Ultraviolet light disinfection of shellfish hatchery sea water: I. Elimination of five pathogenic bacteria

Filtered sea water was seeded with one of five oyster-pathogenic bacteria to obtain a final concentration of 10⁵ cells/ml. UV irradiation at a dose of 93 312–155 520 μW s^?1 cm^?2 rendered the contaminated sea water safe for rearing fertilized eggs of Crassostrea virginica. Fertilized oyster eggs were reared until the larvae began to set in filtered, UV-irradiated sea water and in filtered, non-UV-treated sea water. The data suggest that when a pathogenic bacterium is present the potential for disease is much greater in non-UV-treated sea water.     

Ultraviolet irradiation is an effective alternative to ozonation as a sea water treatment to prevent Kudoa neurophila (Myxozoa: Myxosporea) infection of striped trumpeter,Latris lineata (Forster)

Myxozoan parasites are known pathogens of cultured finfish. Kudoa neurophila n. comb. (Grossel, Dyková, Handlinger & Munday) has historically infected hatchery‐produced striped trumpeter, Latris lineata (Forster in Bloch and Schneider), a candidate species for seacage aquaculture in Australia. We examined the efficacy of four water treatment methods to prevent K. neurophila infection in post‐larval (paperfish) and juvenile striped trumpeter. Treatments included dose‐controlled ultraviolet irradiation [hydro‐optic disinfection (HOD)], ozone with conventional UV (ozone), mechanical filtration at 25 μm and then foam fractionation (primary filtration), and 50‐μm‐filtered sea water (control). In post‐larvae (initially 10.3 ± 2.7 g, mean ± SD, 259 days post‐hatching, dph), the infection prevalence (PCR test) after 51 days was 93 ± 12% in the control, 100 ± 0% in primary filtration and 0 ± 0% in both ozone and HOD. Likewise, in juveniles (initially 114 ± 18 g, 428 dph), prevalence was 100 ± 0% in the control and primary filtration treatments with no infection detected in ozone and HOD. Concurrently, there was a 50–100% reduction in heterotrophic bacteria and 100% reduction in presumptive Vibrio sp. in sea water HOD and ozone treatments. HOD with a dose of ≥44 mJ cm^?2 UV was as effective as ozonation at >700 mV ORP for 10 min, in preventing K. neurophila infection.     

Effect of ultraviolet (UV) radiation on the abundance and respiration rates of probiotic bacteria

Effects of ultraviolet radiation (UV) on probiotic bacteria (Bacillus subtilis and B. licheniformis) were tested in two experiments, with the following treatments: (i) UV treatment – using fluorescent and UV‐lamps and (ii) Control – CTRL, using fluorescent lamps. Bacterial abundance and respiration were evaluated every 24 h for 3 days for Experiment 1, and at 0, 6 and 24 h for Experiment 2. In the Experiment 1, total UV dose was 4 336.41 mW cm^?2. UV treatment presented small respiration rates only on day 3, while in the CTRL oxygen consumption was always high. On all days, the abundance of the Bacilli exposed to UV was significantly smaller than that of the CTRL. The second experiment, with total UV dose of 1 445.47 mW cm^?2, presented oxygen consumption in the UV treatment only during the first 6 h. In the CTRL, oxygen consumption increased from the beginning due to the bigger abundance Bacilli cells. Small coccus‐shaped bacteria ocurred in the UV treatment of both experiments. It may be concluded that exposure to UV, normally used for water disinfection, can inactivate probiotic bacteria.     

A single-dose pharmacokinetic study of oxolinic acid and vetoquinol, an oxolinic acid ester, in Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C

The pharmacokinetic properties of the antibacterial agent oxolinic acid were studied after intravenous, intraperitoneal and oral administration to 1.5–3.0 kg Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C. Following intravenous injection, the plasma drug concentration-time profile showed two distinct phases. The terminal elimination half-life was estimated to be 52 h, whereas total body clearance (Cl_T) was determined to be 0.044 L kg^–1 h^–1. The volume of distribution at steady state, V_d(ss), was calculated to be 3.0 L kg^–1, indicating good tissue penetration of oxolinic acid in Atlantic halibut. The peak plasma concentration (C_max) and the time to peak plasma concentration (T_max) were estimated to be 1.2 and 2.7 μg mL^–1, and 21.5 and 80 h, respectively, following oral administration of medicated feed or intraperitoneal injection. The corresponding bioavailabilities were calculated to be 15% and 92%, respectively. Oral administration of vetoquinol, the carbitol ester of oxolinic acid, increased the bioavailability of oxolinic acid to 64% and the total bioavailability (oxolinic acid + vetoquinol) to 82%, whereas C_max and T_max values of 6.7 μg mL^–1 and 14.5 h, respectively, for oxolinic acid, and 1.0 μg mL^–1 and 6.3 h, respectively, for vetoquinol were obtained. Based on a minimum inhibitory concentration (MIC) of 0.0625 μg mL^–1 for susceptible strains, a single intraperitoneal injection of 25 mg kg^–1 of oxolinic acid maintains plasma levels in excess of 0.25 μg mL^–1, corresponding to four times the MIC value, for ≈12 days. The corresponding values for a single oral dose of 25 mg kg^–1 of oxolinic acid and vetoquinol were 5 and 10 days, respectively. For resistant strains with a MIC of 1 μg mL^–1, a single oral dose of vetoquinol (25 mg kg^–1) maintained plasma levels in excess of 4 μg mL^–1 for 34 h.     

Contribution of microorganisms to the biofilm nutritional quality: protein and lipid contents

The nutritional quality of biofilm, a microbial community associated to an organic matrix, was evaluated in artificial substrate (polyethylene screen) in net cages during 30 days in the Patos Lagoon estuary, Southern Brazil. During this period, samples of biofilm were collected each 5 days for analysis of chlorophyll a, microorganisms abundance, dry weight, protein and lipid contents. During the study, chlorophyll a varied from 0.38 to 2.75 μg cm^?2; dry weight between 7.16 and 17.63 mg cm^?2; protein content from 0.43 to 1.76 mg cm^?2 and lipid concentration between 1.21 and 4.23 mg cm^?2. The variation of lipid in the biofilm was closely related to the abundance of free heterotrophic bacteria (34.25–56.54 × 10⁶ cells cm^?2), filamentous cyanobacteria (7.5–15.9 × 10⁶ filaments cm^?2), flagellates (6.92–12.89 × 10⁶ cells cm^?2) and mainly nematodes (29–1,414 organisms cm^?2), while protein content varied similarly to the abundance of unicellular centric diatoms (52.10–179.81 × 10³ cells cm^?2), and nematodes. This information will allow a better management of food supply to raised aquatic organism with the utilization of natural productivity in the culture systems, with considerable decrease in production costs.     

Variation in clearance and ingestion rates by larvae of the black-lip pearl oyster (Pinctada margaritifera, L.) feeding on various microalgae

Clearance rate (CR) and ingestion rate (IR) of different sizes (89, 125 and 188 μm shell length) of Pinctada margaritifera larvae were determined when feeding on various microalgae. The microalgae tested were the diatoms, Chaetoceros muelleri and C. simplex, and flagellates, Tahitian Isochrysis aff. galbana, Pavlova lutheri and P. salina at 5 or 10 cells μL^–1. Both CR and IR of microalgae tested in this study increased with increasing larval size; but at all larval sizes, diatoms resulted in lower CR and IR. Of the microalgae tested, P. margaritifera larvae showed greatest CR and IR with the two Pavlova spp. Maximum CR for P. salina was 10.5, 21.2 and 29.7 μL h^–1 for larvae with shell lengths of 89, 125 and 188 μm, respectively. The highest IR values for P. margaritifera larvae with shell lengths of 89, 125 and 188 μm were 8.7, 81.0 and 165.7 cells·larva^–1 h^–1, respectively. CR and IR of P. salina were approximately five times higher than those recorded for C. muelleri and C. simplex.     

Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC₅₀) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log₁₀ CFU mL^?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log₁₀ CFU g^?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log₁₀CFU mL^?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.     

Artificial gynogenesis in Cynoglossus semilaevis with homologous sperm and its verification using microsatellite markers

Effective methods for induction of gynogenetic diploids in Cynoglossus semilaevis are needed to initiate monosex culture. An effective protocol to induce half‐smooth tongue sole gynogenesis using homologous sperm was developed in this study. A UV dose of 50 mJ cm^?2 was found to be the most effective for genetic inactivation of tongue sole sperm. Treatment optima for cold shocks were 5 °C for 20–23 min at 5 min after fertilization and the hatching rate of gynogenetic diploids was 10.0%. Microsatellite analysis at locus Csou 6 revealed that there was no genetic contribution from the paternal genome in 24 progenies of a meiotic gynogenetic family. Polymerase chain reaction demonstrated that only four individuals of 24 meiotic gynogenetic diploids produced the female‐specific band of about 205 bp. The female/male ratio of gynogenetic diploids was significantly different from the theoretical ratio of 1:1. It is possible that there are some recessive lethal genes in W chromosome.     

Experimental evidence on prevention of infection by the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon using ultraviolet disinfection of rearing water

In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery‐reared juveniles. This study examined the susceptibility of the two parasites to wide‐ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small‐scale rearing system over a 28‐day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 10⁵ μW s/cm² for I. salmonis and ≥8.40 × 10⁵ μW s/cm² for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 10⁶ μW s/cm² succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery‐reared juvenile chum salmon.     

PCR-DGGE analysis of bacterial communities in <Emphasis Type="Italic">funazushi</Emphasis>, fermented crucian carp with rice,during fermentation

Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan. It is characterized by a unique cheese-like flavor and characteristic sour taste. We analyzed the changes in the microbial community during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCR-DGGE) and by plate counts. The plate counts showed that lactic acid bacteria reached 8.0 log₁₀ CFU/g within 7 days of fermentation initiation before decreasing slowly to 4.0 log₁₀ CFU/g during the remainder of 1-year study period. PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and 30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L. acetotolerans. This is the first identification of L. acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive. This is the first report of PCR-DGGE being used to assess the microbial community in funazushi. This technique was also found to be effective in profiling microbial diversity.     

Sustainable water treatment in aquaculture – photolysis and photodynamic therapy for the inactivation of Vibrio species

Species of the genus Vibrio have been recognized as one of the most significant pathogens in aquaculture farming, causing mass mortality of farmed stocks. Photodynamic Antimicrobial Chemotherapy (PACT) with singlet oxygen (¹O₂) has been identified as a powerful and sustainable water treatment method for pathogen eradication. In this study, the efficiencies of photolytic and photodynamic disinfection protocols were studied with two Vibrio species, Vibrio parahaemolyticus and Vibrio owensii. The selected microorganisms were successfully cultivated in marine broth and irradiations were performed with ~10⁸ bacteria mL^?1. Treated samples were monitored for bacterial regrowth for up to 7 days. Photolysis experiments were initially conducted with UV‐A, UV‐B for up to 2 h and visible (VIS) light for up to 24 h. Of these, only irradiation with UV‐B light for at least 45 min was efficient in controlling Vibrio. Irradiations with VIS light were subsequently repeated under PACT conditions in dose?response experiments with two water‐soluble porphyrins, [T₄(MePy)P] and [TPPS₄]. Disinfections of samples were successful for both porphyrin types at minimum concentrations of 10 μM and 24 h of irradiation.     

Contributions to the larval biology of the red-lipped conch, Strombus luhuanus L. 1758, with respect to seed production for mariculture

Spawning behaviour and embryology of the red-lipped conch, Strombus luhuanus L. 1758 (Strombidae, Gastropoda), was investigated from 4 April to 19 May 1991, at Okinawa, southern Japan. At the laboratory and at a water temperature of 22.5-23.5^oC, veliger larvae developed 92 h after spawning. In all, 2140 larvae were examined for morphometric data. Growth and development was monitored at different water temperatures (23, 28 and 33^oC), in natural sea water filtered through 150-μm, 60-μm and 1-μm screens and when fed various combinations of food organisms, namely Chaetoceros sp., Dunaliella sp. and Pavlova sp. The minimum duration of the pelagic period of the larvae was 14.5 days. Infestation by parasites was the main cause of high larval mortality before the age of 10-12 days if the water was not filtered at a minimum of 60 μm. Inappropriate food diversity was the most significant source of mortality beyond this age. The maximum age reached during all rearing experiments was 16 days. Under optimized feeding conditions and in natural sea water filtered at 1-60 μm, the pelagic period of S. luhuanus larvae lasted 16.5 to 17.4 days (95% confidence limits). Optimum water temperature was 23-28^oC. A stepwise increment of filter sizes and a contemporary provision of a combination of specific supplementary food organisms is advised through grow-out of the larvae.     

A new approach to kelp mariculture in Chile: production of free-floating sporophyte seedlings from gametophyte cultures of Lessonia trabeculata and Macrocystis pyrifera

Substantial amounts of Macrocystis and Lessonia are traditionally harvested and exported from Chile as raw material for alginate. Because of intense mariculture of abalone (Haliotis ssp.), herbivorous molluscs that feed on brown kelps, pressure on local populations of Macrocystis and Lessonia has increased to critical levels within the past 5 years, strongly supporting efforts to produce algae maricultured biomass. Here, we present our results on the development of new techniques for large‐scale kelp mariculture in Chile. We have abandoned the traditional technique of direct spore seeding onto inoculation lines. Instead, we used gametophyte cultures that were manipulated to enter gametogenesis and to produce synchronous batches of 10⁴–10⁵ embryos. Juvenile sporophytes were cultured under permanent aeration and agitation, floating unattached in contamination‐free glass bottles up to 10 L, plexiglass cylinders and 800 L greenhouse tanks. When holdfast initials were formed at a size of 8 cm, the sporophytes were spliced into Nylon rope fragments and transferred to the sea. Twelve months after initiation of gametogenesis in the laboratory, Macrocystis pyrifera attained 14 m length and 80 kg fresh weight m^?1 line in the sea. For Lessonia trabeculata 6 months after gametogenesis initiation, 0.25 kg fresh weight m^?1 was attained in the sea.     

Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

Induced spawning of Asian catfish, Clarias batrachus (Linn.): effect of various latency periods and SGnRHa and domperidone doses on spawning performance and egg quality

An experiment was conducted to induce ovulation in Asian catfish, Clarias batrachus, by a single injection of SGnRHa (d ‐Arg⁶, Trp⁷, Leu⁸, Pro⁹, Net) in combination with domperidone. The effects of latency periods, 11, 14, 17, 20 and 23 h, and doses of inducing agent, 10 μg SGnRHa+5 mg domperidone, 20 μg SGnRHa+10 mg domperidone, 30 μg SGnRHa+15 mg domperidone and 40 μg SGnRHa+20 mg domperidone kg^?1 body weight, were studied on the total egg output, stripping response, fertilization, hatching and normal larval production. The highest (P<0.05) number of eggs were stripped at 23 h of post injection of 20 μg SGnRHa+10 mg domperidone kg^?1 female body weight. The highest (P<0.05) stripping response was observed when the females were stripped at 20 and 23 h latency, at all dose levels of the inducing agent. The eggs stripped at 11 h latency did not fertilize, and hence did not hatch irrespective of administration of any dose levels of the inducing agent. The fertilization and hatching per cent of eggs had significantly increased (P<0.05) with increase in latency period to 14–23 h at a dose of 20 μg SGnRHa+10 mg domperidone. The latency period of 14–17 h, and dose of 20 μg SGnRHa+10 mg domperidone and 30 μg SGnRHa+15 mg domperidone kg^?1 of female, was found to be suitable to obtain best spawning performance, and good‐quality egg and larval production in C. batrachus.     

Optimizing stocking density and microalgae ration improves the growth potential of tropical black‐lip oyster,Saccostrea echinata,larvae

The combined effects of stocking density and microalgae ration on survival and size of Saccostrea echinata larvae were studied in two‐factor experiments for the major developmental stages: D‐veliger (1‐day posthatch [dph], Experiment 1), umbonate (12 dph, Experiment 2), and eyed (19 dph, Experiment 3) larvae. Larvae were stocked into replicate sets of four 10‐L aquaria with ambient 1‐μm filtered sea water (28 ± 1.5°C and 36 ppt) and cultured for four days at densities of 0.5, 2, 5, 7, or 10 larvae/mL and provided with microalgae rations at each of five densities (cells larvae^?1 day^?1); 0, 1, 3, 5, or 8 × 10³ (D‐veliger larvae, Experiment 1); 0, 5, 12, 18, or 25 × 10³ (umbonate larvae, Experiment 2); and 0, 15, 30, 40, or 60 × 10³ (eyed larvae, Experiment 3). Microalgae rations for each larval life stage were selected on the basis of increasing food requirement with larval size and comprised a 2:1:1 mixture of Chaetoceros calcitrans, Tisochrysis lutea, and Pavlova spp., calculated on an equal dry‐weight basis. Contour plots were generated from larval survival and larval size (dorso‐ventral measurement [DVM]) data to determine optimal culture conditions. Larvae showed high survival (54–100%) over a wide range of both treatment parameters across all life stages, confirming broad tolerance limits for this species. The interaction effects of larval stocking density and microalgae ration on larval size were significant (p < 0.001) across all life stages. Results indicate that maximum larval size (DVM) is achieved when S. echinata are cultured at: 6–8 larvae/mL and fed 5–6 × 10³ cells larvae^?1 day^?1 for D‐veligers (mean DVM >80 μm), at 2–8 larvae/mL and fed 11–25 × 10³ cells larvae^?1 day^?1 for umbonate larvae (mean DVM > 190 μm), and at 1–4 larvae/mL and fed 15–40 × 10³ cells larvae^?1 day^?1 for eyed larvae (mean DVM >230 μm). Results will help refine current hatchery methods for S. echinata supporting further development toward commercial aquaculture production of this species.     

Towards sustainable exhibits – application of an inorganic fertilization method in coral reef fish larviculture in an aquarium

Coral reef fish are collected from the wild and exhibited in aquaria worldwide. Some of the fish spawn in captivity; however, the eggs are usually neglected. In this study, we collected the eggs spawned naturally in the exhibit tanks, hatched and cultured them indoor in 2000‐L fibreglass tanks (initial density = 18 000 egg tank^?1). We applied an inorganic fertilization method commonly used in freshwater fish culture in raising these coral reef fish larvae. We maintained inorganic phosphorus concentration at 100 μg P L^?1 and inorganic nitrogen at 700 μg N L^?1 daily in the fertilized group (n = 4), while the control tanks (n = 4) were fed with rotifers (10 ind mL^?1). Chlorophyll a at particle sizes of both 0.45–20 μm and >20 μm, as well as NH₃‐N, NO₃‐N, and PO₄‐P concentrations were significantly higher in the fertilized group than the control. Zooplankton in the size groups of 10–50 μm (mainly flagellates) and 50–100 μm (mainly ciliates) were abundant (about 10~60 ind mL^?1) during 3–7 days in fertilized tanks. The average larval fish survival rate at 21 day after hatch in fertilized group was consistently higher than the control in two trials. The experiments demonstrated that the inorganic fertilization approach can be successfully adapted for coral reef fish culture in an aquarium to achieve sustainable exhibits.     

Larval calcification and growth of veligers to early pediveliger of the queen conch Strombus gigas in mesocosm and laboratory conditions

The goals of this study were to evaluate growth, development, and calcification process of veligers of Strombus gigas grown in natural conditions in mesocosm versus laboratory conditions. In this study, larvae bred in mesocosm conditions had a good growth rate (33.3±12.40 μm.day^?1) when fed with natural phytoplankton in natural flowing seawater versus a lower growth rate of 8.8±5.20 μm.day^?1 for larvae reared in laboratory conditions and fed with a monoalgal diet of Nannochloropsis oculata. Physicochemical parameters did not explain the difference on larval growth in both culture systems according to the principal component analysis done. Raman microspectrometry carried out on conch larvae grown in mesocosm and lab conditions allowed us to emphasize the biosynthesis of calcium carbonate species and their structure type evolution (amorphous, aragonite, or calcite structure) as a function of the breeding time by detecting and identifying in the spectra the characteristic Raman bands of CO₃ chemical groups and lattice vibrations. This analytical method seems to indicate that crystalline CaCO₃ structures are not detected in the recorded spectra on larvae reared in laboratory conditions during the early stages of the shell construction of 1 to 8 days. For this reason, light and current flow have also been considered in the discussion that can help to explain the differences found in this study. Contrarily, the Raman spectra acquired on larvae grown in mesocosms exhibit characteristic bands of aragonite (CO₃ double peak at 697–701 cm^?1 and lattice vibrations at 170 and 220 cm^?1) from the third day of breeding. The best shell growth and shell calcification pointed out in larvae grown in mesocosm compared to laboratory conditions are probably due to the nutrient amounts present in the food available in mesocosm.

     

Toxicity of the aquaculture pesticide cypermethrin to planktonic marine copepods

The acute and sublethal toxicity of cypermethrin, the active ingredient in the sea lice treatment formulation Excis^®, to non‐target planktonic marine copepods was determined. The comparative sensitivities of three life stages (nauplii, copepodites, adults) of four common marine copepods (Acartia clausi, Pseudocalanus elongatus, Temora longicornis and Oithona similis) were assessed in 48‐h exposures, followed by a recovery period in toxicant‐free sea water. The cyclopoid copepod, O. similis, was most affected by cypermethrin, with EC₅₀ values ranging from 0.14 to 0.24 μg L^?1 for nauplii and adults respectively. With the exception of T. longicornis nauplii, the calanoid copepods (A. clausi, P. elongatus and T. longicornis) responded similarly to cypermethrin. Overall, 48‐h EC₅₀ values ranged from 0.12 μg L^?1 (T. longicornis nauplii) to >5 μg L^?1 (P. elongatus adults). For all species, nauplii and copepodite EC₅₀ values were lower than those of the adults. The primary toxic effect, immobilization, was generally irreversible. A sublethal test with adult A. clausi females, involving pulse exposures over 4 days measured a significant increase in egg production at the higher concentrations (1.58 and 5 μg L^?1). Concentrations causing acute toxicity to planktonic copepods were lower than the recommended sea lice treatment concentration of 5 μg L^?1 cypermethrin, indicating the potential for toxic effects in the field. However, acute toxicity values were higher than the Environmental Quality Standard of 0.016 μg L^?1 for dispersing treatment plumes, suggesting that cypermethrin released to the marine environment following sea lice treatments is unlikely to affect adversely planktonic copepods.     

Determination of minimal concentration of Piscirickettsia salmonis in water columns to establish a fallowing period in salmon farms

A highly sensitive real‐time PCR procedure to detect and quantify the number of Pisciricketsia salmonis units in seawater samples from affected farm sites has been developed. The purpose was to determine a fallowing period that would allow safe restocking of the target farm with new fish. Bacterial load was determined in water samples by comparing the obtained amplification values against a standard curve generated by the amplification of known concentrations of the ITS‐ribosomal component of P. salmonis DNA, cloned in a suitable vector. The standard curve was linear over the range of 10¹–10¹⁰ log units. Target samples were taken every 10 days over a 40‐day period, at 5 m depth and at the surface. In a highly affected area of southern Chile, the number of bacterial units in farm water decreased to zero at day 50. Therefore, a fallowing period of 50 days post‐removal of cages of affected fish appears to be appropriate before restocking. This procedure could be adapted to control disease problems because of other pathogens in fish farm waters.