Gerald R. Stephenson

Comparative effects of pH and Vision® herbicide on two life stages of four anuran amphibian species

Vision, a glyphosate-based herbicide containing a 15% (weight:weight) polyethoxylated tallow amine surfactant blend, and the concurrent factor of pH were tested to determine their interactive effects on early life-stage anurans. Ninety-six-hour laboratory static renewal studies, using the embryonic and larval life stages (Gosner 25) of Rana clamitans, R. pipiens, Bufo americanus, and Xenopus laevis, were performed under a central composite rotatable design. Mortality and the prevalence of malformations were modeled using generalized linear models with a profile deviance approach for obtaining confidence intervals. There was a significant (p < 0.05) interaction of pH with Vision concentration in all eight models, such that the toxicity of Vision was amplified by elevated pH. The surfactant is the major toxic component of Vision and is hypothesized, in this study, to be the source of the pH interaction. Larvae of B. americanus and R. clamitans were 1.5 to 3.8 times more sensitive than their corresponding embryos, whereas X. laevis and R. pipiens larvae were 6.8 to 8.9 times more sensitive. At pH values above 7.5, the Vision concentrations expected to kill 50% of the test larvae in 96-h (96-h lethal concentration [LC50]) were predicted to be below the expected environmental concentration (EEC) as calculated by Canadian regulatory authorities. The EEC value represents a worst-case scenario for aerial Vision application and is calculated assuming an application of the maximum label rate (2.1 kg acid equivalents [a.e.]/ha) into a pond 15 cm in depth. The EEC of 1.4 mg a.e./L (4.5 mg/L Vision) was not exceeded by 96-h LC50 values for the embryo test. The larvae of the four species were comparable in sensitivity. Field studies should be completed using the more sensitive larval life stage to test for Vision toxicity at actual environmental concentrations.

Chemical and biomonitoring to assess potential acute effects of Vision herbicide on native amphibian larvae in forest wetlands.

In conjunction with operational forest herbicide spray programs in Ontario, Canada, chemical and biological monitoring studies were conducted in 51 different wetlands to quantify the probability and magnitude of contamination by a glyphosate herbicide formulation (Vision). Wetlands were classified as oversprayed, adjacent, or buffered in relation to the operational target spray blocks. Results show that vegetated buffers significantly mitigated against exposure and thus potential for acute effects. Aqueous concentrations of glyphosate in buffered wetlands were below analytical limits of quantitation (0.02 mg acid equivalent [a.e.]/L) in 14 of 16 cases, with mean concentration (0.03 +/- 0.02 mg a.e./L) significantly (p < 0.05) less than that of either adjacent (0.18 +/- 0.06 mg a.e./L) or oversprayed wetlands (0.33 +/- 0.11 mg a.e./L). Biomonitoring with caged amphibian larvae showed no significant differences among mean mortality (48 h) of either Rana pipiens (p = 0.194) or Rana clamitans larvae (p = 0.129) exposed in situ to Vision under these various wetland conditions. Percent mortality was not significantly (p = 0.05) correlated with exposure concentrations for either amphibian species tested. Results suggest that exposures typically occurring in forest wetlands are insufficient to induce significant acute mortality in native amphibian larvae.

Effects of Vision® herbicide on mortality, avoidance response, and growth of amphibian larvae in two forest wetlands

The effects of Vision (glyphosate, 356 mg acid equivalents (a.e.)/L) on mortality, avoidance response, and growth of larval amphibians (Rana clamitans and Rana pipiens) were investigated using in situ enclosures deployed in two forest wetlands of northern Ontario, Canada. In addition to untreated controls, Vision was applied to yield initial concentrations ranging from 0.29 to 14.3 mg a.e./L (0.94-46.1 mg/L of Vision). Resultant 96-h median lethal concentration (LC50) values ranged from 2.70 to 11.5 mg a.e./L (8.71-37.1 mg/L of Vision) depending on the species or site involved. Substantial mortality and incidences of abnormal avoidance response occurred only at concentrations exceeding the expected environmental concentrations (EEC) (1.43 mg a.e./L, or 4.61 mg/L of Vision) as calculated by Canadian regulatory authorities. The concentration dependence of larval growth rate and maximum size varied depending on site and species. Mean growth rates and maximum sizes exposed to 1.43 mg a.e./L (EEC) treatments were the same or greater than controls. Experimental site and biotic/abiotic factors therein, such as pH and suspended sediments, substantially affected the expression of Vision herbicide toxicity in the amphibian larvae tested. Overall, results suggest that the silvicultural use of Vision herbicide in accordance with the product label and standard Canadian environmental regulations should have negligible adverse effects on sensitive larval life stages of native amphibians.

The distribution of nine pesticides between the juice and pulp of carrots and tomatoes after home processing

The distribution of nine pesticides between the juice and pulp of carrots and tomatoes during home culinary practices was investigated. Tomato and carrot pulp contained a higher percentage of all pesticide residues, except for mancozeb in tomatoes. Although there was a difference in the relative distribution of the pesticides between the commodities with greater amounts present in the pulp of tomatoes, the pesticides followed a similar trend in both. A relationship between the pulp/juice distribution and water solubility of the pesticide was apparent. Pesticides with the highest water solubility were present to a greater extent in the juice. An exception was noted in the case of diazinon and parathion, which were present in higher amounts in the pulp than their water solubility would suggest. The percent residue in the pulp ranged from 56.4 to 75.2% for carrots, and 49.7 to 95.4% for tomatoes. Residues in the juice prepared from washed commodities ranged from not detected to 0.83 microgram/g. Washing of the produce removed more residue from carrots than from tomatoes, but it did not affect the relative distribution of the residues. The behaviour and fate of the chemical varied with the pesticide as well as the crop.

Foliar Interception and Retention Values after Pesticide Application. A Proposal for Standardized Values for Environmental Risk Assessment (Technical Report)

In performing risk assessments for plant protection products by applicants or regulators in relation to the registration of the products, an important aspect to take into account is the foliar interception and retention of the active substance of the product on the plant. An overview is given of the approaches to this item in several parts of the world. The relevant circumstances and influencing variables, such as growth phase, planting density, and some physicochemical characteristics (e.g., vapor pressure and Henrys coefficient) are dealt with. Finally, a proposal is presented for how to take into account the phenomenon of foliar interception and retention in the initial phase, first tier, of the risk assessment process.

Estimation of Pesticide Exposure to Greenhouse Application Using Video Imaging and Other Assessment Techniques

Pesticide exposure in greenhouse applicators was measured using the video imaging technique to assess exposure (VITAE) along with dermal patches, air monitoring, and biological assessment techniques. The exposure of five males to pesticides during high- and low-volume application methods in commercial greenhouse operations was evaluated. Failure to use precautionary handling methods when using low-volume applications resulted in the highest level of dermal tracer deposition. Results demonstrated nonuniform deposition of tracer/pesticide mixtures on various body regions, supporting earlier work that questioned the assumption of uniform deposition when assessing exposure with the dermal patch technique. By combining the tracer with an oil-based concentrate, it remained uniformly suspended in the spray solution, and deposition ratios remained constant. Estimates of pirimicarb exposure using the VITAE method were highly correlated with excretion of urinary metabolites (r2 = 0.93). The immediate visual results provided by the VITAE system to applicators proved to be a powerful educational tool in helping them adopt precautionary application techniques. The need to employ protective operating procedures when handling pesticides was demonstrated, no matter how brief the exposure period.

The effect of CDAA (N,N,-diallyl-2-chloroacetamide) pretreatments on subsequent CDAA injury to corn (Zea mays L.)

Abstract The effects of CDAA ( N,N -diallyl-2-chloroacetamide) pretreatment on subsequent CDAA injury to corn were examined and compared with the effects of the herbicide protectant R-25788 ( N,N ,-diallyl-2,2-dichloroacetamide). In addition, the effects of CDAA pretreatment on subsequent CDAA metabolism were determined. It was found that 5μ M CDAA protected corn from injury by 200 μ M CDAA when given as a 2.5- or 1-day pretreatment. R-25788 at similar concentrations did not protect corn from subsequent R-25788 injury. Pretreatment with CDAA increased GSH levels of corn roots by 61% within 1 day, and these levels did not increase with a longer 2.5-day pretreatment with CDAA. GSH- S -transferase activity was assayed spectrophotometrically using CDNB (1-chloro-2,4-dinitrobenzene). A 1-day pretreatment with CDAA increased the root GSH- S -transferase activity by 35%, but did not affect shoot GSH- S -transferase activity. A 2.5-day pretreatment resulted in a 50% increase in root GSH- S -transferase activity but no response of the shoot enzyme was observed. Even longer pretreatments with CDAA did not result in any further increases in enzyme activity. When corn roots pretreated with CDAA for 2.5 days were excised and incubated with radiolabeled CDAA, they exhibited greater rates of uptake and metabolism than did nonpretreated roots. With in vitro studies, a fairly high rate of nonenzymatic degradation of CDAA was observed. However, the enzymatic rate was always double that of the nonenzymatic rate under the experimental conditions used. It is concluded that elevations in the GSH levels and GSH- S -transferase activities of corn roots following CDAA pretreatments may be involved in the protection of corn from subsequent CDAA injury.

THE IMPACT OF INSECTICIDES AND HERBICIDES ON THE BIODIVERSITY AND PRODUCTIVITY OF AQUATIC COMMUNITIES

Relyea (2005a), asserts that current application rates for the glyphosate formulation Roundup are highly lethal to many amphibian species. We strongly disagree with this conclusion since it is based on inappropriate generalization and extrapolative inference from a single test that is arguably irrelevant to most real-world scenarios. We contend that the application rates and resultant aqueous exposure concentrations used in this experiment are quite atypical of those associated with dominant uses of glyphosate formulations in agriculture, forestry, and industrial sectors. Two specific statements in the Relyea paper serve as focal points for concern: 1) This study represents one of the most extensive experimental investigations of pesticide effects on aquatic communities and offers a comprehensive perspective on the impacts of pesticides when non-target organisms are examined under ecologically relevant conditions. 2) Collectively, the available data indicate that, contrary to conventional wisdom, current application rates of Roundup can be lethal to many species of amphibians. Both statements exemplify a tendency for overgeneralization and excessive extrapolation. We submit that no single experiment represents an extensive investigation of pesticide effects on aquatic organisms. Neither could it offer a comprehensive perspective on such a broad topic area. Bartell et al. (1992), notes that ecological risk estimation is complex and requires, as a minimum, full characterization of concentration–response relationships and determination of the range of real-world exposure concentrations relevant to major use patterns. Resultant risk estimates are based on the probability that significant deleterious effects may be generated under real-world exposure regimes. A comprehensive perspective would require review and critical analysis of hundreds of previously published papers. None of these fundamental requirements are met in this publication. We certainly agree with the author on the general value of mesocosm studies in assessing potential effects of pesticides in aquatic systems. However, as discussed previously (e.g., Touart and Slimak 1989, Thompson 2004), mesocosm testing should be considered as one element in a multi-tiered research program that generates data, which when considered in whole, may be sufficient for effective estimation of ecological risk. The industry and complexity involved in conducting a single mesocosm study does not confer broad spectrum inference potential. Moreover, results must be considered in relation to relevant pre-existing data and with due consideration for the ‘‘weight of scientific evidence’’ principle. These criteria are also not met by the Relyea (2005a) publication. This is demonstrated by the fact that a number of directly relevant papers (e.g., Berrill et al. 1997, Lajmanovich et al. 2003, Chen et al. 2004, Edginton et al. 2004, Howe et al. 2004, Thompson 2004, Thompson et al. 2004, Wojtaszek et al. 2004) comprising a significant proportion of the preexisting body of knowledge on potential effects of glyphosate herbicides to amphibians were neither cited nor discussed in the paper. This omission is particularly disquieting given the author’s explicit statement (Relyea 2005a:625) that ‘‘Prior tests of glyphosate on amphibians have been rare,’’ the fact that several of these papers draw opposing conclusions, and the author’s assertion as to what the ‘‘collective data’’ on this topic indicate. In our view, neither the specific results of this experiment nor the collective data support the conclusion that Roundup is likely to be lethal to many species of amphibians under environmentally realistic use scenarios. This viewpoint is based on the general lack of relevance of experimental results to typical use patterns and application rates, and available data on measured concentrations in lentic systems associated with the predominant use of glyphosate formulations in agriculture, forestry or industrial sectors. In addition, several methodological issues and design flaws result in substantial uncertainty in extrapolation of test results to effects that might be expected in natural aquatic systems. To facilitate cross-comparisons and avoid confusion, all application rates and aqueous concentration values in this letter have been converted to units of glyphosate acid equivalents (a.e.). In the case of formulations containing the isopropylamine (IPA) salt, as for the test material in the Relyea study, the appropriate conversion factor is based on the molecular weight ratio of glyphosate acid to isoproplyamine salt (356/480 1⁄4

Comparative metabolism of atrazine and EPTC in proso millet (Panicum miliaceum L.) and corn

Abstract The purpose of this study was to examine the differential activities of proso millet ( Panicum miliaceum L.) and corn ( Zea mays L.) with respect to atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- S -triazine] and EPTC ( S -ethyldipropyl thiocarbamate) metabolism. GSH- S -transferase was isolated from proso millet shoots and roots. When assayed spectrophotometrically using CDNB (1-chloro 2,4-dinitrobenzene) as a substrate, the shoot enzyme had only 10% of the activity of corn shoot enzyme while the root enzyme had 33% the activity of corn root enzyme. However, when proso millet shoot GSH- S -transferase was assayed in vitro using 14 C-ring-labeled atrazine, it degraded the atrazine to water-soluble products at the same rate as the corn shoot enzyme. Incubation of excised proso millet and corn roots with [ 14 C]EPTC indicated that uptake of EPTC was similar in both plants. However, proso millet metabolized the EPTC to water-soluble products at only half the rate of corn. Glutathione levels of proso millet roots were 35.9 μg GSH/g fresh wt, compared with 65.4 μg GSH/g fresh wt for corn. However, a 2.5-day pretreatment with R-25788 (N,N-diallyl-2-2-dichloroacetamide) elevated proso millet GSH levels to 62.7 μg GSH/g fresh wt. R-25788 did not elevate the activity of proso millet GSH- S -transferase, in contrast to its effects on corn. We conclude that differences in response to atrazine and EPTC in proso millet and corn are a result of their differential metabolism.

Estimating pirimicarb exposure to greenhouse workers using video imaging

Exposure of greenhouse chrysanthemum workers to the pesticide pirimicarb was measured by the Video Imaging Technique to Assess Exposure (VITAE) along with air monitoring and biological assessment techniques. Workers at five commercial chrysanthemum operations were video imaged prior to debudding plants which had been treated with a fluorescent tracer and the pesticide, pirimicarb, 36–48 h earlier. After 1–4 h of debudding, workers were again imaged and the rates of tracer deposition determined with the VITAE system. Tracer deposition from contact with treated foliage was found to increase in a linear fashion over 4 h. Greatest deposition occurred on the hands and arms and represented 42% and 20% of total exposure, respectively. No detectable air residue samples of pirimicarb were found while workers were debudding plants. Monitoring of four workers for pirimicarb and its urinary metabolites revealed no detectable residues following 4 h of contact with foliage, which had been treated 48 h earlier.