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. 2022 Oct;83(3):284-294.
doi: 10.1007/s00244-022-00959-y. Epub 2022 Oct 3.

Determining Toxic Potencies of Water-Soluble Contaminants in Wastewater Influents and Effluent Using Gene Expression Profiling in C. elegans as a Bioanalytical Tool

Antoine Karengera  1   2 , Ilse Verburg  2   3 , Mark G Sterken  4 , Joost A G Riksen  4 , Albertinka J Murk  5 , Inez J T Dinkla  2
Affiliations

Determining Toxic Potencies of Water-Soluble Contaminants in Wastewater Influents and Effluent Using Gene Expression Profiling in C. elegans as a Bioanalytical Tool

Antoine Karengera et al. Arch Environ Contam Toxicol. 2022 Oct.

Abstract

With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode's xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Volcano plots showing the distribution of gene expression changes and p-values. Each dot represents a spot on the microarray, as analyzed by three linear models. On the x-axis the effect is given (a negative sign indicates lower expression over increasing concentrations, a positive sign higher expression over increasing concentrations), on the y-axis the − log10(p value) obtained from the linear model. These effect plots show an obvious distinction between wastewater samples before and after treatment in a WWTP. Colors provide a visual guide for the thresholds of − log10(p) > 4 and − log10(p) > 5. A Hospital samples, B nursing home samples, C community samples, D WWTP influent samples, E WWTP effluent samples
Fig. 2
Fig. 2
Comparison of gene expression profiles in nematodes treated with (waste)water samples. Sampling points are shown in A, including wastewater Community (C), Hospital (H), Nursing home wastewater (N), WWTP influent (I), WWTP effluent (E) and surface water (SW) receiving the treated effluent. B Is a heatmap showing the up- (red–orange) and down-regulation (blue) of C. elegans genes after exposure to different (waste)water samples. There is a clear difference between gene expression patterns before and after wastewater treatment
Fig. 3
Fig. 3
Principal component analysis (PCA) for variation in gene expression. The first two principal components PC1 and PC2 combined captured 56.6% of the variance and mainly separate the surface water and effluent samples from the other samples
Fig. 4
Fig. 4
Differences and similarities of genes expression profiles in nematodes after exposure to different wastewater samples. The Venn diagram shows that from the 1756 DEGs (up- or downregulated) in one or more of the polluted samples (i.e., hospital, nursing home, community, and influent), the majority (69%) of these genes were specific to community and/or nursing home wastewaters. The overlap of 209 DEGs (approx. 11%) were found in all polluted samples
Fig. 5
Fig. 5
Expression fold change range of differentially expressed genes (DEGs) in the nematodes treated with wastewater samples. Bar charts display the number of DEGs in each fold-change range (i.e., < twofold, twofold–fivefold, fivefold–tenfold, and > tenfold) of the transcription levels induced in the nematodes treated with the samples originating from community (C), nursing home (N), hospital (H), WWTP influent, WWTP effluent (E), or surface water (SW)
Fig. 6
Fig. 6
Some significantly upregulated genes for which enriched terms could be obtained [false discovery rate (FDR) < 0.05]. Full results of functional enrichment analysis are provided in Tables S3 and S4 of supplementary information
Fig. 7
Fig. 7
Validation of gene expression microarray results by reverse transcription polymerase chain reaction (RT-qPCR) for 15 _target genes in two independent biological replicates using the RNA template from microarray samples. Negative values indicate downregulation and positive values upregulation of the _target genes relative to two housekeeping genes (tbg-1 and par-5) used to normalize the expression fold changes
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