2026 NJC H2 Bio Prelim P4 (MS)

©NJC 2026 9477/04/Preliminary Examinations [Turn over NATIONAL JUNIOR COLLEGE, SINGAPORE PRELIMINARY EXAMINATION CANDIDATE NAME MARK SCHEME BIOLOGY CLASS 2bi REGISTRATION NUMBER BIOLOGY Paper 4 Practical 9477/04 2 September 2026 2 hour 30 minutes You must answer on the question paper. You will need: The materials and apparatus listed in the confidential instructions INSTRUCTIONS • Answer all questions • Use a black or dark blue pen. You may use an HB pencil for any diagrams or graphs. • Write your name, Biology class and registration number in the boxes at the top of the page. • Write your answer to each question in the space provided. • Do not use an erasable pen. Do not use correction fluid or tape. • You may use an approved calculator. • Write the details of the shift and laboratory in the boxes provided. INFORMATION • The total mark for this paper is 50. • The number of marks for each question or part question is shown in brackets [ ]. Shift 1 2 3 Laboratory BI23 BI24 CM43 CM44 For Examiner’s Use 1 / 31 2 / 19 Total / 50 This document consists of 16 printed and 4 blank pages.

©NJC 2026 9477/04/Preliminary Examinations [Turn over 2 1 Beetroot contains red pigments in its cells. When beetroot is put in ethanol, the red pigment is released from the beetroot tissue, and the solution changes to a red colour. In this question, you will investigate the effect of different concentrations of ethanol on the release of red pigments from beetroot tissue. (a) Predict how increasing the concentration of ethanol will affect the release of red pigments from beetroot tissue. Explain your prediction. prediction More red pigments will be released from beetroot tissue with increasing ethanol concentration; explanation Higher ethanol concentration causes greater membrane damage/disruption, increasing membrane permeability, so more red pigment diffuses out; Accept: greater rupture / breakdown / dissolution / degradation / damage / disruption of the cell surface membrane (and tonoplast) Reject: reference only to damage/rupture/lysis of the cell, with no mention of a membrane, e.g. “greater cell rupture” or “more cell lysis”. Examiner’s comment: • [common error] Many candidates mentioned a difference in water potential / concentration gradient or effective collision between substrates and enzymes and so did not gain credit for the 2nd point. [clarification] - A concentration gradient only explains the direction of diffusion. It does not explain why increasing ethanol concentration causes more leakage. - Water potential mainly affects water movement by osmosis, not pigment leakage. • For candidates who understood the tonaplast and plasma membrane damage by ethanol, the comparative link (i.e., greater damage with higher ethanol concentration) was often omitted. [2] FYI (detailed explanation for understanding the basis of the experiment) • Ethanol (an organic solvent) disrupts cell membranes by dissolving the non-polar hydrophobic tails of the phospholipid bilayer and denaturing membrane proteins, which causes the membrane to lose its structural integrity and become highly permeable. • When applied to beetroot tissue, this disruption destroys both the plasma membrane and the tonoplast (the membrane surrounding the central vacuole). • As a result, the water-soluble red pigment (betacyanin) leaks out of the vacuole and into the surrounding ethanol solution, turning it red.

©NJC 2026 9477/04/Preliminary Examinations [Turn over 3 You are required to: • prepare different concentrations of ethanol • put beetroot tissue into the different concentrations of ethanol for 5 minutes • record the intensity of colour for different concentrations of ethanol • use your results to estimate the concentration of a solution of ethanol of unknown concentration You are provided with: • 100.0 cm3 of 100% ethanol, labelled E • 10.0 cm3 of an unknown ethanol concentration, labelled U • 300.0 cm3 of distilled water, in a container labelled W • 2 beetroot cylinders, in a Petri dish labelled B Relevance to answering Q1c – the beetroot cylinders were prepared by using a cork borer to punch through the beetroot. The use of the cork borer ensures consistent diameter (and hence the cross-section area) of the beetroot cylinder. Candidates are required to cut the cylinders into 30 discs of 2mm thickness. Ethanol is flammable and beetroot tissue can stain clothing. You are advised to wear suitable eye protection. If any solution comes into contact with your skin, was off immediately with tap water.

©NJC 2026 9477/04/Preliminary Examinations [Turn over 4 (b) (i) You will carry out proportional dilutions of the 100% ethanol to obtain a range of concentrations in which the concentration of the ethanol is reduced by 25% between each successive dilution. You will prepare 20.0 cm3 of each concentration, using E and W. Complete Table 1.1 to show how you will prepare the different concentrations of ethanol solution. Table 1.1 percentage concentration of ethanol volume of E / cm3 volume of W / cm3 0 0.0 20.0 25 5.0 15.0 50 10.0 10.0 75 15.0 5.0 100 20.0 0.0 1. Correct calculation and correct volume from ethanol and distilled water 2. Correct precision (1 decimal place for volume) [2] Examiner’s comment: • Most candidates were able to get full credit.

©NJC 2026 9477/04/Preliminary Examinations [Turn over 5 Read steps 1–10 before starting the investigation. Proceed as follows. 1 Label five boiling tubes with the concentrations listed in b(i). 2 Prepare the concentrations of ethanol in plastic containers, using your method in (b)(i). 3 Put 10.0 cm3 of each concentration of ethanol into the appropriately labelled boiling tubes. 4 Place one of the beetroot cylinders from the Petri dish labelled B on the white tile and blot dry with a paper towel. Cut 25 disc-shaped pieces of beetroot, each 2 mm in length, from the beetroot cylinder. 5 Put the beetroot discs into a small beaker. Put enough W to cover the discs in the beaker. Swirl and pour W into the beaker labelled waste. 6 Repeat step 5 until W remains colourless. 7 Put the discs on a white tile and blot dry with a paper towel. 8 Put five discs into each of the boiling tube. Leave for 5 minutes. 9 After 5 minutes, pour the ethanol from each boiling tube into a clean test-tube, leaving the beetroot discs in the boiling tube. Label the test-tube with the appropriate ethanol concentration. 10 Record the colour intensity of each concentration of ethanol using the symbols from Fig. 1.1 in an appropriate table in (b)(ii). Fig. 1.1

©NJC 2026 9477/04/Preliminary Examinations [Turn over 6 (ii) Record your results in an appropriate table. [4] Concentration of ethanol / % Colour intensity 0 + 25 ++ 50 ++++ 75 +++++ 100 ++++++ H: Heading for independent variable (percentage concentration of ethanol OR concentration of ethanol / %) + before heading for dependent variable (colour intensity) + no units in body of table P: Whole number for ethanol concentration and results recorded using scale in the key T: Colour intensity increases with increasing ethanol concentration C: Readings for all samples Examiner’s comment: • Most candidates were able to gain full credit. 11 Repeat steps 3–9 using the unknown concentration, U. Record your result in (b)(iii). (iii) Record the result for U. Accept values from 25 to 75% [1] Note: No units for this dependent variable The actual concentration of U is 50% ethanol. For assessment, accept values from 25 to 75%.

©NJC 2026 9477/04/Preliminary Examinations [Turn over 7 (iv) Estimate the concentration of ethanol in U, using your results recorded in (b)(ii) and (b)(ii