indicate the physiological condition of the animals However, as the osmoregulatory capability of crustaceans changes with ontogenetic development (Sandifer et al., 1975; Charmantier et al., 1988), only animals with a similar morphological development should be used for comparison For instance, differences in gill development at early developmental stages may affect the capability of the animals to tolerate changes in salinity (Ribeiro, 1998) and hence invalidate the results of the salinity stress test In this regard, our results suggest that the salinity stress test was not sensitive enough to distinguish both groups of larvae and therefore should have its use restricted to later developmental stages In contrast, the ammonia stress test was able to differentiate the larval groups, proving to be an appropriate tool to evaluate larval quality One possible application of the ammonia test would be in broodstock nutrition studies Previously it was shown that 8-day-old larvae originating from females with different nutritional backgrounds could be distinguished by an ammonia stress (Cavalli et al., 1999a) This result was in line with the superior broodstock performance observed in some of the production parameters considered (fecundity and hatching rate) In a similar study, the response of M rosenbergii females to the dietary supplementation of phospholipids was assessed and no significant differences were detected in any of the performance parameters measured (Cavalli et al., 1999b) In this case, the ammonia test also found no differences in the 24-h LC50 values of larvae from the different treatments Also, one should not rule out possibilities for more practical applications, such as the measurement of fry quality before leaving the commercial hatchery and thus before submitting them to the stress of transportation and/or transfer to nursery or grow-out ponds In this case, however, as an alternative to the methodology presented here, the procedures for the ammonia test could be simplified to minimise the needs for labour, equipment and time For instance, reliable results may 342 R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 be obtained with fewer ammonia concentrations The use of a single concentration might also be feasible (Cavalli et al., 1999a), but this would require a previous knowledge of the LC50 values for each larval or postlarval stage (as ammonia tolerance varies with development) and the maintenance of similar pH levels for the different treatments (to ensure that the larvae from different groups are exposed to similar NH3 concentrations) Finally, it is concluded that the ammonia test is a sensitive and reproducible criterion for the establishment of larval quality, even for the early stages of development One can assume that this test could also be used as a general larval quality method for other aquatic species, perhaps even as a predictive indicator of larval viability Regardless of the species under consideration, further work is warranted to examine the precise relationship between larval quality, as evaluated by the ammonia test, and the future performance under grow-out conditions Acknowledgements This work was partially supported by a grant from the Brazilian Council for Science and Technology (CNPq), which is gratefully acknowledged The authors express their gratitude to Nopadol Phuwapanish, Department of Fisheries, Thailand, for providing the broodstock prawns, and to Geert Vandewiele and Petra Rigole for their technical assistance References Ako, H., Tamaru, C.S., Bass, P., Lee, C.S., 1994 Enhancing the resistance to physical stress in larvae of Mugil cephalus by the feeding of enriched Artemia nauplii Aquaculture 122, 81 – 90 Albert, A., 1973 Selective Toxicity Chapman and Hall, London Arellano, E., 1990 Fatty acid composition of wild and cultured Penaeus 6annamei as a method to evaluate postlarval quality In: Abstracts of the World Aquaculture Society Meeting National Research Council Canada, Ottawa, p 50 Armstrong, D.A., Chippendale, D., Knight, A.W., Colt, J.E., 1978 Interaction of ionized and un-ionized ammonia on short-term survival and growth of prawn larvae, Macrobrachium rosenbergii Biol Bull 154, 15 – 31 Bauman, R.H., Jamandre, D.R., 1990 A practical method for determining quality of Penaeus monodon (Fabricius) fry for stocking in grow-out ponds In: New, M., Saram, H., Singh, T (Eds.), Technical and Economic Aspects of Shrimp Farming Proceedings of the Aquatech 90 Conference Infofish, Kuala Lumpur, pp 124 – 131 Bray, W.A., Lawrence, A.L., Lester, L.J., 1990 Reproduction of eyestalk-ablated Penaeus stylirostris fed various levels of total dietary lipids J World Aquacult Soc 21, 41 – 52 Briggs, M.R.P., 1992 A stress test for determining vigour of post-larval Penaeus monodon Fabricius Aquacult Fish Manage 23, 633 – 637 Browdy, C., 1992 A review of the reproductive biology of Penaeus species: perspectives on controlled shrimp maturation systems for high quality nauplii production In: Wyban, J (Ed.), Proceedings of the Special Session on Shrimp Farming, World Aquaculture Society Meeting World Aquaculture Society, Baton Rouge, LA, pp 22 – 51 Cavalli, R.O., Lavens, P., Sorgeloos, P., 1999a Performance of Macrobrachium rosenbergii broodstock fed diets with different fatty acid composition Aquaculture 179, 387 – 402 Cavalli, R.O., Menschaert, G., Lavens, P., Sorgeloos, P., 1999b Maturation performance, offspring quality and lipid composition of Macrobrachium rosenbergii females fed increasing levels of dietary phospholipids Aquacult Int., submitted Charmantier, G., Charmantier-Daures, M., Bouraricha, N., Thuet, P., Aiken, D.E., Trilles, J.P., 1988 Ontogeny of osmoregulation and salinity tolerance in two decapod crustaceans: Homarus americanus and Penaeus japonicus Biol Bull 175, 102 – 110 Coutteau, P., Sorgeloos, P., 1995 Intercalibration exercise on the qualitative and quantitative analysis of fatty acids in Artemia and marine samples ICES Coop Res Rep 211, 30 pp Devresse, B., Romdhane, M., Buzzi, M., Rasowo, J., Le´ger, P., Brown, J., Sorgeloos, P., 1990 Improved larviculture outputs in the giant freshwater prawn Macrobrachium rosenbergii fed a diet of Artemia nauplii enriched with w3-HUFA and phospholipids World Aquacult 21 (2), 123 – 125 Dhert, P., Lavens, P., Sorgeloos, P., 1992 Stress evaluation: a tool for quality control of hatchery-produced shrimp and fish fry Aquacult Eur 17 (2), – 10 Emerson, K., Russo, R.C., Lund, E.R., Thurston, R.V., 1975 Aqueous ammonia equilibrium calculations: effect of pH and temperature J Fish Res Bd Can 32, 2379– 2383 FAO, 1998 Aquaculture Production Statistics 1987– 1996 FAO Fisheries Circular No 815, Revision 10 FAO, Rome, 197 pp R.O Ca6alli et al / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 333–343 Fegan, D., 1992 Recent developments and issues in the penaeid shrimp hatchery industry In: Wyban, J (Ed.), Proceedings of the Special Session on Shrimp Farming, World Aquaculture Society Meeting World Aquaculture Society, Baton Rouge, LA, pp 55 – 70 Folch, J., Lees, M., Stanley, G.H.S., 1957 A simple method for the isolation and purification of total lipids from animal tissues J Biol Chem 266, 497 – 509 Fromm, P.O., Gillette, J.R., 1968 Effect of ambient ammonia on blood ammonia and nitrogen excretion of rainbow trout (Salmo gairdneri ) Comp Biochem Physiol 26, 887 – 896 Greenberg, A.E., Clesceri, L.S., Eaton, A.D., 1992 Standard Methods for the Examination of Water and Wastewater, 18th edn American Public Health Association, American Water Works Association and Water Pollution Control Federation, Washington, USA Hamilton, M.A., Russo, R.S., Thurston, R.V., 1977 Trimmed Spearman Karber Method for estimating median lethal concentrations in toxicity bioassays Environ Sci Technol 11, 714 – 719 Kinne, O., 1976 Cultivation of marine organisms: water quality management and technology In: Kinne, O (Ed.), Marine Ecology, vol III, part Wiley-Interscience, New York, pp 79 – 300 Maddox, M.B., Manzi, J.J., 1976 The effects of algal supplements on static system culture of Macrobrachium rosenbergii (de Man) larvae Proc World Maricult Soc 7, 677 – 698 Merchie, G., 1996 Artemia: use of nauplii and metanauplii In: Lavens, P., Sorgeloos, P (Eds.), Manual on the Production and Use of Live Food for Aquaculture FAO Fish Tech Paper 361 FAO, Rome, pp 137 – 163 Merchie, G., Lavens, P., Radull, J., Nelis, H., De Leenheer, A., Sorgeloos, P., 1995 Evaluation of vitamin C-enriched Artemia nauplii for larvae of the giant freshwater prawn Aquacult Int 3, 355 – 363 343 Nelis, H.J., De Leenheer, A., Merchie, G., Lavens, P., Sorgeloos, P., 1997 Liquid chromatographic determination of vitamin C in aquatic organisms J Chromatogr Sci 35 (7), 337 – 341 Ribeiro, F.A.L.T., 1998 The postlarval development, growth and nutrition of the Indian white prawn Penaeus indicus Ph.D Thesis, University of Wales, 176 pp Romdhane, M.S., Devresse, B., Le´ger, P., Sorgeloos, P., 1995 Effect of feeding w-3 HUFA enriched Artemia during a progressively increasing period on the larviculture of freshwater prawns Aquacult Int 3, 236 – 242 Samocha, T.M., Guajardo, H., Lawrence, A.L., Castille, F.L., Speed, M., McKee, D.A., Page, K.I., 1998 A simple stress test for Penaeus 6annamei postlarvae Aquaculture 165, 233 – 242 Sandifer, P.A., Hopkins, J.S., Smith, T.I.J., 1975 Observations on the salinity tolerance and osmoregulation in laboratory-reared Macrobrachium rosenbergii postlarvae (Crustacea, Caridea) Aquaculture 6, 103 – 114 Singh, I.S.B., Philip, R., 1995 A simple device for the separation of weak larvae of Macrobrachium rosenbergii (de Man) Aquacult Res 26, 225 – 227 Tackaert, W., Abelin, P., Le´ger, P., Sorgeloos, P., 1989 Stress resistance as a criterium to evaluate quality of postlarval shrimp reared under different feeding procedures In: Proceedings III Simpo´sio Brasileiro sobre Cultivo de Camara˜o Joa˜o Pessoa, Brazil, pp 393 – 403 Tomasso, J.R., 1994 Toxicity of nitrogenous wastes to aquaculture animals Rev Fish Sci 2, 291 – 314 Uno, Y., Kwon, C.S., 1969 Larval development of Macrobrachium rosenbergii (de Man) reared in the laboratory J Tokyo Univ Fish 55 (2), 179 – 190 Watanabe, T., Kitajima, C., Fujita, S., 1983 Nutritional values of live organisms used in Japan for mass propagation of fish: a review Aquaculture 34, 115 – 143 Ways, P., Hanahan, D.J., 1964 Characterization and quantification of red cell lipids in normal man J Lipid Res 5, 318 – 328 ... aimed to assess the feasibility of a short-term ammonia toxicity test as a larval quality evaluation criterion The freshwater prawn M rosenbergii was chosen as a model species since it easily reproduces... was analytically detectable in the larval tissues, the ammonia test was able to distinguish both groups of larvae, i.e as early as larval stage Furthermore, this pattern of differentiation remained... groups of larvae with distinct quality properties as study material for the ammonia testing was considered achieved Comparison of the ammonia tolerance (LC50) of larvae at the same stage of morphological