**2.5 Collection of effluents sample**

The coloured polluted sewage water was collected from the municipality channel of the Preonagar, Mathpara and Harishnagar, North 24 Parganas (22.61680 N, 88.40290 E), West Bengal, India in the winter season from three locations at the depth of 5–7 inches from five random points within the municipality drains of each location. The dirty water was filtered by muslin cloth so many times to remove all visible muddy materials present in the water sample and later on storage having been done in a clean container for conducting physiochemical analysis followed by use in cytological studies of the experimental onion crop.

#### **2.6 Physiochemical parameters**

The heavy metals (Manganese, Nickel, Copper, Zinc, Cadmium, Chromium, Iron and Lead) and chloride responsible for different chromosomal mutations were estimated for their concentration (mg/l) in the water sample of the different parts of North 24 Parganas (N), West Bengal (**Table 1**).

*Sample assay.*

Onion (*A. cepa* L.)

Onion (*A. cepa* L.) bulbs of about the same size (42 mm) weighing about 33 gm and 10 months old were selected by removing the loose outer scale, older roots without damaging meristematic tissues and scrapped the bottom part with the help of a blade so that root primordia were formed in wastewater samples of different locations (treatments) and distilled water (control condition). Keep in mind that the bottom part of each bulb slightly emerged out of the water. The incubated time of the sample in each treatment was 48 hours at the temperature of 22°C in dark conditions.

#### **2.7 Chromosome preparation**

The squash preparation demonstrated by Sharma and Sharma [13] was used for the chromosome preparation of the treated onion roots. The following methods were used to investigate the root meristematic tissue exhibiting mitotic cell division:
