Strength of methods assessment for aquatic primary producer toxicity data: A critical review of atrazine studies from the peer-reviewed literature.

Improving the quality of pesticide toxicity studies is a shared goal in ecotoxicology and a priority for risk assessors. Using the herbicide atrazine and testing on primary producers as a case study, we developed and applied a transparent scoring system for assessing the quality of peer-reviewed studies. The exercise also highlights where data gaps exist for planning future work. We determined that, while a large number of studies (147) present experimental data fitting basic inclusion criteria, only a small proportion provide sufficient information on the test substance, test organism, and test results to be considered of sufficient quality (i.e., a minimum score of >8 out of 16, meaning no critical study weaknesses identified) that would allow recommendation for their use in decision-making. Optimal studies for use in first tier risk assessment were further identified for each taxonomic group as the highest-scoring study scoring >8, that also used the technical grade active ingredient, reported an EC50 for a population-level endpoint (e.g. cell density, dry weight), and an exposure period in line with standard tests (≤96-h for algae, ≤14-d for macrophytes). Ultimately, 22 freshwater studies (four periphyton, ten macrophytes, and eight phytoplankton) achieved scores >8. Only one study with marine phytoplankton scored >8, and no studies met the risk assessment inclusion criteria for marine/estuarine periphyton or macrophytes. This indicates a potential research need with respect to toxicity data for salt-water species. Finally, registrant studies were evaluated, and in many cases, were the most appropriate for risk assessment, with the greatest scores observed for their respective species relative to those reported in the peer-reviewed literature. This exercise highlights the importance of defining and identifying well-performed toxicity tests, illuminating knowledge gaps, and reporting high quality data in support of the risk assessment process outside of the standard regulatory framework.

A freshwater mesocosm study into the effects of the neonicotinoid insecticide thiamethoxam at multiple trophic levels.

Thiamethoxam is a neonicotinoid insecticide used widely in agriculture to control a broad spectrum of insect pests. To assess potential risks from this compound to non-target aquatic organisms, an outdoor mesocosm study was performed. Mesocosms (1300 L) were treated once with a formulated product with the active substance (a.s.) thiamethoxam at nominal concentrations of 1 (n = 3), 3 (n = 3), 10 (n = 4), 30 (n = 4), and 100 (n = 2) µg a.s./L, plus untreated controls (n = 4). Primary producers (phytoplankton), zooplankton, and macroinvertebrates were monitored for up to 93 days following treatment. Thiamethoxam was observed to have a water column dissipation half-life (DT50) of ≤1.6-5.2 days in the mesocosms. Community-based principal response curve analysis detected no treatment effects for phytoplankton, zooplankton, emergent insects, and macroinvertebrates, indicating a lack of direct and indirect effects. A number of statistically significant differences from controls were detected for individual phytoplankton and zooplankton species abundances, but these were not considered to be treatment-related due to their transient nature and lack of concentration-response. After application of 30 µg a.s./L, slight temporary effects on Asellus aquaticus could not be excluded. At 100 µg a.s./L, there was an effect with no clear recovery of Asellus observed, likely due to their inability to recolonize these isolated test systems. A statistically significant but transient reduction in the emergence of chironomids by day 23 at the 100 µg a.s./L treatment was observed and possibly related to direct toxicity from thiamethoxam on larval stages. Therefore, a conservative study specific No Observed Ecological Adverse Effect Concentration (NOEAEC) is proposed to be 30 µg a.s./L. Overall, based on current concentrations of thiamethoxam detected in North American surface waters (typically <0.4 µg/L), there is low likelihood of direct or indirect effects from a pulsed exposure on primary producers, zooplankton, and macroinvertebrates, including insects, as monitored in this study.

Response of the mayfly (Cloeon dipterum) to chronic exposure to thiamethoxam in outdoor mesocosms.

Thiamethoxam is a widely used neonicotinoid insecticide that has been detected in surface water monitoring programs in North America and Europe. This has led to questions about its toxicity to nontarget insects, specifically those with an aquatic life stage. To address the uncertainty associated with possible impacts from environmental exposures, a chronic (35-d) outdoor mesocosm study with a formulated product containing thiamethoxam was conducted. The specific focus of the study was the response of mayflies (Ephemeroptera), which have been reported to be particularly sensitive in laboratory studies. A range of concentrations (nominally 0.1, 0.3, 1.0, 3.0, and 10.0 µg/L thiamethoxam), plus untreated controls were tested, and the abundance and emergence of mayflies (Cloeon dipterum) were assessed weekly for 35 d. Mean measured time-weighted average exposures were within 6% of nominal over the duration of the study, with the mean half-life of thiamethoxam in each treatment ranging from 7 to 13 d. Statistically significant reductions in both larval abundance and adult emergence were observed at 10.0, 3.0, and 1.0 µg/L following 1, 2, and 3 wk of exposure, respectively. Exposure to 0.1 and 0.3 µg/L thiamethoxam had no statistically significant effect on larval mayfly abundance or adult emergence at any point in the study. These findings support a 35-d no-observed-effect concentration (NOEC) of 0.3 µg thiamethoxam/L for mayflies (C. dipterum) under chronic conditions. Furthermore, because the 95th percentile of environmental concentrations has been reported to be 0.054 µg/L, these results indicate that populations of C. dipterum and similarly sensitive aquatic insects are unlikely to be significantly impacted by thiamethoxam exposure in natural systems represented by the conditions in our study. Environ Toxicol Chem 2018;37:1040-1050. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Comprehensive characterization of the acute and chronic toxicity of the neonicotinoid insecticide thiamethoxam to a suite of aquatic primary producers, invertebrates, and fish.

Thiamethoxam is a neonicotinoid insecticide used widely in agriculture to control a broad spectrum of chewing and sucking insect pests. Recent detection of thiamethoxam in surface waters has raised interest in characterizing the potential impacts of this insecticide to aquatic organisms. We report the results of toxicity testing (acute and chronic) conducted under good laboratory practices for more than 30 freshwater species (insects, molluscs, crustaceans, algae, macrophytes, and fish) and 4 marine species (an alga, a mollusc, a crustacean, and a fish). As would be anticipated for a neonicotinoid, aquatic primary producers and fish were the least sensitive organisms tested, with acute median lethal and effect concentrations (LC50/EC50) observed to be ≥80 mg/L in all cases, which far exceeds surface water exposure concentrations. Tested molluscs, worms, and rotifers were similarly insensitive (EC50 ≥ 100 mg/L), except for Lumbriculus sp., with an EC50 of 7.7 mg/L. In general, insects were the most sensitive group in the study, with most acute EC50 values < 1 mg/L. However, the crustaceans Asellus aquaticus and Ostracoda exhibited a sensitivity similar to that of insects (acute EC50 < 1 mg/L), and the midge larvae Chaoborus sp. were relatively insensitive compared with other insects (EC50 > 5.5 mg/L). The most sensitive chronic response was for Chironomus riparius, with a 30-d no-observed-effect concentration (NOEC; emergence) of 0.01 mg/L. Observed toxicity to the tested marine organisms was comparable to that of freshwater species. We used the reported data to construct species sensitivity distributions for thiamethoxam, to calculate 5% hazard concentrations (HC5s) for acute data (freshwater invertebrates), and compared these with measured concentrations from relevant North American surface waters. Overall, based on acute toxicity endpoints, the potential acute risk to freshwater organisms was found to be minimal (likelihood of exceeding HC5s < 1%). Environ Toxicol Chem 2017;36:2838-2848. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Influence of light, nutrients, and temperature on the toxicity of atrazine to the algal species Raphidocelis subcapitata: Implications for the risk assessment of herbicides.

The acute toxicity of herbicides to algae is commonly assessed under conditions (e.g., light intensity, water temperature, concentration of nutrients, pH) prescribed by standard test protocols. However, the observed toxicity may vary with changes in one or more of these parameters. This study examined variation in toxicity of the herbicide atrazine to a representative green algal species Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) with changes in light intensity, water temperature, concentrations of nutrients or combinations of these three parameters. Conditions were chosen that could be representative of the intensive corn growing Midwestern region of the United States of America where atrazine is used extensively. Varying light intensity (4-58µmol/m(2)s) resulted in no observable trend in 96-h EC50 values for growth rate. EC50 values for PSII yield generally increased with decreasing light intensity but not significantly in all cases. The 96-h EC50 values for growth rate decreased with decreases in temperature (20-5°C) from standard conditions (25°C), but EC50 values for PSII yield at lower temperatures were not significantly different from standard conditions. Finally, there was no clear trend in 96-h EC50 values for both endpoints with increases in nitrogen (4.1-20mg/L) and phosphorus (0.24-1.2mg/L). The 96-h EC50 values for both endpoints under combinations of conditions mimicking aquatic systems in the Midwestern U.S. were not significantly different from EC50 values generated under standard test conditions. This combination of decreased light intensity and temperature and increased nutrients relative to standard conditions does not appear to significantly affect the observed toxicity of atrazine to R. subcapitata. For atrazine specifically, and for perhaps other herbicides, this means current laboratory protocols are useful for extrapolating to effects on algae under realistic environmental conditions.

Effects of atrazine on egg masses of the yellow-spotted salamander (Ambystoma maculatum) and its endosymbiotic alga (Oophila amblystomatis).

Embryonic growth of the yellow-spotted salamander (Ambystoma maculatum) is enhanced by the presence of the green alga Oophila amblystomatis, in the egg capsule. To further assess potential impacts of herbicides on this relationship, A. maculatum egg masses were exposed to atrazine (0-338 µg/L) until hatching (up to 66 days). Exposure to atrazine reduced PSII yield of the symbiotic algae in a concentration-dependent manner, but did not significantly affect visible algal growth or any metrics associated with salamander development. Algal cells were also cultured in the laboratory for toxicity testing. In the 96-h growth inhibition test (0-680 µg/L), ECx values were generally greater than those reported for standard algal test species. Complete recovery of growth rates occurred within 96-h of transferring cells to untreated media. Overall, development of A. maculatum embryos was not affected by exposure to atrazine at concentrations and durations exceeding those found in the environment.

Optimization of culturing conditions for toxicity testing with the alga Oophila sp. (Chlorophyceae), an amphibian endosymbiont.

Eggs of the yellow-spotted salamander (Ambystoma maculatum) have a symbiotic relationship with green algae. It has been suggested that contaminants that are preferentially toxic to algae, such as herbicides, may impair the symbiont and, hence, indirectly affect the development of the salamander embryo. To enable testing under near-standard conditions for first-tier toxicity screening, the authors isolated the alga from field-collected eggs and identified conditions providing exponential growth rates in the apparent asexual phase of the alga. This approach provided a uniform, single-species culture, facilitating assessment of common toxicity end points and comparison of sensitivity relative to other species. Sequencing of the 18s ribosomal DNA indicated that the isolated alga is closely related to the recently described Oophila amblystomatis but is more similar to other known Chlamydomonas species, suggesting possible biogeographical variability in the genetic identity of the algal symbiont. After a tiered approach to culturing method refinement, a modified Bristol's media with 1 mM NH4 (+) as nitrogen source was found to provide suitable conditions for toxicity testing at 18 °C and 200 µmol m(-2) s(-1) photosynthetically active radiation (PAR) on a 24-h light cycle. The validity of the approach was demonstrated with Zn(2+) as a reference toxicant. Overall, the present study shows that screening for direct effects of contaminants on the algal symbiont without the presence of the host salamander is possible under certain laboratory conditions.

Response of the green alga Oophila sp., a salamander endosymbiont, to a PSII-inhibitor under laboratory conditions.

In a rare example of autotroph-vertebrate endosymbiosis, eggs of the yellow-spotted salamander (Ambystoma maculatum) are colonized by a green alga (Oophila sp.) that significantly enhances salamander development. Previous studies have demonstrated the potential for impacts to the salamander embryo when growth of the algae is impaired by exposure to herbicides. To further investigate this relationship, the authors characterized the response of the symbiotic algae (Oophila sp.) alone to the photosystem II (PSII) inhibitor atrazine under controlled laboratory conditions. After extraction of the alga from A. maculatum eggs and optimization of culturing conditions, 4 toxicity assays (96 h each) were conducted. Recovery of the algal population was also assessed after a further 96 h in untreated media. Average median effective concentration (EC50) values of 123 µg L(-1) (PSII yield), 169 µg L(-1) (optical density), and 299 µg L(-1) (growth rate) were obtained after the 96-h exposure. Full recovery of exposed algal populations after 96 h in untreated media was observed for all endpoints, except for optical density at the greatest concentration tested (300 µg L(-1) ). Our results show that, under laboratory conditions, Oophila sp. is generally less sensitive to atrazine than standard test species. Although conditions of growth in standard toxicity tests are not identical to those in the natural environment, these results provide an understanding of the tolerance of this alga to PSII inhibitors as compared with other species.

Sensitivity of a green alga to atrazine is not enhanced by previous acute exposure.

Exposure to atrazine in small lotic systems can be episodic, with short-term pulses (peaks) followed by lower, decreasing concentrations. Algae and macrophytes recover rapidly from pulsed exposure to atrazine, but reported observations of population response to subsequent exposures are minimal and inconclusive. Consequently, the sensitivity of Pseudokirchneriella subcapitata to atrazine following a pulsed exposure was assessed. Exposure concentrations reflected amplifications of those observed in streams from highly vulnerable watersheds in regions of intense use. Initial pulsed atrazine exposure at 0, 150 or 300 µg/L for 24-h was followed by 72-h exposure to 0, 5, 10, 25, or 50 µg/L. Measured responses were cell density, growth rate, chlorophyll-a, and maximum quantum yield of photosystem II. Algal recovery was rapid and prior pulsed exposure to atrazine did not significantly affect subsequent sensitivity (EC10s, EC25s) for any endpoint, indicating no changes in tolerance at the population level for this species.

Interactions between atrazine and phosphorus in aquatic systems: effects on phytoplankton and periphyton.

It has been proposed that the herbicide atrazine may increase rates of parasitic trematode infection in amphibians. This effect may occur indirectly as a result of increased biomass of periphyton and augmented populations of aquatic snails, which are the trematode's primary larval host. Evidence has also shown that nutrients alone may induce the same indirect responses. Since both atrazine and nutrients commonly enter surface waters from agricultural run-off, their spatial and temporal co-occurrence are highly probable. In light of recent wide-spread declines in amphibian populations, a better understanding of the role of atrazine in the proposed ecological mechanism is necessary. A microcosm study was conducted to quantify biomass of phytoplankton and periphyton over a range of atrazine and phosphorus concentrations (from 0 to 200 µg L(-1) each) using a central composite rotatable design. Over 10 weeks, biomass and water chemistry were monitored using standard methods. Regression and canonical analyses of the response surfaces for each parameter were conducted. We found significant effects of atrazine and phosphorus on dissolved oxygen, pH, and conductivity throughout the study. Additions of phosphorus mitigated the apparent inhibition of these photosynthetic indicators caused by atrazine. Despite these changes, no consistent treatment-related differences in algal biomass were observed. These results indicate that the indirect impacts of atrazine on total growth of periphyton and likely, subsequent effects on aquatic snails, are not expected to be ecologically significant at the concentrations of atrazine tested (up to 200 µg L(-1)) and over a range of nutrient conditions commonly occurring in agroecosystems.

Atrazine does not affect algal biomass or snail populations in microcosm communities at environmentally relevant concentrations.

The herbicide atrazine is a photosynthetic inhibitor used around the world in agricultural applications. Contamination of surface waters adjacent to treated areas can directly reduce growth of nontarget aquatic autotrophs, but the severity of impacts is highly dependent on species sensitivity and exposure concentration. Secondary effects resulting from macrophyte or phytoplankton decline may include an expansion of the more tolerant periphyton community. Recently, this shift in the autotrophic community has been proposed as a mechanism for increased rates of parasite infections in amphibians via augmented populations of aquatic snails which act as intermediate hosts to larval trematodes. To further clarify this relationship, an outdoor microcosm study was conducted to examine the effects of atrazine on primary production and snail populations over a range of environmentally relevant concentrations. In July 2009, 15 experimental ponds were treated to achieve initial concentrations of 0, 1, 10, 30, and 100 µg/L atrazine. Over a period of 73 d, measures were taken of macrophyte, phytoplankton, and periphyton biomass, growth, and fecundity of caged snails (Physella spp. and Stagnicola elodes) and free-living snails (Physella spp.). Except for declines in macrophyte biomass at the highest treatment level, no consistent relationships were found between atrazine concentration and any measured parameter. Comparison of these results with previous findings highlights the variability of responses to atrazine exposure between similarly constructed freshwater communities, even at concentrations up to 20 times higher than sustained environmental levels.