Patel et al.

(Azuma et al. 1996, Wilcke et al. 2000 and Krauss et al.

Materials and Methods

2005).

Growth Conditions and Acenapthene Treatment

Production from anthropogenic activities such as coking

of coal, distillation of wood, operation of the gas works

Axenicculturesof Scytonema , Chlorellavulgaris Beijrinck

and oil refineries, runoff from asphalt pavements,

and Desmodesmus subspicatus Chodat procured from

vehicular emission, petroleum spills, and the incomplete

the National Facility for Blue-Green Algae (IARI, New

combustion of fossil fuels and organic matter (Canet

Delhi, India) were grown at 25±2ºC in BG-11 medium

et al., 2001). The accumulation of hydrocarbons in the

(Rippka et al., 1979) under an illumination of 3,000 Lux

seas and oceans has become a source of considerable

light with a photoperiod of 14:10 (L/D). PAH treatment

environmental concern, especially in recent decades. In the

was carried out on the cultures in the logarithmic phase

last century, human activities have resulted in substantial

of growth, Acenapthene, obtained from Sigma Aldrich

additional hydrocarbon inputs to the oceans (Shaw, 1981).

(Mumbai, India). Following a series of experiments to

The growing demand for oil has been accompanied by an

determine the LC50 dose, 2 ml of the inoculums from

increase in oil pollution due to spillage in both freshwater

previously grown logarithmic cultures was introduced

and marine ecosystems. When an oil spill occurs in the

into a freshly prepared autoclaved modified BG-

sea, the oil spreads on the water surface and drifts by

11 medium for D. subspicatus, BG-11 medium for

wind and currents, the low-boiling fraction evaporates,

Scytonema sp. and Zarrouk’s medium (Zarrouk, 1996) for

and the low-boiling aromatic fraction dissolves easily in

C. vulgaris in which Acenapthene had been added at final

the water (Gunkel et al., 1980).

concentrations of 0 (control), 0.5, 1.0, 5.0, 10, 20, 40,

60 and 80 ppm to make up a total volume of 20 ml. The

The non-target effects of organic pollutants on autotrophic

LC50 can be defined as the standard measure of toxicity

microalgae and cyanobacteria have been reviewed

that leads to 50% reduction in the sample population of

by many researchers (Ramakrishnan et al., 2011 and

a specific test organism in a specified period of exposure

Venkateswarlu, 1993). Recently, the phycoremediation

to a compound. The final LC50 value determined was

by microalgae has been attempted for various pollutants

given in Table 1. To prevent PAH degradation, stock

(Mallick, 2002, Mehta et al., 2005 and Olguín, 2003). The

solutions were prepared just prior to each experiment

data about the effect of PAHs on different physiological

by dissolving the Acenapthene in HPLC grade Acetone.

and biochemical aspects of photosynthetic activity in

Each experiment was conducted in triplicate and all

microalgae are still insufficient in spite of the fact that

spectrophotometer reading was taken in UV-VIS-NIR

photosynthetic parameters are recognized as reliable

spectrophotometer, Lambda-19, Perkin Elmer.

indicators of many biotic stresses (Huang et al., 2010

and Mallakin et al., 2002). Recently several methods

have been developed for measuring of PAHs impact on

Table 1: On the basis of a series of experiments for LC50,

the effective doses were resulted.

microalgae based on evaluation of its primary metabolites,

photosynthesis pigments and enzymatic activities. Miral

Species

Lower LC50 LC50

Higher LC50

Patel et al., 2014; 2014) had also studied the effect of PAHs

on several biological organisms treated with different

C. vulgaris

1.25 ppm

2.5 ppm

5.0 ppm

PAHs. An attempt has, therefore, been made in the present

D. subspicatus

1.5 ppm

3.0 ppm

6.0 ppm

study to determine the chronic effects of Acenapthene on

Scytonema sp.

2.5 ppm

5.0 ppm

10 ppm

pigments like total chlorophyll, carotenoids, phycocyanin,

allophycocyanin, and phycoerythrene, as well as on the

Pigment Analysis

biochemical compounds, C/N ratio and nitrate reductase,

The growth of the test organism was determined in terms

succinate dehydrogenase and glutamate synthetase of

of total chlorophyll. Total Chlorophyll and carotenoids

the two important microalgal species C. vulgaris and D.

were measured spectrophotometrically in cell lysates

subspicatus and cynobacterial species Scytonema .

after extraction in 80% acetone (Jeffrey and Humphrey,

468