Toxicity analysis of three different types of microplastics - polyethylene, polypropylene, and polystyrene - immersed in the benthic and pelagic regions of Muskegon Lake, Michigan

1. Toxicity identification evaluation of microplastics immersed in Muskegon Lake, Michigan
Abstract
Microplastics pollute aquatic ecosystems and develop biofilms on them,
which enhances the sorption of other contaminants to the microplastic, such
as persistent organic pollutants. This poses a potentially serious threat to
organisms that ingest them. We investigate the toxicity of biofilms eluted
from three different types of microplastics – polyethylene, polystyrene, and
polypropylene – placed in the pelagic and benthic layers in Muskegon Lake.
The toxicity was assessed by measuring the length and developmental stage
of the nematode Caenorhabditis elegans. Approximately 10 synchronized L1
nematodes were exposed to each sample in a 96 well microliter plate at 20
degrees Celsius. After 72 hours, worms were heat-killed and dyed with Rose
Bengal. Worms were imaged and their lengths were determined using ImageJ
software. The elutriate reduced development in a dose dependent manner.
Elutriates were modified to exclude different classes of toxicants – ammonia,
organic pollutants, particulates matter etc. Preliminary findings showed
biofilm toxicity was reduced the most after being modified by filtration and
C18-solid phase extraction for the two highest doses – 25% and 50%.
Biofilms eluted from plastics in the benthic locations were more toxic
compared to biofilms from plastics in the pelagic location. Ongoing toxicity
identification evaluation seeks to identity the physicochemical characteristics
of the biofilms, and the identity of toxicants.
Methods
Results
Jeannie Kane, Sofia Vozza, & Babasola Fateye, Department of Biomedical Sciences, Grand Valley State University
Figure 1. (a) Study site: Muskegon Lake. In situ exposure of microplastics were
placed in specially designed containers (b) , to keep them in place while
allowing lake water to flow through. Containers were placed at pelagic (F) and
near benthic (C & H) locations in Muskegon Lake in summer 2018 and retrieved
after 3 months (c).
Picture Credit: https://www.gvsu.edu/wri/director/plastic-debris-as-an-exposure-
source-for-persistent-88.htm.
• Suspend beads in K. Medium
• Vortex on high 10 min
• Obtain eluted biofilm (elutriate)
Development assay, 72 hours
Figure 2. (a) Sequential toxicity identification and evaluation (TIE). Biofilm eluted from microplastics
were modified sequentially (NFESC, 2003). (b). Schematic for toxicity assays (morphometry and
survival) in Caenorhabditis elegans. Briefly, 10 synchronized larval (L1) staged C. elegans were
exposed to unmodified and modified microplastic elutriate. After for 72 hours, worms were then heat
killed, stained with Rose Bengal, and length measured (Hoss et al, . 2011; Donken & Dusselberry, 1993)
Conclusions
To rule out classes of toxicants, a step wise approach was taken as in Figure 2.
For a 25% dilution of biofilm, toxicity levels remained the about the same for the
biofilm itself, and after the addition of sodium thiosulfate and EDTA. After
filtration, toxicity was greatly reduced, and reduced more after C18 SPE (Table 1
& Figure 3). This outcome indicates toxicity is likely due to particulate factors
and organic pollutants. Further investigative methods may be used to further
characterize the toxicants.
Literature Cited
NFESC. 2003. Guide for Planning and Conducting Sediment Pore Water Toxicity Identification
Evaluations (TIE) to Determine Causes of Acute Toxicity at Navy Aquatic Site. Retrieved from
https://www.navfac.navy.mil/content/dam/navfac/.
Delatolla ...Tufenkji N. Rapid and reliable quantification of biofilm weight and nitrogen content of
biofilm... polystyrene beads. Water Res. 2008;42: 3082-3088
Höss S ...Transpurger, W.. Toxicity of ingested cadmium to the nematode Caenorhabditis elegans.
Environ Sci Technol. 2011;45(23):10219-10225. Donkin & Dusenbery. A soil toxicity test using the
nematode C elegans and an effective method of recovery. Arch Environ Contam Toxicol. 1993;25(2):145-
151
We thank Drs. Rick Rediske and Al Steinman, AWRI for the microplastics & GVSU Office
Undergraduate Research for funding.
Figure 3. There was a dose dependent toxicity in most elutriates. The graph above
shows the acute toxicity of 4 dilutions of the elutriate from polypropylene
microplastics retrieved after 3 months in location H.
Table 1. Relative impact of sequential elutriate modification on toxicity.
Modification: metal chelation - sodium thiosulfate and/or EDTA was added; filtration: glass fiber
microfilter; Solid phase extraction: C18 HLB column. *** P< 0.001 (t-test) ; ** P< 0.01; * P<
0.05; NS Not significant ; Δ 25%; # 50%
(a)
(a) (b)
(c)
(b)
Elute
biofilm
3
months
Elutriate
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Unknown
substances
Microorganisms
Absorbed/dissolved
organic pollutants
Associated inorganic chemicals
Macroorganisms