Alejandra Llanos-Rivera

The effect of the combination of marine toxins produced by algal blooms, in conjunction with varying environmental characteristics on organisms in the water column, is a poorly explored research field. Pelagic fish species of commercial importance, such as anchoveta (Engraulis ringens) in central Chile, may be exposed to these combined factors in a climate change scenario. This is observed from documented changes in the length of the upwelling season, frequency of upwelling events, and the increased frequency of atmospheric rivers affecting the southern spawning zone of this species. This study evaluated the integrity of hair cells in neuromasts, mechanosensory organs present in fish larvae, under exposure to different combinations of the algal bloom-produced okadaic acid OA (1 ng mL⁻¹), and two temperature (12 and 14°C), and salinity treatments (historically more frequent: 34 PSU- 12°C; expected: 32 PSU - 14°C). Viable hair cells were counted in newly hatched larvae from the Biobío region, central Chile. Results showed a significant decrease in the average number of viable hair cells per neuromast (from 6.1 ± 1.6 to 4.0 ± 1.2) under lower salinity treatments (32 PSU) compared to normal conditions. Additionally, a seasonal trend was observed with fewer viable cells (from 7.4 ± 1.2 to 4.4 ± 1.1) as the fish species’ reproductive period progressed. The combined effect of OA exposure and modifications with the environmental factors also resulted in a significant decrease of up to 70% in the number of viable hair cells in larvae exposed to OA and high temperatures, indicating damage influenced by the toxin along with a synergistic and/or additive role of temperature. These findings reveal how the lipophilic toxin okadaic acid, produced by harmful algal blooms, interacts with abiotic environmental factors affecting coastal ecologically and socio-economically important organisms. This emphasizes the need to consider multiple factors when studying the effects of marine toxins.

The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1–0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

Despite the invaluable role of anesthetics as a tool for ensuring animal welfare in stressful situations, there is currently a lack of anesthetic drugs that meet the requirements of intensive aquaculture. In response to the growing interest in anesthetic substances of natural origin, this study evaluated the physiological and health impact of an anesthetic based on an extract of the microalga Heterosigma akashiwo on juvenile salmon (Salmo salar) exposed for a period of 72 h. To simulate a condition closer to reality where fish are subjected to stimuli (e.g., transport), the animals were exposed to 50 mg L⁻¹ of algal extract and to physical stress. Functional, physiological, and histological parameters were evaluated in blood and tissues at different sampling periods (0, 24, and 72 h). There was no mortality and the induction and recovery times observed were within the established criteria for anesthetic efficacy. The anesthetic extract did not induce any side effects, such as stress or metabolic damage, indicating that this extract is a viable option for supporting fish welfare during deleterious events. This study provides information to support that the anesthetic extract tested, derived from H. akashiwo, is a promising candidate drug for operations requiring sedation (e.g., Salmonid transport).

The anchoveta (Engraulis ringens) and sardine (Strangomera bentincki) are coastal pelagic species with important spawning areas off the coast of Chile. The discharge of secondary-treated effluents from a kraft pulp plant near one of these spawning areas has raised environmental concerns. Therefore, effluent effects on the development of anchoveta and sardine eggs were assessed by in vitro exposure. Eggs were sampled between 2007 and 2010 off Talcahuano, Chile. Subsequent toxicity tests (96 h duration, 12 °C) were performed using increasing effluent concentrations, a filtered seawater control, and two potassium dichromate concentrations (to verify consistent embryonic sensitivity). Egg mortality and hatching success were evaluated. For anchoveta, mortality (9.9 ± 7.1%) did not significantly differ among groups in five toxicity tests except the final toxicity test that showed significant differences in mortality (5.6% control vs 27.8% in 100% effluent). For sardines, no differences in mortality existed between the effluent dilutions (2.6 ± 3.6%) and control (6.3 ± 3.9%). Notably, anchoveta egg survival and hatching success rates were inconsistent, i.e., the highest rates of hatching failure occurred on the same sampling date with the highest rates of survival for the 100% effluent group (72%). In conclusion, the obtained results indicate that (i) anchoveta egg mortality and hatching failure increase only under 100% effluent exposure, coinciding with decreased egg quality near the end of spawning season and (ii) high effluent dilutions not significantly increase sardine and anchoveta egg mortalities. Nevertheless, the recorded adverse effects to the hatching process should be studied in greater detail, particularly considering interspecific variability and the complexity of reproductive processes, especially during early development.

Fish oil is used in the production of feed for cultured fish owing to its high polyunsaturated fatty acid content (PUFA). The over-exploitation of fisheries and events like “El Niño” are reducing the fish oil supply. Some marine microorganisms are considered potentially as alternative fatty acid sources. This study assesses a strain of Rhodotorula sp. (strain CNYC4007; 27% docosahexaenoic acid (DHA) of total fatty acids), as feed for fish larvae. The total length and ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) ratio of Danio rerio larvae was determined at first feeding at six and 12 days old (post-yolk absorption larvae). Larvae fed with microencapsulated Rhodotorula sp. CNYC4007 had a significantly higher RNA/DNA ratio than control group (C1). At six days post-yolk absorption group, the RNA/DNA ratio of larvae fed with Rhodotorula sp. bioencapsulated in Brachionus sp. was significantly higher than control group fed with a commercial diet high in DHA (C2-DHA). Finally, at 12 days post-yolk absorption, the RNA/DNA ratio was significantly higher in larvae fed with Rhodotorula sp. CNYC4007 and C2-DHA (both bioencapsulated in Artemia sp. nauplii) than in control group (C1). These results suggest that Rhodotorula sp. CNYC4007 can be an alternative source of DHA for feeding fish at larval stage, providing a sustainable source of fatty acids.

It has been proposed that current levels of ultraviolet B (UVB) radiation could cause lethal or sublethal effects on fish embryos located in the upper layers of the water column. Observed levels of UVB off central Chile (36°S, 73°W) indicate that planktonic fish embryos could be exposed to harmful UVB radiation. From July 2011 to January 2012 embryos from anchoveta (Engraulis ringens) and common sardine (Strangomera bentincki) were used to test experimentally whether the UVB levels in central Chile produce lethal or sublethal effects in epipelagic fish embryos. Simultaneously, whether the embryos might be exposed to harmful UVB levels in the field was investigated. Our experimental results show that UVB may cause a decrease in hatching success, changes in buoyancy and embryonic malformations. These results, along with the observed vertical distribution of embryos and UVB radiation levels in the field during late spring suggest that lethal and sublethal effects may be occurring in the embryos of both species.

Variations in the rate of embryonic malformations of the anchoveta Engraulis ringens in a spawning area off central–southern Chile were assessed. Eggs in stages of development subsequent to blastodisc formation were collected and preserved. Several severe malformations were identified and grouped by: (1) proliferation of disorganized tissue in early and late developmental stages; (2) irregular blastopore closure; and (3) notochordal deformation, of which the second had the highest incidence among the embryos. The average rate of malformation was 5.64%, a value close to that reported for other Clupeiformes and below the rate of 10%, indicating the influence of purely endogenous factors. The expected relationship between temperature variations and malformation rates was not observed, probably because the natural temperature range experienced by the embryos was above that at which severe malformation had previously been observed in the laboratory. The relationship between egg size and malformation rates over the spawning season was not determined; however, a relationship between egg size and malformation rates was detected among the samples from each sampling date, which suggests that malformations may be attributable to endogenous factors rather than to environmental factors acting directly on the eggs in the plankton.

A series of six 96-h static bioassays were performed to validate the use of anchoveta (Engraulis ringens) embryos as test organisms for ecotoxicological studies. The standardization protocol utilized potassium dichromate (K₂Cr₂O₇) as a reference toxicant and egg mortality as the endpoint. The results indicated that the mean sensitivity of anchoveta embryos to potassium dichromate was 156.1 mg L⁻¹ (range: 131–185 mg L⁻¹). The statistical data analysis showed high homogeneity in LC50 values among bioassays (variation coefficient = 11.02%). These results demonstrated that the protocol and handling procedures implemented for the anchoveta embryo bioassays comply with international standards for intra-laboratory precision. After secondary treatment, an effluent from a modern Kraft pulp mill was tested for E. ringens embryo toxicity, finding no significant differences from the controls.