Environmental Concentrations of Sulfonamides Can Alter Bacterial Structure and Induce Diatom Deformities in Freshwater Biofilm Communities

Abstract

Since the early 1920s, the intensive use of antibiotics has led to the contamination of the aquatic environment through diffuse sources and wastewater effluents. The antibiotics commonly found in surface waters include sulfamethoxazole (SMX) and sulfamethazine (SMZ), which belong to the class of sulfonamides, the oldest antibiotic class still in use. These antibiotics have been detected in all European surface waters with median concentrations of around 50 ng L–1 and peak concentrations of up to 4–6 μg L–1. Sulfonamides are known to inhibit bacterial growth by altering microbial production of folic acid, but sub-lethal doses may trigger antimicrobial resistance, with unknown consequences for exposed microbial communities. We investigated the effects of two environmentally relevant concentrations (500 and 5,000 ng L–1) of SMZ and SMX on microbial activity and structure of periphytic biofilms in stream mesocosms for 28 days. Measurement of sulfonamides in the mesocosms revealed contamination levels of about half the nominal concentrations. Exposure to sulfonamides led to slight, transitory effects on heterotrophic functions, but persistent effects were observed on the bacterial structure. After 4 weeks of exposure, sulfonamides also altered the autotrophs in periphyton and particularly the diversity, viability and cell integrity of the diatom community. The higher concentration of SMX tested decreased both diversity (Shannon index) and evenness of the diatom community. Exposure to SMZ reduced diatom species richness and diversity. The mortality of diatoms in biofilms exposed to sulfonamides was twice that in non-exposed biofilms. SMZ also induced an increase in diatom teratologies from 1.1% in non-exposed biofilms up to 3% in biofilms exposed to SMZ. To our knowledge, this is the first report on the teratological effects of sulfonamides on diatoms within periphyton. The increase of both diatom growth rate and mortality suggests a high renewal of diatoms under sulfonamide exposure. In conclusion, our study shows that sulfonamides can alter microbial community structures and diversity at concentrations currently present in the environment, with unknown consequences for the ecosystem. The experimental set-up presented here emphasizes the interest of using natural communities to increase the ecological realism of ecotoxicological studies and to detect potential toxic effects on non-target species.

Contrasting Effects of Environmental Concentrations of Sulfonamides on Microbial Heterotrophic Activities in Freshwater Sediments

“…Whereas βglucosidase and phosphatase activities (Figures 2A,C) were not sensitive to low levels of SMZ (<4 µg SMZ kg −1 dw), leucine aminopeptidase activity (Figure 2B) was found to be transiently stimulated at both low and high levels of SMZ exposure after 14 days of exposure (p < 0.05). This transient microbial response had also been observed previously by Kergoat et al (2021) in periphytic biofilms exposed to mean SMZ concentrations ranging from about 106 to 157 ng SMZ L −1 . To our best knowledge, there is no other published study investigating the chronic effect of SMZ on the leucine aminopeptidase activity.…”

“…Most of the microbial activities increased over time, reflecting microbial growth and activity. As previously reported in several studies assessing the effects of SMX and SMZ on aquatic microbial biofilms (Yergeau et al, 2010(Yergeau et al, , 2012Kergoat et al, 2021;Paumelle et al, 2021), the functional responses observed in the present experiment varied markedly according to the antibiotic tested.…”

“… Yergeau et al (2010 , 2012) showed that more environmentally relevant concentrations of SMZ or SMX (500 ng L –1 of either) could affect community composition and the meta-transcriptome of river biofilms exposed for 8 weeks. More recently, Kergoat et al (2021) also observed changes in the bacterial and diatom composition of river biofilms exposed for 4 weeks to 500 and 5,000 ng L –1 of SMX or SMZ (tested individually at these two nominal concentrations). Moreover, they observed slight and transitory effects on microbial heterotrophic functions such as the phosphatase and leucine aminopeptidase enzymatic activities ( Kergoat et al, 2021 ).…”

“…They hypothesized that the β-glucosidase increase was due to the use of an external C supply in the antibiotic treatments and/or to an adaptative response to resist antibiotics (Paumelle et al, 2021). By contrast, Kergoat et al (2021) observed no change in the β-glucosidase activity of periphytic biofilms exposed for 28 days to SMX concentrations between 0.2 and 4 µg L −1 . Moreover, Liu et al (2016) observed a negative effect of SMX on the β-glucosidase activity of soil microbial communities, but the concentrations they used (5 and 50 mg SMX kg −1 dw soil) were far higher than the environmental concentrations tested here.…”

“…Only a few studies have investigated the effects of SMZ and SMX on aquatic microbial communities ( Grenni et al, 2018 ). Most have focused on periphytic biofilms, which are complex assemblages that develop on inert substrata, such as pebbles or cobbles ( Yergeau et al, 2010 , 2012 ; Johansson et al, 2014 ; Kergoat et al, 2021 ). Using Biolog Ecoplates, Johansson et al (2014) estimated that the effective concentrations (CE) of SMX inhibiting 10 and 50% of total carbon source utilization by marine biofilms after 4 days of exposure were respectively, close to 14 μg L –1 (CE 10 ) and 272 μg L –1 (CE 50 ).…”