Responses of Toxic Algae to Environmental Variables in the Pearl River Estuary and Hong Kong Waters

珠江口和香港水域中的有毒藻類在環境因子變化下的響應研究

Student thesis: Doctoral Thesis

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Award date9 Oct 2019

Abstract

Toxic algae are algal species that are fast-growing under favorable conditions and are capable of producing bio-toxins, which can result in harmful algal blooms (HABs). They can have a direct impact on their competitors, consumers, and ultimately the marine ecosystem as a whole. HABs can cause large-scale fish kills, and severely poison marine mammals and humans following inhalation of algal toxins or consumption of contaminated seafood. HABs have occurred worldwide with increasing frequency in the past few decades. Toxic algae have been emerging threats to the marine ecosystems and human health.

Algal toxins are secondary metabolites produced by toxic algae. They can accumulate in clams, fish and cetaceans, and their effect can be biomagnified through the marine food web. Based on the chemical properties of various algal toxins, they can be classified as hydrophilic algal toxins (HATs) or lipophilic algal toxins (LATs). LATs include the okadaic acid (OA) group, pectenotoxin (PTX) group, yessotoxin (YTX) group, azaspiracid (AZA) group, brevetoxin (BTX) group, gymnodimine (GYM) group, and spirolide (SPX) group. They are produced by dinoflagellates mainly and are responsible for diarrhetic shellfish poisoning, neurologic shellfish poisoning, ciguatera fish poisoning, and azaspiracid shellfish poisoning. These algal toxins have been detected in algae, shellfish, seawater, and sediment samples around the world.

In recent decades, the responses of toxic algae to various environmental variables has attracted increasing attention from researchers. It has been reported that algal distribution has spatial and temporal variations. The physiological processes and the toxin production in algae can be affected by different environmental conditions. Many physical, chemical and biological factors, such as temperature, salinity, pH, light, nutrients, pollutants, water movement, and bacteria, can affect algal growth and toxin production in algae. But the mechanisms of algal toxin production are still largely unknown. Studying the responses of various toxic algae under different environmental conditions could provide useful information on possible effects of climate change on toxic algae. This would also contribute to understanding the toxin-producing mechanisms in toxic algae.

In previous studies, toxic algae that can produce LATs were found in the Pearl River Estuary (PRE) and Hong Kong (HK) waters. In HK waters, Coolia malayensis has been found as the dominant benthic dinoflagellate, especially in winter. They are toxic to Artemia larvae according to previous research. However, there is no information on the toxin levels in the environmental samples in the PRE and HK area, and there has been little research on how the toxic algae in this area respond to variations in environmental conditions. Therefore, this study included studies on field samples and controlled trials in the laboratory to verify the responses of toxic algae to environmental variables.

The present study had four main aims: (1) to verify the toxins produced by toxic algae from HK waters; (2) to examine the levels of typical LATs in seawater samples collected in the PRE and HK waters; (3) to reveal the major environmental variables affecting toxic-algae activities in the PRE and HK waters; and (4) to determine the effects of temperature and salinity on the growth, toxicity, and toxin production of a certain toxic algal species.

In this study, a robust liquid chromatography−mass spectrometry (LC-MS) method was employed to quantify nine typical LATs - OA, dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), YTX, AZA1, AZA2, PTX2, GYM, and SPX - in algal and seawater samples, coupled with effective pre-treatment methods based on published methods. The relationship between environmental variables and toxin distribution in the PRE and HK waters was studied via multivariate analysis. The effects of temperature and salinity on the physiology (growth curves, maximum quantum yield of PSII, phaeo-pigments), toxicity (brine shrimps bioassay), and toxin production (putative LATs in algal extracts) of HK local species C. malayensis were investigated in controlled experiments.

The typical LATs in monocultures of 14 benthic algal strains collected from HK coastal waters were quantified using the LC-MS method. The algal species included four strains of Ostreopsis cf. ovata, two strains of C. malayensis, one strain of C. tropicals, one strain of C. canariensis, one strain of Fukuyoa ruetzleri, one strain of Amphidinium thermaeum, one strain of Prorocentrum mexicanum, one strain of Prorocentrum dentatum, one strain of Prorocentrum cf. lima, and one strain of Prorocentrum sp. HK Type 2. The algal strain of Prorocentrum cf. lima was confirmed with toxin production of OA, DTX1, and five analogues of OA group toxins. OA and DTX1 were also detected in one C. malayensis strain. A putative analogue of YTX was detected in another C. malayensis strain, and a putative AZA2 analogue was detected in the strain of F. ruetzleri.

The levels of typical LATs in seawater samples collected from 60 sampling locations in the PRE and HK waters were investigated. Five types of LATs were detected in the seawater samples: OA, DTX1, YTX, PTX2, and GYM. The highest average levels of OA, DTX1, GYM, and total toxins were observed in the eastern PRE waters, while the highest average levels of PTX2 and YTX were found in HK waters. Distance-based linear model analysis was used to assess the relationship between various environmental variables and toxin distribution. The results showed that salinity played the most important role in the toxin distribution of the PRE waters. Besides, dissolved oxygen, chlorophyll a, bacteria community richness, and dissolved organic carbon also have significant relationships with toxin compositions. In HK waters, soluble Si, temperature, and salinity showed significant relationships with toxin compositions, especially soluble Si in seawater. Thus, the diatoms in HK waters might largely affect the toxin distribution in this area. Two environmental parameters, salinity and temperature, were selected for laboratory experiments.

The responses of C. malayensis to temperature variations were studied at a range of 16−28℃. The highest algal abundance and specific growth rate of this strain were recorded at 24℃. Significantly higher Fv/Fm (maximum quantum yield of PSII) and total phaeo-pigments value were observed in the log phase of C. malayensis cultures under temperature treatment of 28℃. The toxicity of C. malayensis had a significant positive correlation with Fv/Fm values in the log phase, but it did not quite correlate with its growth features. The highest toxicity was recorded at 28℃. A putative OA analogue and two AZA analogues were detected in C. malayensis extracts under seven temperature treatments. Interestingly, the highest toxin contents were all detected at 26℃.

The responses of C. malayensis to salinity variations were investigated in a range of 20−40‰. The highest growth rate of C. malayensis occurred at 35‰, while the highest total phaeo-pigment amount in the log and stationary phases occurred at 40‰ and 20‰, respectively. The changing trends of total pigments in the log phase was significantly different from that in the stationary phase along with changes in salinity. The pigment amounts in the log phase were higher than in the stationary phase generally. C. malayensis showed higher toxicity under high-salinity condition (40‰) and lower toxicity under low-salinity condition (20‰) comparing to middle-salinity conditions (25−35‰). The putative OA and AZA1 analogues observed in temperature exposure experiments had not been detected in salinity exposure experiments.

In summary, in the present study, four out of 14 algal species were detected with LATs and putative LAT analogues: Prorocentrum cf. lima (OA group), C. malayensis (OA, YTX, and AZA group), and F. ruetzleri (AZA group). Five types of LATs (OA, DTX1, YTX, PTX2, and GYMA) were detected in seawater samples collected from the PRE and HK waters. The distribution patterns varied among toxins. Salinity played the most important role in toxin distribution of the PRE waters, while diatom might largely contribute to the toxin distribution in HK waters. In laboratory experiments, C. malayensis could survive at a temperature range of 16−28℃ and a salinity range of 20−40‰, with significant effects observed on the physiological activities and toxicity. Under elevated temperature and salinity conditions, the toxicity of C. malayensis to Artemia larvae increased. The toxin production of C. malayensis might closely related to pigment-related physiological activities in log phase like photosynthesis. It is highly probable that under climate change conditions, the growth and toxicity of C. malayensis will change accordingly. The continuous monitoring of OA and related toxins in seawater is highly recommended. There is also a need for further studies on responses of other toxic algal species to typical environmental variables in this area especially under the climate change scenarios.

    Research areas

  • lipophilic algal toxins, Pearl River Estuary, Hong Kong, temperature, salinity, Coolia malayensis