Effect of starving and feeding on some haematological and physiological responses of the Nile catfish, <Emphasis Type="Italic">Clarias gariepinus</Emphasis> exposed to copper at extreme seasons

The lethal concentration for 50% of fish for 96h (96h LC₅₀) of copper (Cu²⁺) was estimated for the Nile catfish (Clarias gariepinus) in extreme seasons, winter and summer, 4.31 and 4.79 mg/l, respectively. The Nile catfish was exposed to 96h LC₅₀ of copper for 7 days in extreme winter and summer. The body indices, haematological parameters as well as some plasma and liver enzyme activities and metabolite level were significantly differed in fish exposed to copper over than those of the control fish. Most of the tested parameters were not significantly different between the control fish of winter and summer (winter, water temperature 18 ± 2°C and summer, 27 ± 2°C). The effect of two ration sizes on copper toxicity in two different seasons on C. gariepinus was justified. It was found that the haematological parameters and the tested plasma activities of enzymes were significantly valid due to season differences. The blood parameters as well as plasma activities of enzymes were significantly differed in fishes fed elevated ration (3%) and exposed to copper challenge. On the other hand, the exploit of low feeding ration (0.5%) along with copper exposure during the examined seasons induced non-significant differences of the tested parameters, from those of the corresponding control. Therefore, the low feeding ration provides some tolerance against the possible water-borne copper exposure.     

Susceptibility of steelhead trout Salmo gairdneri Richardson to redmouth infection Yersinia ruckeri following exposure to copper

Abstract. Steelhead trout were exposed to sublethal concentrations of copper and inoculated with Yersinia ruckeri. Copper exposure at 7 and 10 μg/1 for 96 h caused more fish to die of infection than control fish (no copper). Infection susceptibility increased with time of exposure to a single dosage of copper (10 μg/1), reaching a maximum at 48 h. Lowering the copper concentration to 5 μg/1 caused the infection susceptibility to occur at 24 h. The infectious dose of Y. ruckeri was lower in fish exposed to 10 μg/1 copper for 48 h than control fish.     

Acute effects of copper on superoxide dismutase, catalase and lipid peroxidation in the freshwater teleost fish, Esomus danricus

Static-renewal bioassays [Methods for acute toxicity tests with fish, macro-invertebrates and amphibians: USEPA, ERS, EPA 660/3 75-009 (1975)] were carried out on Esomus danricus exposed to sub-lethal (0.55 mg/l) and lethal (5.5 mg/l) concentrations of copper. The 96-h median lethal concentration (LC₅₀) was 5.5 mg/l. Biochemical stress responses, such as visceral superoxide dismutase (SOD) and catalase (CAT) activities, were measured during this 96-h period. Malondialdehyde, a product of lipid peroxidation, was present at elevated levels in the visceral tissue of copper-exposed fish. Copper was found to be highly toxic to the fish and induced significant declines (p < 0.05–0.001) in all of the biochemical profiles studied, demonstrating a linear and positive correlation with both the concentration and duration of exposure to copper. In E. danricus, CAT appeared to be more sensitive to copper exposure (p < 0.001) than SOD at both lethal and sub-lethal levels. These results indicate that antioxidant responses can be employed as biomarkers of oxidative stress for this species in aquatic environments contaminated with copper.     

急性氨氮暴露对大弹涂鱼炎性反应相关基因表达的影响

为研究急性氨氮胁迫对大弹涂鱼炎性反应相关基因表达的影响,实验挑选初始体质量为(15.14±0.05) g的健康大弹涂鱼幼鱼180尾,进行96 h的急性氨氮胁迫实验。结果显示,大弹涂鱼96 h氨氮半致死浓度为8.99 mg/L总氨氮(0.11 mg/L非离子氨,T-AN);氨氮胁迫后TNF基因的mRNA表达量分别于12和96 h时显著上调,96 h时表达量达到0 h时的2倍;IL-1基因的mRNA表达量12 h时显著上调,为0 h时表达量的6倍;氨氮胁迫后IL-6基因的mRNA表达量分别于12和96 h时显著上调,表达量达到0 h时的1.5倍;氨氮胁迫后IL-8基因的mRNA表达量在24 h时出现显著下调。研究表明,大弹涂鱼96 h氨氮半致死浓度为8.99 mg/L总氨氮;半致死浓度的氨氮胁迫48 h后,TNF、IL-1、IL-6和IL-8基因的mRNA表达量持续升高,推测过度炎性应激可能是导致鱼类氨中毒死亡的原因之一。     

Alterations in blood electrolytes of a freshwater catfish <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis> in response to treatment with a botanical pesticide, <Emphasis Type="Italic">Nerium indicum</Emphasis> leaf extract

The present study aimed at investigating the effects of Nerium indicum leaf extract on the blood electrolytes of Heteropneustes fossilis for short- and long term. Fish were subjected to Nerium indicum leaf extract for short term (11.27 mg/L i.e. 0.8 of 96 h LC₅₀) and long term (2.81 mg/L i.e. 0.2 of 96 h LC₅₀). Fish were killed on each time intervals from control and experimental (Nerium indicum) groups after 24, 48, 72, and 96 h in short-term exposure and after 7, 14, 21, and 28 days in long-term experiment. Blood samples were analyzed for calcium and inorganic phosphate levels. Acute exposure of Nerium indicum leaf extract caused a progressive decrease in the serum calcium levels after 48 h in fish H. fossilis, which persists till the close of the experiment (96 h). The serum inorganic phosphate levels remain unaffected till 48 h in the Nerium indicum leaf extract–exposed fish. After 72 and 96 h, the levels exhibit a decrease. Chronic Nerium indicum leaf extract treatment provoked a decrease in serum calcium levels at day 14. This decrease continues till 28 days. The serum phosphate level of the Nerium indicum leaf extract–treated fish decreases on day 14 and 21. However, on day 28, the levels become close to the normal values. We conclude that Nerium indicum leaf extract exposure alters the blood electrolytes of the fish, thus causing physiological disturbances which might affect seriously the normal vital functions, growth rate, reproduction, and their survival in nature.     

The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum)

The anaesthetic effects of clove-oil-derived eugenol were studied in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Acute lethality and the effects of multiple exposures to eugenol were measured. The estimated 8-96 h LC₅₀ for eugenol was found to be approximately 9 p.p.m. Times to induction and recovery from anaesthesia were measured and compared with MS-222 under similar conditions. Eugenol generally induced anaesthesia faster and at lower concentrations than MS-222. The recovery times for fish exposed to eugenol were six to 10 times longer than in those exposed to similar concentrations of MS-222. Clove oil eugenol was determined to be an acceptable anaesthetic with potential for use in aquaculture and aquatic research. Doses of 40-60 p.p.m. eugenol were found to induce rapid anaesthesia with a relatively short time for recovery in juvenile trout.     

Acute toxicity of ammonia in Pacu fish (Piaractus mesopotamicus,Holmberg, 1887) at different temperatures levels

Piaractus mesopotamicus juveniles (total length 12 ± 0.5 mm) were exposed to different concentrations of ammonia‐N (un‐ionized plus ionized ammonia as nitrogen), using the static renewal method at different temperature levels (15, 20 and 25°C) at pH 7. The 24, 48, 72, 96 h LC₅₀ values of ammonia‐N in P. mesopotamicus juveniles were 5.32, 4.19, 3.79 and 2.85 mg L^?1 at 15°C; 4.81, 3.97, 3.25 and 2.50 mg L^?1 at 20°C; and 4.16, 3.79, 2.58 and 1.97 mg L^?1 at 25°C respectively. The 24, 48, 72, 96 h LC₅₀ values of NH₃‐N (un‐ionized ammonia as nitrogen) were 0.018, 0.014, 0.013, 0.009 mg L^?1 at 15°C temperature; 0.023, 0.019, 0.016 and 0.012 mg L^?1 at 20°C; 0.029, 0.026, 0.018 and 0.014 mg L^?1 at 25°C. The temperature increase from 15 to 25°C caused an increase of ammonia‐N susceptibility by 21.80%, 9.55%, 31.92% and 30.87%, after 24, 48, 72 and 96 h exposure respectively. Furthermore, we found that exposure of fish to ammonia‐N caused an elevation in total haemoglobin and blood glucose with an increase of 2 mg L^?1 concentration. Ammonia levels tolerated, especially in different temperatures levels, have important implications for the management of aquaculture.     

Acute and chronic effects of aqueous ammonia on marbled spinefoot rabbitfish,Siganus rivulatus (Forsskål 1775)

Ammonia is a metabolite of aquatic organisms which might reach deleterious levels in intensive fish farms. The aim of the present study was to determine median lethal concentrations (96‐h LC₅₀) of total ammonia nitrogen (TA‐N) on marbled spinefoot rabbitfish (Siganus rivulatus) and chronic effects of TA‐N on survival, growth and behaviour of juvenile rabbitfish over a 50 day period. In the first experiment, fish were exposed to 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mg L^?1 TA‐N for 96 h and survival evaluated. In the second experiment, 12 fish were stocked per 50‐L tank and treated with one of 0, 2, 4, 6, 8, 10 and 12 mg L^?1 TA‐N with three replicate tanks per treatment. Survival and growth were determined and histopathological alterations of gills due to chronic ammonia exposure were studied by light and electron microscopy. The 96‐h LC₅₀ values were 16–18 mg L^?1 TA‐N. In the chronic exposure experiment, fish reared in water with 0 mg L^?1 TA‐N had 100% survival and had 50% weight increase in 50 days. Fish at 2 and 4 mg L^?1 TA‐N all died whilst fish in 6, 8, 10 and 12 mg L^?1 TA‐N survived and grew albeit less than in treatment 0 mg L^?1. Gills from ammonia treated fish displayed severe histological and ultrastructural alterations including hyperplasia, hypertrophy and fusion of secondary lamellae, aneurysms and presence of pleomorphic altered cells. Chronic exposure to ammonia is deleterious to marbled spinefoot rabbitfish and low concentrations of ammonia appear to kill the fish in <50 days whilst fish can survive for more than 50 days at concentrations between 6 and 12 mg L^?1 TA‐N.     

Acute toxicity of ammonia in Meagre (Argyrosomus regius Asso, 1801) at different temperatures

Argyrosomus regius (3.0 ± 0.9 g) were exposed to different concentrations of ammonia in a series of acute toxicity tests by the static renewal method at three temperature levels (18, 22 and 26°C) at a pH of 8.2. Low temperature clearly increased the tolerance of the fish to total ammonia nitrogen (TAN) and unionized ammonia (NH₃) (P < 0.05). While the 96‐h LC₅₀ values of TAN were 19.79, 10.39 and 5.06 mg L^?1, the 96‐h LC₅₀ of NH₃ were 1.00, 0.70 and 0.44 mg L^?1 at 18, 22 and 26°C respectively. The safe levels of NH₃ for A. regius was estimated to be 0.10, 0.07 and 0.04 mg L^?1 at 18, 22 and 26°C respectively (P < 0.05). This study clearly indicates that A. regius is more sensitive to ammonia than other marine fish species cultured on the Mediterranean and Eastern Atlantic coasts.     

Acute Toxicity of Ammonia and Nitrite to Sea Bream,Sparus aurata (Linnaeus, 1758), in Relation to Salinity

Sea bream, Sparus aurata, is one of the most important fish species that is commonly cultured in the Mediterranean and the eastern coasts of the Atlantic Ocean. The life cycle of sea bream in its natural habitat passes through hyposaline and hypersaline lagoons. It is important to determine the tolerance of the fish to nitrogenous compounds for aquaculture at maximum stocking densities. In the present study, a series of acute experiments were performed to evaluate the effect of salinity on ammonia and nitrite toxicity to sea bream. The fish were exposed to different ammonia and nitrite concentrations according to the static renewal methodology at three different salinities (10, 20, and 30 ppt) and at a temperature of 20 C and a pH of 8.2. The toxic effect of total ammonia nitrogen (TAN) and nitrite nitrogen (NO₂‐N) decreased with increasing salinity levels (P < 0.001). Acute toxicity (96‐h lethal concentration 50 [LC₅₀]) values of TAN were determined to be 5.93, 11.72, and 19.38 mg/L at 10, 20, and 30 ppt salinity, respectively. The 96‐h LC₅₀ values of NO₂‐N were determined to be 370.80, 619.47, and 806.33 mg/L at 10, 20, and 30 ppt salinity, respectively. Results indicate that sea bream is less tolerant to ammonia but more tolerant to nitrite compared with some other fish species.     

Ammonia‐, Sodium Chloride‐, and Calcium Sulfate‐induced Changes in the Stress Responses of Jundiá, Rhamdia quelen,Juveniles

Salt (NaCl) and gypsum (CaSO₄) are used as water additives to mitigate fish stress and improve specimen survival. High stocking densities and the transportation of fish can increase aqueous ammonia, which can, in turn, alter fish cortisol secretion. The objectives of this study were to assess the effects of salt, gypsum, and aqueous ammonia on some stress‐induced physiological responses of jundiá, Rhamdia quelen, juveniles induced by captivity and handling, and to determine the lethal ammonia concentration for this species. Jundiá juveniles were subjected to the following five treatments: water only, water + ammonia (0.4 mg/L), water + NH₃ + NaCl (6 g/L), water + NH₃ + gypsum (150 mg/L), and water + NH₃ + NaCl + gypsum. Blood samples were taken after intervals of 1, 5, 24, and 96 h, and the concentrations of cortisol, glucose, chloride, ammonia, and hematocrit were determined. The NH₃ LC₅₀ value after 96 h of exposure (LC_50?96h) was measured to be 1.9 mg/L NH₃. Either salt or gypsum reduced both cortisol and glucose levels during most of the experimental period, but the combination of both reduced these levels even further. The combined use of NaCl and CaSO₄ demonstrates a synergic effect on mitigating stress responses induced by handling and aqueous ammonia in jundiá juveniles.     

Effects of Nitrite Exposure on Growth and Survival of Sea Bass,Lates calcarifer,Fingerlings in Various Salinities

Sea bass, Lates calcarifer, fingerlings were acclimated to 0. 15, and 32 ppt, and the toxic effects of nitrite exposure were assessed. The 96-hour median lethal concentrations (96-hour LC₅₀ for nitrite were estimated to be 14.5 mg/L at 0 ppt, 105 mg/L at 15 ppt and 93 mg/L at 32 ppt salinity. Chronic exposure to a nitrite concentration equivalent to 10% of the respective 96 hour LC₅₀ resulted in marked growth reduction: growth being reduced in the order of 0 ppt > 32 ppt > 15 ppt. In nitrite-free water, growth rate for fish raised at a salinity of 15 ppt was higher compared to fish raised at salinities of 0 ppt and 32 ppt, a phenomenon which probably reflected the advantage of a reduction in osmoregulatory work in an iso-osmotic environment.     

Chronic effects of NeemAzal on biochemical parameters of grass carp,Ctenopharyngodon idella

The aim of this study was to assess the chronic effects of a bio‐pesticide (NeemAzal) on some biochemical parameters of grass carp (Ctenopharyngodon idella) under laboratory conditions. At first, 96 h LC₅₀ of the pesticide was determined using probit analysis software and was found to be 0.73 mg L^?1 of the active ingredient (azadirachtin) of the pesticide. Fresh fish were then divided into three treatments and a control and were exposed to sublethal concentrations (10%, 20% and 30% of the 96 h LC₅₀ value) of the pesticide for a month. The biochemical examination of the blood showed a remarkable decrease in values of protein, albumin, AST, ALT, ALP and LDH (P < 0.05).     

Acute and Sublethal Effects of Ammonia on Striped Bass and Hybrid Striped Bass

Bioassays in static water (mean ± SD; temperature, 20–22 C; pH, 8.2–8.4; alkalinity, 205 ± 10 mg/L CaCO₃; total hardness, 220 ± 10 mg/L CaCO₃) were used to determine median lethal concentrations (LC₅₀) of un-ionized ammonia (NH₃-N) for striped bass Moronc saxatilis and hybrid striped bass M. saxatilis × M. chrysops. The 96 h LC₅₀ for striped bass was 1.01 ± 0.24 mg/L NH₃-N₃ and was significantly higher than the LC₅₀ for hybrid striped bass (0.64 ± 0.05 mg/L NH₃-N). The effects of sublethal ammonia were evaluated after fish were exposed for 96 h to 0.0, 0.25, or 0.5 mg/L NH₃-N or to additional exposure to oxygen depleted water (about 2.0 mg O₂/L). Plasma ammonia of striped hass did not change as sublethal ammonia increased, but exposure to oxygen depletion caused a decrease in plasma ammonia. In contrast, plasma ammonia of hybrid striped bass increased as environmental ammonia increased, and increased further after exposure to oxygen depletion. Plasma cortisol levels of striped bass were significantly higher and more variable than cortisol levels of hybrid striped bass; additional exposure to oxygen depletion increased this variability, but these responses may be due to the stress of handling and confinement. Mean differences also existed between species for hemoglobin and hematocrit, while differences in variability occurred for osmolality and oxygen depletion rates. Striped bass tolerated ammonia better than hybrid striped bass but were more susceptible than hybrid striped bass to the additional stress of oxygen depletion. Most changes in physiological characteristics were relatively independent of environmental ammonia, but they were affected by oxygen depletion challenge.     

Experimental Infection of Rainbow Trout,Oncorhynchus mykiss,and Hybrid Striped Bass,Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD₅₀ for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 10⁴ pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.     

Acute Toxicity and Sublethal Effects of Nitrite on Selected Hematological Parameters and Tissues in Dark-banded Rockfish, Sebastes inermis

Acute toxicity and sublethal effects of nitrite in dark‐banded rockfish, Sebastes inermis (83.3 ± 7.2 g), were studied under static conditions for a period of 96 h. The acute toxicity of nitrite evaluated for the 96‐h lethal concentration (LC₅₀) was 700 mg/L. The sublethal effects on selected hematological parameters of S. inermis, such as total erythrocyte count (TEC), hemoglobin, plasma glucose, and serum protein content, were measured after 0, 6, 12, 24, 48, 72, and 96 h of exposure to 0, 50, 100, 200, 400, and 700 mg/L of nitrite. Sublethal nitrite caused progressive reduction in the TEC, hemoglobin, and serum protein content in fish depending on the nitrite concentration and exposure period. The 96‐h exposure resulted in a 14–42% reduction in TEC and 25–33% reduction in hemoglobin content for 100–700 mg/L of nitrite compared to the control. A dose‐related reduction in plasma glucose (25.7–34.2%) was observed for concentrations of 200–700 mg/L of nitrite during 48 h of exposure, followed by an increase through 96 h. A significant reduction in serum protein (7.3–12.6%) was observed for 200–700 mg/L of nitrite after 96 h of exposure. Abnormal histological changes in skin, gill, liver, and kidney tissue were observed in fish exposed to 700 mg/L of nitrite after 96 h of exposure compared to the control. Although no mortality of S. inermis occurred at 500 mg/L of nitrite, all hematological parameters adversely responded to a nitrite dose of 200 mg/L for 96 h. These results showed that although acute toxicity concentration of nitrite in S. inermis is higher than 700 mg/L, sublethal concentrations of nitrite also negatively affect hematological parameters.     

Toxicity of Four Chemotherapeutic Agents to Rabbitfish Siganus rivulatus

As aquaculture evolves from extensive pond culture to intensive tank and cage systems, chemical dips and baths are increasingly being used to treat a concomitant increase in ectoparasitic and bacterial infestations. Some of the main disease‐causing agents are ectoparasites on the skin and gills of fish. Consequently, application of chemotherapeutics is increasing in aquaculture industries in order to control outbreaks of parasitic infestations. However, the toxic effect of commonly used chemotherapeutic agents on specific aquacultured fish species is often unknown. The present work was performed to test the effect of four commonly used chemical treatments on rabbitfish, Siganus rivulatus. The lethal concentrations for 50% of population (LC₅₀) of formalin, copper sulfate, potassium permanganate, and hydrogen peroxide for S. rivulatus juveniles treated for 1 h were assessed. Formalin and potassium permanganate tolerance values were determined by calculating 72‐h LC₅₀ values through probit analysis. The 72‐h LC₅₀ values for the formalin toxicity tests were 551.0 and 1.68 mg/L, respectively. LC₅₀ of copper sulfate and hydrogen peroxide could not be determined from the concentrations tested but were found to be >3 and >700 mg/L, respectively. Accordingly, treatment concentrations of formalin and potassium permanganate used for other fish species could be lethal to S. rivulatus, but the species appears to be quite tolerant to copper sulfate and hydrogen peroxide.     

Toxicity of Peracetic Acid to Fish: Variation among Species and Impact of Water Chemistry

There has been strong interest in the use of peracetic acid (PAA) in aquaculture as it can be used to disinfect water and hard surfaces and thereby eliminate or lower the burden of fish pathogens. Unfortunately, there has been little research on the toxicity of PAA to fish. Twelve species of fingerling fish that are important to aquaculture were exposed to PAA for 24 h in static toxicity bioassays in well water. These fish were: fathead minnow, Pimephales promelas; black‐nose crappie, Pomoxis nigromaculatus; bluegill, Lepomis macrochirus; blue tilapia, Oreochromis aureus; channel catfish, Ictalurus punctatus; golden shiner, Notemigonus crysoleucas; goldfish, Carassius auratus; grass carp, Ctenopharyngodon idella; largemouth bass, Micropterus salmoides; rainbow trout, Oncorhynchus mykiss; sunshine bass, Morone chrysops × M. saxatilis; and walleye, Sander vitreus. Median lethal concentration (LC₅₀) values were estimated with the trimmed Spearman–Karber method using nominal PAA concentrations. The mean 24‐h LC₅₀ values ranged from 2.8 to 9.3 mg/L PAA. Fathead minnow were very sensitive and blue tilapia were very tolerant to PAA exposure; LC₅₀ values of other species tested were within the range of 4.1–6.2 mg/L PAA. More importantly, the 24‐h no‐observed‐effect concentration (NOEC) ranged from 1.9 to 5.8 mg/L PAA; the NOEC would be considered as the safe range for culturists to investigate the use of PAA. Decreased alkalinity/hardness increased the toxicity of PAA, while a small increase of dissolved organic content had no effect on PAA toxicity. Results of the present study are important information on the safe application of PAA for the aquaculture industry.     

Acute toxicity of nitrite to mud crab Scylla serrata (Forsskål) larvae

Early larval stages of mud crab Scylla serrata were exposed to different concentrations of nitrite (40, 80 and 160 mg L⁻¹ and a control, without added nitrite) and three salinity levels (25, 30 and 35 g L⁻¹) using a static renewal method. No interactive effect of nitrite and salinity was detected. Estimated LT₅₀ in 96‐h toxicity tests decreased in all stages with increasing nitrite concentrations in all salinity levels. The 96‐h LC₅₀ values of nitrite‐N were 41.58, 63.04, 25.54, 29.98 and 69.93 mg L⁻¹ for zoea 1, 2, 3, 4 and 5 respectively. As the larvae grew, they showed a progressive increase in tolerance to nitrite. The toxicity of nitrite to larvae increased with exposure time. The median lethal concentration was not affected by salinity. The chloride component of salinity within 25–35 g L⁻¹ did not seem to be as effective in alleviating toxicity as has been reported in other crustacean species. Based on 96‐h LC₅₀ and an application factor of 0.1, the ‘safe level’ of rearing mud crab larvae was calculated to be 4.16, 6.30, 2.55, 2.99 and 6.99 mg L⁻¹ nitrite‐N for zoea 1, 2, 3, 4 and 5 respectively.