Study area and sampling sites
The study was conducted in the Shadegan wetland, which is located on the lower Jarrahi River in the Province of the Khuzestan, at the head of the Persian Gulf (Iran). The sixteen study sites were selected from the upstream and downstream areas of the Shadegan wetland (Fig.1). Station 1 (Darkhovin) (30° 46.357′N and 48° 27.431′E), Station 2 (Drissie) (30°35,476′N and 48° 30.553′E), Station 3 (Malch) (30°42.699′N and 48°28.744′N) were located towards the Northern portion of the Shadegan wetland. The Station 4 (30°41.008′N and 48°32.445′E), 5 (30°44.590′N and 48°44.646′E), 6 (30°39.158′N and 48°31.933′E), 7 (30°38.705′N and 48°32.574′E), 8(30°39.678′N and 48°31.370′E) and 9 (30°40.055′N and 48°30.535′E) were in the wetland. Station 10 (Aboudi Creek)( 30°60.993′N and 48°65.569′E), Station 11 (Duragh Creek) (30°32.466′N and 48°49.652′E), Station 12 (30°88.053′N and 48°51.521′E), Station 13 (Mard Creek) (30°50.424′N and 48°31.856′E), Station 14 (Shadegan Port)(30°53.632′N and 48°68.901′E) and Station 15 (Abadan)( 30°44.520′N and 48°26.456′E) were located towards the southern portion. In this study, three different periods were used flood seasons in autumn and winter with rains in the Zagros Mountains causing extensive flooding throughout the Shadegan wetland, the dry season (months of summer), and spring as the wet season (21).
Environment variables
At each site, nine replicate surface water samples were collected between 2017 to 2019 Twice in each flood season (January to March and October to November), dry season (Jun 15 to September), and wet season (April to Jun 15) using a 1.5 polyethylene plastic container. It was then sieved through a 20 mm plankton net and stored in the dark in ice to be transported to a laboratory for chemical and biological analysis (22). The water quality parameters including temperature, water pH, salinity, and electrical conductivity were measured in-situ by a calibrated digital multi-parameter (YSI Environmental Model-556 MPS) (Meters Portable HQ40d, Hach). The DO meter (HQ 40b model-Hach) was used for measuring Dissolved oxygen concentration (DO). Water level and meteorological data were collected from synoptic stations in Khuzestan province (2017-2019).
Nutrient concentration
The dissolved inorganic carbon (DIC), dissolved total carbon (DTC), and dissolved organic carbon (DOC) concentration in the water sample was determined using an elemental analyzer (Vario EL III, Germany). Then, the water samples were filtered with 0.45-μm organic microporous membrane filters. A segmented continuous flow analyzer (Futura, France) was used to determine dissolved total nitrogen (DTN), nitrate-nitrogen (NO3- -N), and ammonia nitrogen (NH4+-N) concentration. The phosphate (PO43- ) concentration was measured using an automatic chemistry analyzer (Smartchem 200, Italy). The digestion methods were used to measure total phosphorus (TP) and total nitrogen (TN) concentration (23).
Phytoplankton
Phytoplankton and water samples used for Chlorophyll-a analysis were collected from the sixteen sampling sites, fixed according to Lugo’s reagent fixation method (from 2017 to 2019). The phytoplankton samples sitting for 48 h were siphoned out of the supernatant. Then, 1 ml of formaldehyde solution was added to the leaving 60 ml of samples at the bottom and stored this sample solution for laboratory test. The eyepiece lens of the wide visual field method was used for determining the species composition, density, and biomass of phytoplankton based on valid previous references (24, 25). The water sample has been vigorously shaken, then 0.1 ml of the sample and 0.1 ml of the counter box were added and covered with glass carefully. Then, counting the number of algae in each field of vision and the number of the field of vision was done using an optical microscope (Magnification 10× and 40×). To calculate the density and biomass of the samples, the average values of the three parallel experiments were applied (24).
Chlorophyll-a
Determination of chlorophyll-a was done spectrophotometrically in 90% acetone according to the Parson et al method (26). The concentration of Chlorophyll a was calculated according to the Lorenzen method (27):
E665b = D665b - D750b (1)
E665a = D665a - D750a (2)
Where D665b and D750b respectively are the absorbance values at 665 and 750 nm wavelengths before acidification and absorbance values at 665 and 750 nm wavelengths following acidification are D665aandD750a respectively.
Chlorophylla concentration(μg/L) = A × K × (E665b- E665a) × v/ V × L (3)
Where A is the specific absorptivity of chlorophyll a within different extraction solvents, the ratio of the optical density before acidification and changes in optical density before and following acidification of pure chlorophyll a is K constant. The extracting solution volume (mL) is the v. The volume of sample (L) is the V. the optical path length of the colorimetric sample cup (cm) is the L.
Statistical analysis
The Kolmogorov-Smirnov test was used to evaluate the data normality. Kruskal-Wallis rank-sum test and pairwise comparisons using Wilcoxon rank-sum test were used to test significant differences in Chlorophyll-a, environmental parameters, and nutrients during the three different water level periods (flood season, dry season, and wet season). To assess relationships among water levels, Chlorophyll-a, nutrients, and phytoplankton species density and biomass, Pearson correlation coefficients were used. All analysis was done in OrginePro 2016 software programs and R version 3.5.3.