In vitro hydroxylation of vitamin D3 and 25-hydroxy vitamin D3 in tissues of Atlantic salmon Salmo salar, Atlantic mackerel Scomber scombrus, Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua

The in vitro metabolism of ¹⁴CD₃ and ³H25OHD₃ was investigated in different tissues from Atlantic salmon Salmo salar , Atlantic mackerel Scomber scombrus , Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua . The tissues analysed were liver, kidney, head kidney, gills, spleen and intestine. The metabolites were extracted in methanol–chloroform and separated by normal-phase high-pressure liquid chromatography (HPLC) followed by scintillation counting. Identification of the metabolites was by comigration with standards on normal and reversed-phase HPLC systems and by protein-binding assays. All tissues from all species analysed produced hydroxylated derivatives identified as 25OHD₃, 24,25(OH)₂D₃ and 1,25(OH)₂D₃. In addition, some unidentified derivatives were recorded, one probably being 25,26(OH)₂D₃. Organs producing great amounts of one metabolite also produced considerable amounts of the other possible derivatives, suggesting a lower degree of specificity in fish organs than in human organs. The predominating metabolite was 24,25(OH)₂D₃ in all organs from salmon and mackerel during incubation with ¹⁴CD₃ and within most organs from all species during ³H25OHD₃ incubation. The latter observation probably results from the need for decreasing rather than increasing the calcium absorption in these species, which live at least some periods of life in a marine environment.     

Tissue distribution of vitamin D3 in Atlantic salmon Salmo salar: effect of dietary level

Atlantic salmon Salmo salar were given three dietary doses of vitamin D₃ for a period of 11 weeks: diet groups I, II and III received a 0.04, 2.21 and 28.68 μg g⁻¹ diet of vitamin D₃, respectively. The tissue distribution of vitamin D₃ was investigated, and between diet groups II and III, the level of vitamin D₃ found in the tissues was increased by a factor similar to the increased level in the feed. No effects were observed on weight gain, survival, plasma level of calcium, red blood cell count or haematocrit, relative to the dietary levels of vitamin D₃. The results showed that while the plasma concentration of 25(OH)D₃ increased between diet groups II and III, the concentration of 1,25(OH)₂D₃ decreased.     

Use of duckweed (Lemna sp.) grown in sugarmill effluent for milkfish, Chanos chanos Forskal, production

Abstract. Oxidation treatment of sugarmill waste using duckweed ( Lemna sp.) as part of the system, to reduce effluent nutrient concentrations and biological oxygen demand (BOD), was evaluated during a 6 month milling season in Negros Oriental, Philippines. Mean ammonia concentration in effluent water was reduced from 0·87 to 0·31 mg/l NH₃-N and orthophosphate from 0·93 to 0·51 mg/l P₂O₅, while mean BOD was reduced from 611 to 143mg/t BOD₅, after treatment. Seasonal mass fish kills in the adjoining bay no longer occurred during the 3 years following introduction of this treatment. The mean duckweed production (dry weight) was 8–8g/m²/day. Duckweed was harvested from a 1·9ha area of the system and transferred to an adjacent milkfish, Chanos chanos Forskal, farm. Its fertilization effect, in terms of lablab production ( lablab is the biological complex of bluegreen algae, diatoms, bacteria and various animals which forms a mat at the bottom or floats in patches), was evaluated in the milkfish ponds. This was compared with ponds fertilized with either inorganic fertilizers or cow manure, in the traditional way. Lablab growth was significantly increased using duckweed, with ash-free dry weight production averaging 32g/m²/day following fertilization with duckweed compared with 4g/m²/day using inorganic fertilizers. Milkfish net production averaged 320 kg/ha/90-day crop in inorganically fertilized ponds. 545kg/ha/90-day crop for cow manure and 820kg/ha/90-day crop in duckweed-fertilized ponds. The system is described and the benefits of this integrated waste treatment-fish production facility are discussed.     

Adsorption of minerals on the isolated mantle of Patinopecten yessoensis

The isolated mantle of scallop ( Patinopecten yessoensis ) was shown to adsorb minerals (Zn, Ca, Mg, Fe, Cu, Ni and Co) to more than 1% upon treatment with mineral solution. The amounts of adsorbed Zn, Ca and Mg were 44.3, 47.4 and 15.5 mg/g dry weight, when the mantle was treated with 5% ZnSO₄, CaCl₂ and MgSO₄ solutions at room temperature, respectively. The amounts of adsorbed Zn, Ca and Mg were dependent on the concentration of mineral solution used. This is the first report showing that the isolated mantle adsorbs nutritionally important minerals such as Ca, Mg and Fe. This finding may lead to the development of new mineral-enriched food supplements.     

Sensitivity of juvenile lobsters, Homarus americanus L., to different concentrations of malachite green during various states of the intermoult cycle

Abstract. Survival of the early stages (egg, larva and juvenile) of the American lobster, Homarus americanus L., reared under controlled conditions has often been threatened by a variety of fungi and microbial epibionts. A preliminary study was conducted for the purpose of defining toxicity levels of malachite green for 5th stage juveniles during each of the following intermoult (or fused intermoult) states: A-B, C₄, D₀ and D₂-D₃ Irrespective of exposure time or concentration, all the lobsters treated during intermoult state A-B died. This contrasts with seemingly good survival of lobsters in intermoult states C₄, D₀ and D₂- D₃ treated at concentrations of 900 mg/1 malachite green for exposure periods of 10 min. The findings suggest that an effective treatment combination for lobsters immersed in malachite green may be one which is employed during intermoult state C₄, for about 6min and at concentrations of about 700–800 mg/l.     

Effects of varying salinities on the toxicity of potassium permanganate to larval and juvenile striped bass, Morone saxatilis (Walbaum)

Abstract. Eight-day-old striped bass, Morone saxatilus (Walbaum) larvae, and one-month-old juveniles were held in varying salinities and exposed to a range of concentrations of potassium permanganate. Lethal concentrations of potassium permanganate for 50% of the larval fish exposed for96h (96h LC₅₀) were 1middot;02, 2 middot;11, 1 middot;41 and 1 middot;73mg/1 for salinities of 0, 3, 6and 9 g/l, respectively; The 96h LC₅₀S for juveniles were 0 middot;96, 3 middot;26, 1 middot;63 and 1 middot;48 mg/l for salinities of 0, 5, 10 and 15g/l, respectively. There were significant differences in 96h LC₅₀ values at different salinities for both life stages. The 96 h LC₅₀ values derived for both life stages revealed a decrease in toxicity as salinity increased from 0 to 3–5g/l. Results indicated potassium permanganate to be toxic in fresh water to striped bass larvae and fingerlings at recommended treatment levels (3 middot;0mg/l).     

Toxicity of Ammonia and Nitrite to Penueus monodon Juveniles

Pcnaeus monodon juveniles (35.4 ± 2.2 mm TL) were exposed to seawater (20 ppt) having different concentrations of total ammonia (NH₃+ NH,+) and nitrite. Median lethal concentration (LC₅₀) was determined with static-renewal tests. The LC₅₀, of total ammonia-N, NH₃-N and niMte-N on shrimps decreased with increase of exposure time. The 24, 48, 72, % and 120 h LC₅₀ were 94.96, 61.09, 47.47 45.58 and 38.00 mg/L total ammonia-N (2.68, 1.73, 135, 1.29 and 1.08 mg/L NH₃-N) and 215.85, 185.33, 88.54, 54.76 and 37.97 mg/L nitrite-N, respectively. The 144h LC₅₀ of total ammonia-N and NH₃-N was 36.71 mg/L and 1.04 mgL. The "threshold" of ammonia and nitrite was found at 144h and 120h respectively, from the toxicity curve approaching asymptote. A "safe value" was 3.7 mg/L total ammonia-N, 0.1 mg/L NH₃-N (20 ppt, pH 7.70, 27C) and 3.8 mg/L nitrite-N for P. monodon juvenile .     

Histopathology of rainbow trout, Salmo gairdneri Richardson, exposed to diurnally fluctuating un-ionized ammonia levels in static-water ponds

Abstract. Rainbow trout, Salmo gairdneri Richardson, were reared intensively in nine aerated static-water ponds. Dissolved oxygen concentrations were maintained above 5 mg/l and un-ionized ammonia (NH₃) levels were monitored in all ponds. Fish were removed periodically and gill, liver and trunk kidney samples were removed for histological examination. Average NH₃ concentrations did not exceed a previously reported chronically toxic level, but average daily maximum concentrations exceeded this level in all but two ponds. Gills exhibited lesions characteristic of those attributed to ammonia exposure in all ponds and fish with the most damaged gills had livers demonstrating reduced glycogen vacuolation. No histological changes were noted in kidney tissue. Because gills were damaged even though average NH₃ levels were quite low, it is recommended that research in static water carefully consider the cyclical nature of NH₃ concentrations.     

Hemolymph Oxyhemocyanin and Protein Levels and Acid-Base Balance in the Tiger Shrimp Penaeus monodon Exposed to Copper Sulfate

Abstract.— Subadult tiger shrimp Penaeus monodon (30.15 ± 2.19 g) were exposed individually in sea water of 25%o to 0 (control), 1.11, 4.76, 10.06 and 19.09 mg/L copper for 24 h. Hemolymph pH, PO₂ (partial oxygen pressure), PCO₂ (partial carbon dioxide pressure), HCO₃, oxyhemocyanin and protein levels were determined. Hemolymph PO₂ increased, whereas hemolymph pH, PCO₂, HC0₃-, oxyhemocyanin. protein and the ratio of oxyhemocyanidprotein levels decreased with increasing concentrations of ambient copper in the range of 1.11 to 19.09 ma/L. It is concluded that P. monodon following 24-h exposure to ambient copper as low as 4.76 mg/L shows a reduction of oxyhemocyanin, protein, and the ratio of oxyhemocyanin to protein, and develops metabolic acidosis in the hemolymph.     

Microbial Phytase Supplementation in Rohu, Labeo rohita, Diets Enhances Growth Performance and Nutrient Digestibility

Abstract.— A feeding trial was conducted for 60 d to study the effect of dietary microbial phytase on growth performance and nutrient digestibility of rohu, Labeo rohita , fingerlings. One hundred and twenty fingerlings (average initial weight: 9.17 g/fish) were equally distributed into five experimental tanks, each with four replicates. Five isonitrogenous (35%) and isocaloric (16.79 kJ/g) diets were prepared from plant-based ingredients, supplemented with microbial phytase at the level of 0, 250, 500, 750, and 1000 U/kg diets and fed to T₀, T₁, T₂, T₃, and T₄ groups, respectively. Weight gain %, food conversion ratio, protein efficiency ratio, and apparent net protein utilization were significantly ( P  < 0.05) improved in groups fed phytase-supplemented diets compared to control, the highest being observed in T₃ group. Maximum apparent digestibility of phosphorus and crude protein was recorded in T₃ group. Bone ash, phosphorus (P), and calcium (Ca) contents were also significantly ( P  < 0.05) increased in phytase-fed groups. However, maximum was recorded in T₃ group. Results from the present study indicated that addition of 750 U microbial phytase/kg diets effectively improved nutrient utilization, bone mineralization, and hence growth of L. rohita fingerlings.     

Biochemical Oxygen Demand in Channel Catfish Ictalurus punctatus Pond Waters

A study of the biochemical oxygen demand (BOD) of waters from ten channel catfish ponds at Auburn, Alabama, revealed that the 5-d BOD (BOD₅) seldom exceeded 8 mg/L and that the ultimate BOD (BOD_u) was usually less than 30 mg/L. Water samples from catfish ponds usually needed to be diluted only 2 or 3 times to permit BOD₅ measurements, and nitrification occurred even during a 5-d incubation period. Catfish pond waters were not extremely high in ammonia nitrogen concentration, and ammonia nitrogen introduced in the ammonium chloride-enriched dilution water caused an appreciable increase in BOD of some samples. Plankton respiration is a major component of carbonaceous BOD (CBOD) in catfish pond waters. Thus, the BOD is not expressed as rapidly during 5-d incubations as in typical waste-water. The ultimate BOD (BOD_u) would be a good measurement of oxygen demand for catfish pond effluents, but it is difficult to measure. Data from this study suggest that BOD_u can be estimated from BOD₅, but the correlation is not strong ( R ²= 0.62). An alternative is to develop a short-term BOD measurement specifically for effluents from channel catfish and other aquaculture ponds. This study suggests that a 10-d BOD conducted without nitrification inhibition or addition of ammonia nitrogen in dilution water might be a better alternative to standard BOD₅ or BOD_u measurements normally used in wastewater evaluation.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

Digestibility in goldfish fed diets with and without chromic oxide and exposed to sublethal concentrations of cadmium

Effects of three sublethal levels of cadmium (40, 120 and 260 μg L⁻¹) on apparent total dry matter (TDM) and apparent protein (PD) digestibility in goldfish, Carassius auratus (L.), over an 8-week experimental period were investigated using diets with and without chromic oxide (0.5%) marker. The %TDM and %PD were estimated based on crude fibre as the marker for the diet without Cr₂O₃, and Cr₂O₃ and crude fibre as markers for the diet with Cr₂O₃. Generally, digestibility estimations were lower in goldfish fed the diet with the Cr₂O₃ marker. Similarly, for the diet with Cr₂O₃ marker, digestibility estimates based on crude fibre were higher than those based on Cr₂O₃. In all treatments (2 diets × control and 3 cadmium levels), the weekly estimations of %TDM and %PD did not differ significantly ( P > 0.05). The effect of cadmium on weekly %TDM and %PD was not that obvious in all treatments. However, the overall mean %TDM and %PD differed significantly ( P < 0.05) between diets and between markers, at all cadmium concentrations. The present study raises doubts regarding the validity of the use of Cr₂O₃ as a marker in digestibility studies in fish, as had been recently suggested by other authors but for different reasons.     

Production of Sunfish Lepomis spp. in Ponds Treated with Controlled-Release Fertilizers

Abstract.– Controlled-release fertilizer in pond water released nutrients according to the pattern reported for exposure in soil. A single application of controlled-release fertilizer (13% N, 13% P₂O_5, 13% K₂O) was compared with multiple applications of conventional, liquid fertilizer (10% N, 34% P₂O₅, 0% K₂O) for augmenting sunfish production in ponds. Liquid fertilizer was used at an annual phosphorus input rate of 39.3 kg/ha; controlled-release fertilizer was applied at 19.7, 9.8, and 4.9 kg phosphorus/ha per yr. Phytoplankton abundance inferred from chlorophyll u and Secchi disk visibility estimates and phytoplankton photosynthesis measured by the oxygen, light-dark bottle method were similar in ponds treated with liquid fertilizer and in ponds that received controlled-release fertilizers at the two highest rates. Net sunfish production did not differ among ponds treated with 39.3 kg phosphorus/ha in liquid fertilizer or 19.7 and 9.8 kg phosphorus/ha applied in controlled-release fertilizer. Controlled-release fertilizer used in this study contained more nitrogen and potassium than needed, but a custom, controlled-release fertilizer with the proper ratio of nutrients can be made. This fertilizer would have the advantage of requiring only a single application at the beginning of the growing season, and amounts of soluble reactive phosphorus in outflow from ponds could be reduced by using controlled-release fertilizers.     

Improved Seed Production of Goldstriped Amberjack Seriola lalandi under Hatchery Conditions by Injection of Triiodothyronine (T3) to Broodstock Fish

Survival of the seed of goldstriped amberjack Seriola lalandi was consistently enhanced by intramuscular injection of triiodothyronine (T₃) to the broodstock fish at a dose of 20 mg/kg body weight. Survival rates at the time of transfer to net cages (35 d after hatching) were 2.6–5.0% for the T₃ treatment while only 0.3–2.1% for the control. Survival rates 36 mo after hatching was estimated to be 12 times higher in the T₃-treated group (1.1%) than in the control (0.09%). T₃ concentration in newly spawned eggs increased from control levels of 0.3–1.0 ng/g to 68–127 ng/g 2 d after injection and subsided with successive spawns. T₃ concentration in the treated larvae was about 360 times higher than in the control upon hatching (29 np/p vs 0.08 ng/g), but became indistinguishable from the control 6 d after hatching. These results indicate injection of T₃ to the broodstock of S. lalndi to be of practical value in aquaculture of the species.     

Density and Water Exchange-Dependent Growth and Survival of Litopenaeus setiferus Postlarvae

This present study was designed to investigate the effects of stocking density and water exchange on the growth rate, survival and performance index of L. setiferus postlarvae under controlled laboratory conditions. The experiment was done with postlarvae (PL10 to PL40) at densities of 50, 150, 250 and 350 shrimp/m² and various different water exchanges rate per day (0, 6, 12 and 18%). The maximum growth rate was obtained for shrimp with 12% water exchange per day at all densities. A reduction of the maximum growth rate was observed in relation to density with the highest values in shrimp stocked in a density of 50 and 150 shrimp/m² (mean value of 0.53 mg/d) and the lowest in shrimp stocked in a density of 350 shrimp/m² (0.24 mg/d). The multiple regression equation obtained to relate performance index (growth rate* survival : PI), shrimp density (X₁) and water exchange (X₂) was: PI = 0.31 + (0.001) X₁+ 0.039 X₂+ 2.28 × 10⁻⁶ X₁²+ (−0.0017) X₂²+ (0.000026)X₁X₂, R ²= 0.78; P > 0.03. According to this equation the optimum shrimp density-water exchange comhination was between 5 to 12% of water exchange at stocking density of between 50 and 150 shrimp/m². Salinity, ammonia-N and nitrite-N increased according to the time spent in tanks without water exchange. With no (0%) water exchange, water quality parameters measured were outside the optimum for L. setiferus postlarvae. The use of optimum density and water exchange in a nursery system for L. setiferus with optimum variables established is proposed.     

Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus

ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus . Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. K _m and k _cat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N -acetylchitopentaose (GlcNAc₅). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N -acetylchitooligosaccharides (GlcNAc_n, n  = 2–6). Both chitinases hydrolyzed GlcNAc_n ( n  = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc₄ > GlcNAc₅ > GlcNAc₆ for the 38 kDa chitinase, and GlcNAc₆ > GlcNAc₅ > GlcNAc₄ for the 42 kDa chitinase. Both the chitinases released p -nitrophenol from p -nitrophenyl GlcNAc_n ( n  = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.     

Nutrients apparent digestibility coefficients of selected protein sources for juvenile Siberian sturgeon (Acipenser baerii Brandt), compared by two chromic oxide analyses methods

Apparent digestibility coefficients (ADCs) of dry matter (ADC_d), crude protein (ADC_p), energy (ADC_e) and amino acids in selected feedstuffs were determined for juvenile Siberian sturgeon (8.38 ± 0.20 g). The tested feedstuffs were fishmeal (FM), meat and bone meal (MBM), poultry by-product meal, hydrolysed feather meal, fermented feather meal solvent-extracted cottonseed meal and soybean meal. ADCs were determined using a reference diet and test diets at 7 : 3 ratios with 5 g kg⁻¹ chromic oxide (Cr₂O₃) as an inert marker. Fish were reared in a recirculating system and fed to apparent satiation five times daily. Cr₂O₃ in diets and faeces samples were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) and acid-digestion colorimetry (AC) methods, respectively. The results showed that ICP-AES method was more accurate for Cr₂O₃ determination than AC method, and the results determined by ICP-AES method were used in this study. ADC_d and ADC_p of seven tested ingredients were lowest for MBM (59.1 and 84.5%) and highest for FM (79.9 and 94.5%); ADC_e of tested ingredients were from 71.8% for SECM to 93.2% for FM. ADCs of amino acid in test ingredients followed similar trend to the ADC_p. The ADCs of individual amino acids varied from 61.6% (histidine in MBM) to 98.8% (valine in FM).     

Toxicity of Un-ionized Ammonia and High pH to Post-larval and Juvenile Freshwater Shrimp Macrobrachium rosenbergii

Post-larval and juvenile Macrobrachium rosenbergii were exposed for 72 h at 29 C to four pH levels (8.5, 9.0, 9.5, 10.0) and four concentrations of un-ionized ammonia-nitrogen (0, 1, 2, and 3 mg/L NH_j-N). Results indicated potentiation between NH₃ and high pH. Juveniles were more tolerant of high pH and NH₃ than post-larvae.
For post-larvae, estimates of 72 h LC50 for pH were 9.43, 9.21, and 8.71 at 0, 1, and 2 mg/L NH₃-N, respectively; 72 h LC50 estimates for NH₃-N were 2.18 and 1.45 mg/L at pH levels of 8.5 and 9.0, respectively. For juveniles, estimates of 72 h LC50 for pH were 9.91, 9.56, 9.04, and 8.76 at 0, 1, 2, and 3 mg/L NH₃-N, respectively; 72 h LC50 estimates for NH₃-N were 2.02 and 0.54 mg/L at pH 9.0 and 9.5, respectively.
In pond culture of M. rosenbergii , high pH levels can cause mortality at stocking. The 72 h data can be used as an indication of safe stocking levels of pH and ammonia. These data suggest that post-larvae should not be exposed to pH > 9.0 nor to NH₃-N > 1 mg/L in the pH range 8.5–9.0 and juveniles should not be exposed to pH > 9.5 nor to NH₃-N > 0 mg/L at pH 9.5, > 1 mg/L at pH 9.0, or > 2 mg/L at pH 8.5.     

Acute LD50 and kidney histopathology following injection of erythromycin (Erythro-200) and its carrier in spring chinook salmon, Oncorhynchus tshawytscha (Walbaum)

We evaluated the toxicity of commercial injectable erythromycin (Erythro-200) and the injectable drug carrier alone (PEG-400, ethyl alcohol and ethyl acetate) in sub-adult spring chinook salmon, Oncorhynchus tshawytscha (Walbaum). Both compounds were toxic to salmon when administered in high dosages. The 50% lethal dose (LD₅₀) of the erythromycin within its carrier (Erythro-200) at 12 °C was estimated at 429 mg erythromycin kg^–1 or 2.145 mL kg^–1 when measured by volume. The LD₅₀ of the drug carrier alone was 2.95 mL kg^–1 by volume or equivalent to 3.21 g kg^–1 of the carrier. We evaluated and scored the histopathology in kidney sections removed from fish 96 h after injection with Erythro-200, the drug carrier alone, or sterile phosphate buffered saline (PBS) that served as a reference control. Kidneys from fish injected with saline had lowest average pathology scores; those injected with the carrier had moderate scores; and the pathology score was highest in tissues from erythromycin-injected fish. In tests at 12 °C, vacuoles were more prominent in proximal and distal tubules of fish injected with higher dosages of 403 and 545 mg erythromycin kg^–1 than in tissues from fish injected with 299 mg kg^–1.