Introduction

Microplastics (MPs) are emerging persistent pollutants in freshwater ecosystems. Algae, as primary producers at the base of the food web, are highly vulnerable to them. While plastics like Polyethylene (PE) and Polypropylene (PP) are highly abundant (Figure 1), Expanded Polyethylene (EPE) foam, which typically floats on the surface, is less commonly studied.

Global warming is hypothesized to have a synergistic toxic effect with MPs. This study investigates the impact of EPE MPs on the growth of the freshwater green algae Chlorella vulgaris across different temperatures (18°C to 30°C) and verifies whether warming exacerbates MP-induced toxicity.

Figure 1: Composition of freshwater microplastics.

Methodology

  • MP Prep: Commercial EPE foam ground to <250μm, stained with Rhodamine B.
  • Culture: C. vulgaris grown to exponential phase at 25°C.
  • Design: Incubated at 18, 20, 25, 30°C for 7 days. Groups: Control, Un-dyed MP, Dyed MP (100mg/L).
  • Metrics: Daily UV-Vis (680nm), hemocytometer cell count, and fluorescence microscopy.
1a. Preparation of MP
Grinding & Staining
1b. Preparation of Algae
Culture to Exp. Phase
⇓
2. MP Exposure & Incubation
4 Temp. Variations (18°C, 20°C, 25°C, 30°C) x 7 Days
⇓
3a. Concentration Metrics
  • UV-vis spec (680nm) → Absorbance
  • Haemocytometer → Optical density
[Days 0-2, 5-7]
3b. Microscopic Interaction
  • Fluorescent microscopy → MP & Algae location/aggregation
[Day 7]

Results

a) Effect of warming on C. vulgaris over 7 days

18°C
20°C
25°C
30°C
Abs
0.11 ↑
0.12 ↑
0.14 ↓
0.13 ↓
Cells
320k ↑
350k ↑
586k ↓
180k ↓

The data reveals a temperature-dependent toxicity threshold. While elevated temperatures (30°C) naturally optimize algal proliferation, the introduction of EPE MPs triggered an initial hormetic response (growth stimulation, Days 1-5). However, prolonged exposure at optimal temperatures led to a sudden collapse in viability by Day 7, suggesting that synergistic stress eventually overwhelms the antioxidant defense mechanisms of C. vulgaris.

Results (Continued)

Figure 3: Algal count at 25°C (Top) and Absorbance at 30°C (Bottom). Note the collapse at Day 7.

b) Effect of Rhodamine B dye on algae

Comparative analysis demonstrated a temperature-sensitive dye interference, peaking at a 2.35% excess inhibition at 25°C. This confirms that Rhodamine B introduces both chemical toxicity and optical shading, which significantly skews spectrophotometric growth readings. Strict un-dyed controls are vital for accurate toxicological assessments.

c) Interaction of MP & algae (Hetero-aggregation)

18°C
20°C
25°C
30°C

Fluorescence imaging confirmed that physical interactions are physically mediated by heat. At sub-optimal temperatures (18-20°C), interactions were minimal. However, at 25-30°C, enhanced Extracellular Polymeric Substance (EPS) secretion acted as a robust bio-adhesive, driving massive hetero-aggregation that physically entraps cells and blocks photosynthetic light.

Discussion

The synergistic effect of global warming and EPE microplastics poses a compounded threat to freshwater primary producers. The observed temperature-driven hetero-aggregation not only accelerates the physical entrapment and shading of algae, but also alters the effective density and buoyancy of the floating MPs. Consequently, rising global temperatures are projected to heavily magnify the baseline toxicity of persistent microplastics in aquatic environments, severely disrupting the base of the food web.

Conclusion

The toxicity of EPE microplastics to Chlorella vulgaris is heavily modulated by environmental temperature. Warming conditions (25-30°C) initially accelerate growth but drastically intensify subsequent oxidative stress and massive hetero-aggregation, ultimately exacerbating overall net toxicity.

These findings signify severe challenges for freshwater ecosystems under climate change, stressing the critical necessity of formally incorporating "temperature elevation" parameters into future microplastic ecological risk assessments.

Acknowledgements

Special thanks to Dr. Iain Stott for his guidance, expertise, and continuous support during this research project at the University of Lincoln. Gratitude is also extended to the laboratory technicians for their essential equipment assistance and maintaining the cell cultures.