Interactions with freshwater biofilms cause rapid removal of common herbicides through degradation - evidence from microcosm studies.

We investigated the role of periphyton biofilms for the fate of three common herbicides, i.e. bentazone, metazachlor and metribuzin, at low, environmental levels and 100 times higher, during a 16 days laboratory experiment. We found that herbicide water concentrations were stable during the first 8 days, whereas substantial declines (>78%) occurred between days 8-16 for all three herbicides. These rapid declines were explained only to a small extent (<8% of the total herbicide loss) by biofilm sorption. As herbicide concentrations in light and dark treatments without biofilms were similar, and the applied light regimen did not cover the UV-spectrum, herbicide photolysis was ruled out as a possible explanation for the observed declines. Furthermore, based on the compounds' characteristics, also volatilization was judged negligible. Therefore, we conjecture that the observed declines in herbicides were due to biodegradation and subsequent evasion of 14CO2 that was driven by enzymatic action from heterotrophic microbes. We reason that heterotrophic microbes used herbicide molecules as labile organic C-sources during C-limitation. Future studies should identify the microbial communities and genes involved in biodegradation in order to understand better the role of biofilms for the self-purification of surface waters.

Pesticide Mixtures Cause Short-Term, Reversible Effects on the Function of Autotrophic Periphyton Assemblages.

In a laboratory experiment we investigated the effects of pesticide mixtures on the structure and function of freshwater biofilms, with focus on their photoautotrophic component. We identified 6 herbicides and 1 fungicide commonly found in Swedish streams at relatively high concentrations and created 3 ternary mixtures that were tested in concentration series ranging from observed environmental concentrations to up to 100 times higher. Biofilms were exposed to these pesticide mixtures for 8 d and then allowed to recover for another 12 d. Our results show a rapid and consistent inhibition of photosynthesis after just 24-h exposure to the highest test concentration of pesticides, as well as in some treatments with lower concentrations (i.e., 10 times the environmental level), on exposure. Interestingly, the observed effects were reversible because biofilm photosynthesis recovered rapidly and completely in clean media in all but one treatment. In contrast to the functional response, no effects were observed on the algal assemblage structure, as assessed by diagnostic pigments. We conclude that the pesticide mixtures induce a rapid but reversible inhibition of photosynthesis, without short-term effects on biofilm structure. Environ Toxicol Chem 2020;39:1367-1374. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Limited effects of pesticides on stream macroinvertebrates, biofilm nematodes, and algae in intensive agricultural landscapes in Sweden.

Pesticides are frequently detected in surface waters, sometimes at levels exceeding ecotoxicological guidelines. We screened for almost 100 pesticides in 32 streams from intense agricultural areas in Southern Sweden, in concert with water chemistry parameters. In addition, we investigated the communities of benthic macroinvertebrates, biofilm nematodes and algae and calculated multiple bioassessment metrics. The number of pesticides found in each stream ranged between 2 and 52, but the sum of Toxic Units (ΣTU) for the mixtures was generally low, and exceeded the European Uniform Principles only in a single sample for algae and in 2% of the samples for Daphnia. Only nematode communities were significantly correlated with the ΣTU, potentially due to their higher pesticide exposure in biofilms. Diatom metrics showed that most streams were impacted by eutrophication and macroinvertebrate metrics showed good status in most streams, whereas the SPEARpesticides (SPEcies At Risk) index, specifically designed to indicate pesticide effects, showed that about half of the samples were at risk. Interestingly, SPEARpesticides was not correlated to ΣTUDaphnia, and this discrepancy suggests that redefining the boundaries for quality classes might be necessary for this index. Moreover, SPEARpesticides was positively correlated with the commonly used macroinvertebrate index ASPT, although disparate results were found for several streams. We argue that this questions the scaling of both metrics and the specificity of their responses. We discuss that the overall good/moderate status of the streams, despite the intense agriculture in the catchments, can be due to the fact that i) a sampling strategy with repeated grab samples did not capture peak pesticide concentrations, thus underestimating acute exposure, ii) pesticide run-off indeed was low, due to measures such as buffer strips, and iii) the nutrient-rich conditions and high sediment loads counteracted pesticide toxicity. We conclude that agricultural land use was the overriding stressor in the investigated streams, including strong effects of nutrients, less apparent effects of pesticides and likely impact of hydromorphological alterations (not specifically addressed in this study).

Assessing microbial contamination and antibiotic resistant bacteria using zebra mussels (Dreissena polymorpha).

Aquatic pollution with faecal bacteria and subsequent consumption of contaminated water or food is a worldwide issue that causes severe health effects (e.g. meningitis, salmonellosis, dysentery). In addition, the excessive use of antibiotics in animal husbandry and human medicine has enhanced the selective pressure on pathogenic bacteria, further increasing human health risks and detrimental effects on natural microbial communities. This urges the need to monitor faecal contamination using a time-integrated approach, as grab water samples can miss pathogen peaks. We tested the ability of zebra mussels (Dreissena polymorpha) to take up and depurate faecal indicator bacteria such as Escherichia coli and intestinal enterococci. Furthermore, we quantified the frequency of antibiotic resistant bacteria in water and mussels both in controlled laboratory tests and under in situ conditions downstream of a sewage treatment plant (STP). Laboratory results show that bacterial indicators in mussels were 132 times higher than their concentration in water, and that mussels retained bacteria up to 2 days after pulse exposure. Field results show decreasing bacterial concentrations in both water and mussels downstream the STP, with maximum E. coli concentrations ranging 173-9 cfu mL-1 in water and 2970-330 cfu g-1 in mussels. Similarly, enterococci ranged 59-4 cfu mL-1 and 1450-240 cfu g-1 in water and mussels, respectively. High proportions of antibiotic resistant E. coli were found in mussels (72%) and water (65%), and slightly lower proportion of resistant enterococci was found in mussels (47%) and in water (34%). Moreover, 33% of the bacteria isolated from mussels were resistant to multiple antibiotics, which emphasizes that resistance is a common feature in surface waters and highlights the need for safe water management. Our results show that zebra mussels provide an efficient, time-integrating tool for quantifying faecal indicators, including resistant and multidrug resistant bacteria.

Metal contamination in harbours impacts life-history traits and metallothionein levels in snails.

Harbours with limited water exchange are hotspots of contaminant accumulation. Antifouling paints (AF) contribute to this accumulation by leaching biocides that may affect non-target species. In several leisure boat harbours and reference areas in the Baltic Sea, chronic exposure effects were evaluated using caging experiments with the snail Theodoxus fluviatilis. We analysed variations in ecologically relevant endpoints (mortality, growth and reproduction) in concert with variation in metallothionein-like proteins (MTLP) levels. The latter is a biomarker of exposure to metals, such as copper (Cu) and zinc (Zn), which are used in AF paints as active ingredient and stabilizer, respectively. In addition, environmental samples (water, sediment) were analysed for metal (Cu and Zn) and nutrient (total phosphorous and nitrogen) concentrations. All life-history endpoints were negatively affected by the exposure, with higher mortality, reduced growth and lower fecundity in the harbours compared to the reference sites. Metal concentrations were the key explanatory variables for all observed adverse effects, suggesting that metal-driven toxicity, which is likely to stem from AF paints, is a source of anthropogenic stress for biota in the harbours.

Mortality and histopathological effects in harbour-transplanted snails with different exposure histories.

Contaminants are important stressors in the aquatic environment and may exert selective pressures on organisms. We hypothesized that snails originating from a metal-contaminated habitat (B) would have increased tolerance to harbour contaminants (e.g. metals from antifouling paints), compared to snails originating from a relatively clean habitat (A). We assessed tolerance to metals in terms of survival and histopathological alterations after 2, 4 and 8 weeks of in situ exposure in three Baltic Sea boat harbours and three reference sites. We also hypothesized that any potential tolerance to contaminants would be associated with differences in genetic diversity between the two snail populations (evaluated as mitochondrial cytochrome c oxidase subunit I, COI). The results show that snails from population A survived to a higher extent compared to population B, possibly indicating either a lack of adaptation to metals in snails B or impaired health condition due to contaminant pre-exposure or a higher resilience of snails A. Moreover, the genetic diversity of COI was low within each population and did not differ between populations. In general, 83% of all the types of histopathological alterations (e.g. lysis and necrosis of gonads and digestive gland or granulocytoma and phagocytosis in the storage tissue, among others) had a higher probability of occurrence among harbour-exposed snails compared to reference-exposed snails, regardless of snail population origin. The only significant difference in histological effects between the two populations was in the frequency of parasite infestations and shell fouling, both being larger for population A than B. Interestingly, the rate of parasite infestations was higher for males than females from population A, whereas no sexual dichotomy was observed for population B. Our results show that exposure to harbour contaminants causes both lethal and sublethal toxicity to snails, and the association between many of the toxic responses and metals substantiates that antifouling substances contribute to the observed effects, although there is a large proportion of variation in our data that remains unexplained.

Biofouling of leisure boats as a source of metal pollution.

The release of harmful metals from antifouling paints to water bodies is a well-known problem. In this study, we measured both the amount of biofouling growth on leisure boats during one season as well as the concentration of metals accumulated by the biofouling matrix. Furthermore, the efficiency of antifouling paints and mechanical boat cleaning as well as the effect of hull colour on biofouling were evaluated. Unlike paint residues, biofouling waste has never been regarded as a source of metal contamination and has previously been neglected in the scientific literature. Our results revealed that the biofouling waste contained very high concentrations of metals, up to 28,000 mg copper/kg dw and 171,000 mg zinc/kg dw, which exceeds the guidance values for least sensitive land use in Sweden by factors of 140 and 340, respectively. This observation is important because the contaminated biofouling waste is commonly disposed of in boatyard soils at the end of each season, thus increasing the levels of metal pollution. Moreover, there was no significant difference in the amount of biofouling if the boats were coated with copper or zinc containing paints or no paint at all, indicating that biocide paints might not be necessary in low-salinity areas such as the Stockholm archipelago. For boats that were not painted at all during the season, those washed on boat washers (mechanically) had on average half of the amount of biofouling compared to boats that were not cleaned mechanically. The results of the study indicate the importance of proper management of biofouling waste as well as the use of more environmentally friendly removal methods for biofouling such as boat washers.

XRF measurements of tin, copper and zinc in antifouling paints coated on leisure boats.

Tributyltin (TBT) and other organotin compounds have been restricted for use on leisure boats since 1989 in the EU. Nonetheless, release of TBT is observed from leisure boats during hull maintenance work, such as pressure hosing. In this work, we used a handheld X-ray Fluorescence analyser (XRF) calibrated for antifouling paint matrixes to measure tin, copper and zinc in antifouling paints coated on leisure boats in Sweden. Our results show that over 10% of the leisure boats (n = 686) contain >400 µg/cm(2) of tin in their antifouling coatings. For comparison, one layer (40 µm dry film) of a TBT-paint equals ≈ 800 µg Sn/cm(2). To our knowledge, tin has never been used in other forms than organotin (OT) in antifouling paints. Thus, even though the XRF analysis does not provide any information on the speciation of tin, the high concentrations indicate that these leisure boats still have OT coatings present on their hull. On several leisure boats we performed additional XRF measurements by progressively scraping off the top coatings and analysing each underlying layer. The XRF data show that when tin is detected, it is most likely present in coatings close to the hull with several layers of other coatings on top. Thus, leaching of OT compounds from the hull into the water is presumed to be negligible. The risk for environmental impacts arises during maintenance work such as scraping, blasting and high pressure hosing activities. The data also show that many boat owners apply excessive paint layers when following paint manufacturers recommendations. Moreover, high loads of copper were detected even on boats sailing in freshwater, despite the more than 20 year old ban, which poses an environmental risk that has not been addressed until now.

New analytical application for metal determination in antifouling paints.

Despite the ban of applying TBT coatings on leisure boats in the late 80s, recent studies show an ongoing spread of TBT from leisure boats, particularly during hull cleaning events. Therefore, countries in EU have adopted expensive measures to clean this wash water. A more cost-efficient measure is to focus directly on the source, i.e. identify leisure boats with high concentrations of TBT and prescribe boat owners to remove the paint. We have developed a new antifouling paint application for a handheld X-ray fluorescence (XRF) analyzer to be used for identifying boats with high area concentrations (µg/cm(2)) of Sn (indication that the hull contains TBT paint residues). Copper and zinc are also included in the application since these metals are used in the vast majority of today's paints. A blind test with up to four layers of TBT-, copper- and zinc-based paints showed good correlation between XRF-measured area concentrations and chemically analyzed concentrations. Future usage of the applications involves identification of boat hulls in particular with high Sn concentrations and also with high Cu and Zn concentrations. This method has the potential to become a useful tool in regulatory management of existence and use of toxic elements on boat hulls.