Transgenerational epigenetic inheritance increases trait variation but is not adaptive.

Understanding processes that can produce adaptive phenotypic shifts in response to rapid environmental change is critical to reducing biodiversity loss. The ubiquity of environmentally induced epigenetic marks has led to speculation that epigenetic inheritance could potentially enhance population persistence in response to environmental change. Yet, the magnitude and fitness consequences of epigenetic marks carried beyond maternal inheritance are largely unknown. Here, we tested how transgenerational epigenetic inheritance (TEI) shapes the phenotypic response of Daphnia clones to the environmental stressor Microcystis. We split individuals from each of eight genotypes into exposure and control treatments (F0 generation) and tracked the fitness of their descendants to the F3 generation. We found transgenerational epigenetic exposure to Microcystis led to reduced rates of survival and individual growth and no consistent effect on offspring production. Increase in trait variance in the F3 relative to F0 generations suggests potential for heritable bet hedging driven by TEI, which could impact population dynamics. Our findings are counter to the working hypothesis that TEI is a generally adaptive mechanism likely to prevent extinction for populations inhabiting rapidly changing environments.

Intraspecific genetic variation is critical to robust toxicological predictions of aquatic contaminants.

Environmental risk assessment is a critical tool for protecting aquatic life and its effectiveness is predicated on predicting how natural populations respond to contaminants. Yet, routine toxicity testing typically examines only one genotype, which may render risk assessments inaccurate as populations are most often composed of genetically distinct individuals. To determine the importance of intraspecific variation in the translation of toxicity testing to populations, we quantified the magnitude of genetic variation within 20 Daphnia magna clones derived from one lake using whole genome sequencing and phenotypic assays. We repeated these assays across two exposure levels of microcystins, a cosmopolitan and lethal aquatic contaminant produced by harmful algal blooms. We found considerable intraspecific genetic variation in survival, growth, and reproduction, which was amplified by microcystins exposure. Finally, using simulations we demonstrate that the common practice of employing a single genotype to calculate toxicity tolerance failed to produce an estimate within the 95% confidence interval over half of the time. These results illuminate the importance of incorporating intraspecific genetic variation into toxicity testing to reliably predict how natural populations will respond to aquatic contaminants.

Cyanotoxins accumulate in Lake St. Clair fish yet their fillets are safe to eat.

Consuming fish exposed to cyanobacterial harmful algal blooms (HABs) may be a major route of microcystin toxin exposure to humans. However, it remains unknown whether fish can accumulate and retain microcystins temporally in waterbodies with recurring seasonal HABs, particularly before and after a HAB event when fishing is active. We conducted a field study on Largemouth Bass, Northern Pike, Smallmouth Bass, Rock Bass, Walleye, White Bass, and Yellow Perch to assess the human health risks to microcystin toxicity via fish consumption. We collected 124 fish in 2016 and 2018 from Lake St. Clair, a large freshwater ecosystem in the North American Great Lakes that is actively fished pre- and post-HAB periods. Muscles were analyzed using the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) Lemieux Oxidation method for total microcystins, which was used to perform a human health risk assessment for comparison against fish consumption advisory benchmarks available for Lake St. Clair. From this collection 35 fish livers were additionally extracted to confirm the presence of microcystins. Microcystins were detected in all livers at widely varying concentrations (1-1500 ng g-1 ww), suggesting HABs are an underappreciated and pervasive stressor to fish populations. Conversely, microcystin levels were consistently low in muscles (0-15 ng g-1 ww) and presented negligible risk, empirically supporting that fillets may be safely consumed before and after HAB events following fish consumption advisories.

Lake Erie fish safe to eat yet afflicted by algal hepatotoxins.

Toxic harmful algal blooms (HABs) pose serious threats to human health and instances of wildlife death have been documented across taxa. However, the extent of toxicological impacts on wildlife species is largely unresolved, raising uncertainty about the repercussions of increasingly severe HABs on the biodiversity and functioning of aquatic ecosystems. Here, we conducted a field study to assess human health risks from consuming fish caught across all stages of a HAB and to determine the pervasiveness of potentially harmful levels of the cosmopolitan toxin microcystin on fish populations. We collected 190 fish in 2015 and 2017 from Lake Erie, a large freshwater ecosystem that is highly productive for fisheries and is an epicenter of HABs and microcystin toxicity events. Fish muscles and livers were analyzed for total microcystins, which was used to conduct a human health risk assessment for comparison against fish consumption advisory benchmarks available for Lake Erie. We found microcystins pose low risks to human health from fillet consumption (mean 1.80 ng g-1 ww) but substantial risks to fish health and recruitment from liver concentrations measured well before and after seasonal bloom events (mean 460.13 ng g-1 ww). Our findings indicate HABs are a previously underappreciated but pervasive threat to fish populations.

Experimental Evidence from the Field that Naturally Weathered Microplastics Accumulate Cyanobacterial Toxins in Eutrophic Lakes.

Freshwater ecosystems with recurring harmful algal blooms can also be polluted with plastics. Thus the two environmental problems may interact. To test whether microplastics influence the partitioning of microcystins in freshwater lakes, we examined the sorption of four microcystin congeners to different polymers of commercially available plastics (low-density polyethylene, polyethylene terephthalate, polyvinyl chloride, and polypropylene). We conducted three experiments: a batch sorption experiment in the laboratory with pristine microplastics of four different polymers, a second batch sorption experiment in the laboratory to compare pristine and naturally weathered microplastics of a single polymer, and a 2-month sorption experiment in the field with three different polymers experiencing natural weathering in a eutrophic lake. This series of experiments led to a surprising result: microcystins sorbed poorly to all polymers tested under laboratory conditions (<0.01% of the initial amount added), irrespective of weathering, yet in the field experiment, all polymers accumulated microcystins under ambient conditions in a eutrophic lake (range: 0-84.1 ng/g). Furthermore, we found that the sorption capacity for microcystins differed among polymers in the laboratory experiment yet were largely the same in the field. We also found that the affinity for plastic varied among microcystin congeners, namely, more polar congeners demonstrated a greater affinity for plastic than less polar congeners. Our study improves our understanding of the role of polymer and congener type in microplastic-microcystin sorption and provides novel evidence from the field, showing that naturally weathered microplastics in freshwater lakes can accumulate microcystins. Consequently, we caution that microplastics may alter the persistence, transport, and bioavailability of microcystins in freshwaters, which could have implications for human and wildlife health. Environ Toxicol Chem 2022;41:3017-3028. © 2022 SETAC.

Fish tissue accumulation and proteomic response to microcystins is species-dependent.

Cyanotoxins including microcystins are increasing globally, escalating health risks to humans and wildlife. Freshwater fish can accumulate and retain microcystins in tissues; however, uptake and depuration studies thus far have not exposed fish to microcystins in its intracellular state (i.e., cell-bound or conserved within cyanobacteria), which is a primary route of exposure in the field, nor have they investigated sublethal molecular-level effects in tissues, limiting our knowledge of proteins responsible for microcystin toxicity pathways in pre-to-postsenescent stages of a harmful algal bloom. We address these gaps with a 2-wk study (1 wk of 'uptake' exposure to intracellular microcystins (0-40 µg L-1) produced by Microcystis aeruginosa followed by 1 wk of 'depuration' in clean water) using Rainbow Trout (Oncorhynchus mykiss) and Lake Trout (Salvelinus namaycush). Liver and muscle samples were collected throughout uptake and depuration phases for targeted microcystin quantification and nontargeted proteomics. For both species, microcystins accumulated at a higher concentration in the liver than muscle, and activated cellular responses related to oxidative stress, apoptosis, DNA repair, and carcinogenicity. However, intraspecific proteomic effects between Rainbow Trout and Lake Trout differed, and interspecific accumulation and retention of microcystins in tissues within each species also differed. We demonstrate that fish do not respond the same to cyanobacterial toxicity within and among species despite being reared in the same environment and diet.

The sustainable agriculture imperative: A perspective on the need for an agrosystem approach to meet the United Nations Sustainable Development Goals by 2030.

The development of modern, industrial agriculture and its high input-high output carbon energy model is rendering agricultural landscapes less resilient. The expected continued increase in the frequency and intensity of extreme weather events, in conjunction with declining soil health and biodiversity losses, could make food more expensive to produce. The United Nations has called for global action by establishing 17 sustainable development goals (SDGs), four of which are linked to food production and security: declining biodiversity (SDG 15), loss of ecosystem services and agroecosystem stability caused by increasing stress from food production intensification and climate change (SDG 13), declining soil health caused by agricultural practices (SDGs 2 and 6), and dependence on synthetic fertilizers and pesticides to maintain high productivity (SDG 2). To achieve these SDGs, the agriculture sector must take a leading role in reversing the many negative environmental trends apparent in today's agricultural landscapes to ensure that they will adapt and be resilient to climate change in 2030 and beyond. This will demand fundamental changes in how we practice agriculture from an environmental standpoint. Here, we present a perspective focused on the implementation of an agrosystem approach, which we define to promote regenerative agriculture, an integrative approach that provides greater resilience to a changing climate, reverses biodiversity loss, and improves soil health; honors Indigenous ways of knowing and a holistic approach to living off and learning from the land; and supports the establishment of emerging circular economies and community well-being. Integr Environ Assess Manag 2022;18:1199-1205. © 2021 SETAC.

Cyanotoxins within and Outside of Microcystis aeruginosa Cause Adverse Effects in Rainbow Trout (Oncorhynchus mykiss).

The global expansion of toxic Microcystis blooms, and production of cyanotoxins including microcystins, are an increasing risk to freshwater fish. Differentiating intracellular and extracellular microcystin toxicity pathways (i.e., within and outside of cyanobacterial cells) in fish is necessary to assess the severity of risks to populations that encounter harmful algal blooms in pre-to-postsenescent stages. To address this, adult and juvenile Rainbow Trout (Oncorhynchus mykiss) were, respectively, exposed for 96 h to intracellular and extracellular microcystins (0, 20, and 100 µg L-1) produced by Microcystis aeruginosa. Fish were dissected at 24 h intervals for histopathology, targeted microcystin quantification, and nontargeted proteomics. Rainbow Trout accumulated intracellular and extracellular microcystins in all tissues within 24 h, with greater accumulation in the extracellular state. Proteomics revealed intracellular and extracellular microcystins caused sublethal toxicity by significantly dysregulating proteins linked to the cytoskeletal structure, stress responses, and DNA repair in all tissues. Pyruvate metabolism in livers, anion binding in kidneys, and myopathy in muscles were also significantly impacted. Histopathology corroborated these findings with evidence of necrosis, apoptosis, and hemorrhage at similar severity in both microcystin treatments. We demonstrate that sublethal concentrations of intracellular and extracellular microcystins cause adverse effects in Rainbow Trout after short-term exposure.

Optimization of an MMPB Lemieux Oxidation method for the quantitative analysis of microcystins in fish tissue by LC-QTOF MS.

Microcystins are toxic heptapeptides produced by cyanobacteria in marine and freshwater environments. In biological samples such as fish, microcystins can be found in the free form or covalently bound to protein phosphatases type I and II. Total microcystins in fish have been quantified in the past using the Lemieux Oxidation approach, where all toxins are oxidated to a common fragment (2-methyl-3-methoxy-4-phenylbutyric acid, MMPB) regardless of their initial amino acid configuration or form (free or protein bound). These studies have been carried out using different experimental conditions and employed different quantification strategies. The present study has further investigated the oxidation step using a systematic approach, to identify the most important factors leading to a higher, more robust MMPB generation yield from fish tissue in order to reduce the method detection limit. Field samples were quantified using an in-situ generated MMPB matrix matched calibration curve by isotope dilution with d3-MMPB via liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF MS). This approach improves method's accuracy by taking into account of potential matrix effects that could affect the derivatization, sample prepation and instrumental analysis steps. The validated method showed 16.7% precision (RSD) and +6.7% accuracy (bias), with calculated method detection limits of 7.28 ng g-1 Performance of the method was assessed with the analysis of laboratory exposed Rainbow Trout (Oncorhynchus mykiss) to cyanobacteria as a positive control, where no microcystins were detected in the pre-exposure fish liver and fillet, low levels in the exposed fillet (65.0 ng g-1) and higher levels in the exposed liver (696 ng g-1). Finally, the method was employed for the analysis of 26 fillets (muscle) and livers of Walleye (Sander vitreus) and Yellow Perch (Perca flavescens) from Lake Erie, showing very low concentrations of microcystins in the fillet and higher concentrations in liver, up to 3720 ng g-1.

Shotgun proteomics analysis reveals sub-lethal effects in Daphnia magna exposed to cell-bound microcystins produced by Microcystis aeruginosa.

Microcystins that are cell-bound within Microcystis have demonstrated the ability to cause lethal and reproductive impairment in Daphnia, who constitute an important part of aquatic food chains and are known to feed on viable cyanobacterial cells. Recent advances in environmental toxicogenomics can be used to better understand the mechanistic effects from exposure to cell-bound microcystins in Daphnia; however, there remains a need to examine the effects of microcystins exposure as a function of dose and time in order to help elucidate the progression of (sub-)lethal effects. This study examines the effects of cell-bound microcystin exposure in Daphnia magna as a function of dose and time with shotgun proteomics in order to measure and provide insightful evidence describing functional mechanisms from, and relationships between, protein populations in response to toxic Microcystis aeruginosa. We further characterize the life-history fitness of D. magna in the presence of toxic exposure by measuring somatic growth rate. Chronic dietary exposure to cell-bound microcystins reduced the somatic growth rate of D. magna. Through proteomics analysis, we identified a significant increase in abundance of proteins related to reproductive success and development, removal of superoxide radicals, and motor activity in D. magna parents exposed to cell-bound microcystins at sub-lethal concentrations. We also identified a significant decrease in abundance of proteins related to apoptosis, metabolism, DNA damage repair, and immunity in D. magna neonates. This information will improve our understanding of the risks posed by cell-bound microcystins to cladocerans in freshwater ecosystems.

Assessing the toxicity of cell-bound microcystins on freshwater pelagic and benthic invertebrates.

Cyanobacterial harmful algal blooms dominated by Microcystis frequently produce microcystins, a family of toxins capable of inflicting harm to pelagic and benthic freshwater invertebrates. Research on the effect of microcystins on invertebrates is inconclusive; from one perspective, studies suggest invertebrates can coexist in toxic blooms; however, studies have also measured negative food-associated effects from microcystins. To test the latter perspective, we examined the reproduction, growth, and survival of laboratory-cultured Ceriodaphnia dubia, Daphnia magna, and Hexagenia spp. exposed to cell-bound microcystins through a series of life-cycle bioassays. Test organisms were exposed to a concentration gradient ranging from 0.5 µg L-1 to 300 µg L-1 microcystins, which corresponds to values typically found in freshwaters during bloom season. Lethal concentrations in C. dubia (LC50 = 5.53 µg L-1) and D. magna (LC50 = 85.72 µg L-1) exposed to microcystins were among the lowest recorded to date, and reproductive effects were observed at concentrations as low as 2.5 µg L-1. Length of D. magna was significantly impacted in microcystin treatments great than 2.5 µg L-1. No lethality or growth impairments were observed in Hexagenia. This information will improve our understanding of the risks posed by microcystins to food webs in freshwaters.

An efficient and affordable laboratory method to produce and sustain high concentrations of microcystins by Microcystis aeruginosa.

Microcystis aeruginosa is a cosmopolitan cyanobacteria that continues to jeopardize freshwater ecosystem services by releasing the hepatotoxin microcystin, which can, in some cases, cause death to aquatic fauna and even humans. Currently, our abilities to understand the mechanisms of microcystin toxicology are limited by the lack of a method for producing high concentrations, which are central to large-scale and long-term research in natural systems. Here we present an efficient and affordable laboratory method to produce high concentrations of microcystins by a toxigenic strain of M. aeruginosa. Through batch culture studies, we yielded microcystins at concentrations that are environmentally relevant to freshwaters around the world (1-300 µg L-1), maintained these concentrations without resupplying fresh medium (further reducing costs), and utilized rate equations to model the relationship between the environmental conditions in the cultures and changes occurring within the M. aeruginosa cells. Our assessment suggests that steady production of microcystins depends on the availability of carbon throughout the experiment. Hence, we recommend the use of tissue culture treated flasks with a vented cap to ensure the production of microcystins is uninterrupted. This method demonstrates that microcystins can be produced in the laboratory at concentrations relevant to freshwater ecosystems. •The method demonstrates M. aeruginosa CPCC 300 is a reliable strain of freshwater cyanobacteria that can yield microcystins at environmentally relevant concentrations.•Validation showed M. aeruginosa CPCC 300 is resilient in carbon-limited situations and may respond to stress by shifting the ratio of microcystin congeners.•Cell culture flasks with vented caps -filled no more than 50 % of the flask volume to allow for sufficient air exchange- are an excellent and cost-effective approach to maintaining cell growth and producing microcystins at a range between 300 to 1200 µg L-1.

Comparative evaluation of four biosolids formulations on the effects of triclosan on plant-arbuscular mycorrhizal fungal interactions in three crop species.

Triclosan (TCS) is an antimicrobial ingredient found in personal care products that include soaps, shampoos, and other sanitation goods. TCS is moderately hydrophobic and has been shown to be resistant to wastewater treatment and thus accumulates in biosolids. Biosolids are commonly applied to agricultural land but little is known about the risk that TCS in biosolids poses to soil fungal communities following land application. The purpose of this study was to characterize the effects of TCS on the symbiotic colonization of roots in three field crops (soybean, corn, and spring wheat) by arbuscular mycorrhizal fungi (AMF) in soils amended with four different types of biosolids (liquid, dewatered, composted, alkaline and hydrolyzed). Crops were grown to maturity in pot-exposure systems under controlled temperature settings. Biosolids treatments were spiked with concentrations of TCS typically found in amended fields. Analysis of AMF colonization by hyphae, and the production of arbuscules and vesicles indicated no significant TCS concentration-dependent effects in the three plant species for any of the biosolids formulations. The data indicate that TCS present in municipal biosolids applied to agricultural lands likely poses minimal risks to AMF or its establishment of a symbiotic relationship in the three species tested.

Evaluating the effects of triclosan on 3 field crops grown in 4 formulations of biosolids.

A growing body of evidence suggests that amending soil with biosolids can be an integral component of sustainable agriculture. Despite strong evidence supporting its beneficial use in agriculture, there are concerns that chemicals, such as pharmaceuticals and personal care products, could present a risk to terrestrial ecosystems and human health. Triclosan is one of the most commonly detected compounds in biosolids. To date, laboratory studies indicate that triclosan likely poses a de minimis risk to field crops; however, these studies were either conducted under unrealistic exposure conditions or only assessed 1 or 2 formulations of biosolids. The purpose of the present study was to characterize the effects of triclosan on field crops in soils amended with 4 different formulations of biosolids (liquid, dewatered, compost, and alkaline-hydrolyzed), containing both background and spiked triclosan concentrations, following best management practices used in the province of Ontario. Three crop species (corn, soybean, and spring wheat) were evaluated using several plant growth endpoints (e.g., root wet mass, shoot length, shoot wet/dry mass) in 70-d to 90-d potted soil tests. The results indicated no adverse impact of triclosan on any crop-biosolids combination. Conversely, amending soil with biosolids either enhanced or had no negative effect, on the growth of plants. Results of the present study suggest little risk of triclosan to crops in agricultural fields amended with biosolids. Environ Toxicol Chem 2017;36:1896-1908. © 2016 SETAC.