EFFECT OF ENVIRONMENTAL FACTORS ON CHRYSOCHROMULINA GROWTH; THE MOST COMMON CAUSE OF FISH MORTALITY IN JEDDAH

Ftoon Ashour^1*, Fotoon Sayegh^1-3

Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Marine Natural Products Research Unit, King Fahd Medical Research Centre, King Abdul Aziz University, Saudi Arabia. Environmental -Deputy, Saudi Ministry of Environment Water and Agriculture (MEWA).

---

ABSTRACT

A phytoplankton bloom, dominated by the prymnesiophyte Chrysochromulina sp., developed in two areas of the red sea coast, Al-Nawras and Al-Arbaeen lagoons (Jeddah, Saudi Arabia) in the fall of October and December 2016. Chrysochromulina sp. dominated at total cell densities of average 3×10⁶ cells.ml^-1, and have caused variable degrees of mortality in fish. Fish gills were examined under a microscope and showed the presence of Chrysochromulina sp cells. This study was conducted to investigate environmental factors that affect the growth characters as a function of different salinity, pH, temperature, and light-regime (light duration and intensity). Light-regime showed the highest effect among all the factors tested, at 10:14h L:D for the light duration with a growth rate of (0.352 μ.d^-1), dry weight of (1552.601 ng), production per dry weight of (542.613 ng.d^-1), and chlorophyll-a content (0.221µg.ml^-1), as for light intensity, the best results were at around 60 to 70 µmol with a growth rate of (0.426 μ.d^-1), dry weight of (2213.086 ng), production per dry weight of (929.419 ng.d^-1), and chlorophyll-a content of (0.205 µg.ml^-1). Other factors were best at 15°C, 40 PSU, and 7 to 8 for the temperature, salinity, and pH, respectively. No acute toxicity was present. Therefore, the fish mortality was most likely related to the morphological aspect of the isolated species by clogging of fish gills accompanied with suitable environmental conditions, grazing and a very low dissolved oxygen level in both lagoons

Keywords: Chrysochromulina, Fish Mortality, Haptophyta, Harmful Algae, Prymnesiophyte, Nanoplankton cell

---

Introduction

On Wednesday 12/ October, fall of 2016, a bloom of Chrysochromulina sp. occurred along the coast of Al-Nawras lagoon in Jeddah city, Saudi Arabia. The bloom killed thousands of sardine fish that entered the bay at the time and suffocated. Another bloom happened on Friday 2/ December 2016, along the coast of Al-Arbaeen lagoon in Jeddah city, Saudi Arabia. It also killed larger fish (milkfish and Tilapia) and some crabs. The Prymnesiophyta or Haptophyta are a group of uninucleate flagellates characterized by the presence of a haptonema between two smooth flagella. The prymnesiophyte genus Chrysochromulina presently embraces about 60 formally described species [1]. Chrysochromulina species have been found to make up 45 to 73.5 % of the identified species, and 2.5 to 50 % of the total number of nanoplankton cells [2]. Blooms threaten the environmental or public health and the fast development of economy, industry, and social life is one of the factors affecting negatively the environment. Sewage input into the coastal areas is the major problem along the coast of Saudi Arabia [3]. Jeddah is a major coastal city that has a network to collect municipal wastewater [4]. Some HABs are harmful by virtue of their sheer biomass, whereas some are capable of producing toxins. Other species are non-toxic to humans but harmful to fish and invertebrates (especially in intensive aquaculture systems) by damaging or clogging their gills [5, 6]. The aim of this study was to investigate several environmental factors in order to reveal possible effective factors in growth and bloom formation (Figure 1).

a)

b)

Figure 1. a) Fish kills at Al-Nawras lagoon on 12 October 2016. b) Fish kills at Al-Arbaeen lagoon on 2 December 2016.

Materials and Methods

Samples collection

Samples were obtained in the fall of 2016 (October and December) from surface water during blooms at the Red Sea coast in Al-Nawras lagoon on 12 October 2016 and in Al-Arbaeen lagoon on 2 December 2016. Using a water sampling bottle. The cells were examined using an Olympus inverted microscope equipped with a digital camera.

Stock culture

After sample collection the isolation was achieved with the serial dilution culture method, using the standard pipette dilution. [7]. The batch culture was grown in a 1-L Erlenmeyer flask with IMR medium [8]. All cultures were grown under laboratory conditions at 22-23 ˚C and a continuous light using Extreme Cool Daylight tubes (PHILIPS TL-D 18W) (Figure 2).

 

a)	b)
Figure 2. Culture flask.

Growth measurement

Three replicates of all samples were used for measurement of dry weight by filtering 300 ml of microalgae, using glass microfiber filter 47 mm (Whatman). The growth rate was measured daily by using a Beckman Coulter Counter Multisizer™3, following the equation:

(1)

Growth rate (GR) was calculated as:

 

(2)

Cell volume was estimated at harvest day using the following equation:

 

(3)

Dry weight was estimated at harvest day by filtering 300 ml through Whatman (GF/F) glass fiber filter size 47 mm (pre-weighted). After filtration, samples were rinsed with cold sterilized distilled water. The filters were dried in an oven (JSR) at 80 ˚C for 4h and then weighed (3 times).

(4)

Dry weight per cell (ng) was calculated by:

 

(5)

For production per dry weight (ng.d^-1) was calculated by:

 

(6)

For chlorophyll-a content determination, 100ml culture was filtered using 25mm Whatman GF/C filters. A solvent was added (90% acetone) and grounded with mortar and pestle until it appeared colorless. After preparation, the extract was centrifuged at 4000 rpm for 10 minutes on a Hettich centrifuge MIKRO 220R. After extraction, the absorbance of the solvent extract was measured at the given wavelengths, between the range of 400 and 700 nm against a solvent blank on a Genesys 10S UV/VIS spectrophotometer [9]. Chlorophyll content was estimated using the following SCOR-UNESCO (1966) equations:

Chlorophyll a:

μg chlorophyll/ml medium = (11.64A663 – 2.16 A645 + 0.10A630) v/ lV

where is:

Axxx = the absorbance at xxx nm, after removing the sample absorbance at 750 nm against a blank of the solvent used.

v = the volume of acetone used (ml).

l = the spectrophotometric cell length (cuvette) (cm).

V = the sample volume (ml).

Physical analysis

The methodology of the physical factors experimentation and the toxicity test were according to [10-13]. Five factors were selected (temperature, pH, salinity, light duration, and light intensity) including five measurements degrees. These factors were measured directly after the sample collection using Apera PC60 multi-parameter, a Refractometer, and a digital lux meter.

In the salinity experiment, growth-medium was diluted with ion-free water (milli-Q) and raised with 0.1 M of NaCl to the following salinities: 8, 18, 28, 38, and 48 PSU. Average PFR was 230 μmol m^-1s^-1, the L:D cycle was 24:00 h, and the temperature was 23°C (± 1.5 °C).

In the temperature experiment, the flasks were placed in 5 different water baths using a tank water heater thermostat and air tubes for circulating the water around the flasks at the following temperatures: 10, 15, 20, 25, and 35°C. The average PFRs was 230 Ilmol m^-1s^-1, the L:D cycle 24:00 h, and the salinity 30 PSU.

In the light experiment, the flasks were placed on shelves in a culture room at the following PFRs: 24:00 h, 12: 12 h, 8:16 h, and 14:10 h LD cycles, the temperature was 23°C and the salinity 30 PSU.

In the intensity experiment, the flasks were placed on shelves at the following chosen levels: 25, 60, 70, 150, and 200 Ilmol m^-1s^-1 for light saturated growth.

In the pH experiment, the growth medium pH was lowered with the addition of 0.1 M HCl and raised with the addition of 0.1 M NaOH to the following pH levels: 6.5, 7, 7.5, 8, and 8.5. The temperature was 23°C and the salinity was 30 PSU.

Toxicity test

Toxicity test was performed with nauplii of waterlife Artemia brine shrimp eggs. The test was carried out in a small Petri dish. About 10 ml of cysts were incubated for 24 h at room temperature in filtered and autoclaved seawater diluted to 25 PSU under gentle aeration and continuous illumination. Hatched nauplii were separated from the non-hatched cysts and incubated for another 24 h. After that, 10 nauplii were added with 10 ml of algal culture in a culture plate. The nauplii were defined as dead if they were immobile for 10 s or longer.

Statistical analysis

Data were entered, coded, cleaned, and analyzed using statistical package for social science (IBM SPSS), version 25. The data were analyzed using descriptive statistics (mean, stander deviation, stander error). A normality test was used to determine sample data had been normally distributed, ANOVA test used for the data with normal distribution while Kruskal-Wallis was used for the data that were not normally distributed. Also, the LSD test was used to indicate where significant differences occurred. All graphs were performed using SigmaPlot software, version 14.0. Three-ways ANOVA for all growth characteristics were as follows: growth rate (μ.d^-1), dry weight (ng.cells^-1, production per dry weight (ng.d^-1), and chlorophyll-a content (μg.ml^-1). In all analyses, the statistical significance was at P ≤0.05.

Results and Discussion

Toxicity

A total of 10 nauplii of Brine shrimp (Artemia) were exposed to 3×10⁶ cells.ml^-1 of the isolated Chrysochromulina sp. No mortality was recorded, therefore, no acute toxicity was detected.

Bloom measurements

When both blooms formed, water and fish samples were analyzed from both lagoons. The results were confirmed by the Saudi Ministry of Environment, Water, and Agriculture according to the JFRC (Jeddah Fisheries Research Center) results. The water analysis showed that all samples had very low Dissolved Oxygen level, which was very much less than the allowable level. Salinity was about 50% less than the normal salinity of the open sea (Table 1). The JFRC fish analysis showed that target viral, bacterial, and parasitic diseases were negative.

Chrysochromulina sp. measurements

Microscopic images show cells appeared saddle shape with coiled haptonema between two long flagella and two parietal golden-brown chloroplasts (Figure 3) also, the Coulter Counter revealed some slight variation in the average cell size from 2.023 to 2.034 µm. Images also show evidence of Chrysochromulina sp. cells present inside the gills of dead fish samples (Figure 4). (Table 2) provides an overview of characters that were used to distinguish some individual species of Chrysochromulina by light microscopy. The sample’s characters mentioned in (Table 1) taken from Al-Nawras lagoon, are similar to Al-Arbaeen lagoon characters including the same saddle shape of the cell, length, width, flagella, haptonema, the position of haptonema, and the lack of toxicity.

 

a)	b)
c)
Figure 3. Locally isolated Chrysochromulina sp. under microscope (×100). (photos were taken by Dr. Al-Sayegh using Leica microscope).