Pamela A. Martin

Aquatic phyto-toxicity of 23 pesticides applied at expected environmental concentrations

Abstract Environment Canada uses an Expected Environmental Concentration (EEC) in evaluating the hazard of pesticides to nontarget aquatic organisms. This concentration is calculated by assuming an overspray of a 15 cm deep waterbody at the label application rate. The EEC of pesticides is then related to the EC50 (concentration causing a 50% reduction in a chosen toxicity endpoint) for a given aquatic test organism. At present, the use of an uncertainty factor is suggested in the literature if only a few species are tested because of important interspecific differences in pesticide sensitivity. The phytotoxicity of the EEC of 23 different pesticides to ten algae (24 h inhibition of 14C uptake) and one vascular plant (7-day growth inhibition) was determined in an effort to examine the question of interspecific sensitivity and its relation to the development of pesticide registration guidelines. Chemicals included five triazine herbicides (atrazine, cyanazine, hexazinone, metribuzin, and simazine), four sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, ethametsulfuron-methyl, triasulfuron), two phenoxyalkane herbicides (2,4-D and MCPA), two pyridine herbicides (picloram and triclopyr), a substituted urea, an amine derivative, and an imidazolinone herbicide (tebuthiuron, glyphosate and imazethapyr, respectively), a bipyridylium (diquat), a hydroxybenxonitrile (bromoxynil), an aldehyde (acrolein) and an acetanilide (metolachlor) herbicide, as well as two carbamate insecticides (carbofuran and carbaryl) and a triazole derivative fungicide (propiconazole). Test organisms were selected based on ecological relevance and present use in test protocols. Organisms included green algae (Scenedesmus quadricauda and Selenastrum capricornutum), diatoms (Nitzschia sp. and Cyclotella meneghiana), cyanobacteria (Microcystis aeruginosa, Oscillatoria sp., Pseudoanabaena sp., Anabaena inaequalis and Aphanizomenon flos-aquae) and a floating vascular plant, duckweed (Lemna minor). The five triazine herbicides, acrolein and diquat inhibited the carbon uptake of all algae, diatoms and cyanobacteria by more than 50%. Two other pesticides, carbaryl and tebuthiuron, caused more than 50% inhibition in 90% of the algae tested. Nine of the 23 pesticides, five of which were triazine herbicides, were therefore highly phytotoxic to algae. Twelve pesticides inhibited growth of duckweed by more than 50%. Once again, all five of the triazine herbicides were among this group, as well as three sulfonylurea herbicides and acrolein, diquat, metolachlor and tebuthiuron. Duckweed was the most sensitive organism tested, being equally affected by all pesticides causing algal phytotoxicity (with the exception of carbaryl), as well as being acutely affected by sulfonylurea herbicides. Green algae were least sensitive to diquat; diatoms and one cyanobacterium were the only organisms that showed sensitivity to glyphosate. Through testing the phytotoxicity of a variety of agricultural pesticides to a wide range of algal taxa, it is evident that there are considerable differences in sensitivity among species and that the use of an uncertainty factor is necessary to provide an acceptable margin of safety in evaluating the hazard presented by these chemicals to the aquatic environment.

Second generation anticoagulant rodenticides in predatory birds: Probabilistic characterisation of toxic liver concentrations and implications for predatory bird populations in Canada.

Second-generation anticoagulant rodenticides (SGARs) are widely used to control rodent pests but exposure and poisonings occur in non-target species, such as birds of prey. Liver residues are often analysed to detect exposure in birds found dead but their use to assess toxicity of SGARs is problematic. We analysed published data on hepatic rodenticide residues and associated symptoms of anticoagulant poisoning from 270 birds of prey using logistic regression to estimate the probability of toxicosis associated with different liver SGAR residues. We also evaluated exposure to SGARs on a national level in Canada by analysing 196 livers from great horned owls (Bubo virginianus) and red-tailed hawks (Buteo jamaicensis) found dead at locations across the country. Analysis of a broader sample of raptor species from Quebec also helped define the taxonomic breadth of contamination. Calculated probability curves suggest significant species differences in sensitivity to SGARs and significant likelihood of toxicosis below previously suggested concentrations of concern (<0.1mg/kg). Analysis of birds from Quebec showed that a broad range of raptor species are exposed to SGARs, indicating that generalised terrestrial food chains could be contaminated in the vicinity of the sampled areas. Of the two species for which we had samples from across Canada, great horned owls are exposed to SGARs to a greater extent than red-tailed hawks and the liver residue levels were also higher. Using our probability estimates of effect, we estimate that a minimum of 11% of the sampled great horned owl population is at risk of being directly killed by SGARs. This is the first time the potential mortality impact of SGARs on a raptor population has been estimated.

Potential endocrine disruption of sexual development in free ranging male northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) from areas of intensive row crop agriculture

Intensive row crop agriculture (IRCA) for corn and soybean production is predominant in eastern and central North America. IRCA relies heavily on pesticide and nutrient inputs to maximize production under conventional systems. In 2003-2005, we assessed the occurrence of a suite of potential endocrine effects in amphibians inhabiting farm ponds and agricultural drains in IRCA areas of southwestern Ontario. Effects were compared to amphibians from two agricultural reference sites as well as four non-agricultural reference sites. Pesticide and nutrient concentrations were also determined in water samples from those sites. Atrazine and metolachlor were detected in most samples, exceeding 1 microg L(-1) at some sites. Blood samples were taken from northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) for analysis of circulating sex steroids and vitellogenin-like protein (Vtg-lp), a biomarker of exposure to environmental estrogens. Gonads were histologically examined for evidence of abnormalities. Some evidence of exposure to endocrine disrupting compounds was apparent from the data. The occurrence of testicular ovarian follicles (TOFS) in male R. pipiens was significantly higher (42%; p<0.05) at agricultural sites, particularly those in Chatham county compared to frogs from reference sites (7%). There was no difference in circulating sex steroid levels between frogs from agricultural and reference sites and sex steroid levels did not correlate with pesticide concentrations in the environment. No differences were detected in the gonadosomatic indices or stage of spermatogenesis between frogs from agricultural and non-agricultural regions (p>0.05). Plasma Vtg-lp was detected in only one male R. pipiens from an agricultural site. Neither gonad size, gonad maturity nor sex steroid levels differed between normal males and those with testicular oocytes. Although the proportion of testicular oocytes did not correlate directly with atrazine concentrations, it did correlate with a mixture of pesticides and nutrients, particularly atrazine and nitrate, while the number of pesticides detected at each site was also important.

Toxicity of hexazinone and diquat to green algae, diatoms, cyanobacteria and duckweed

Abstract Hexazinone and diquat are two broad-spectrum contact herbicides used in a variety of crop and non-crop applications. Both pesticides are highly water soluble and persistent in the aquatic system. Hexazinone is mobile in soil and, thus, the potential for leaching into ground water and for overland runoff into surface water is high; diquat, however, is rapidly bound by soil and sediment to a biologically inactive form, at least temporarily. Toxicity to green algae (two species), diatoms (two species) and cyanobacteria (five species) was determined using inhibition of 14 C uptake. Toxicity to the floating vascular plant, duckweed, was tested by measuring seven-day growth inhibition. Test organisms exhibited large differences in sensitivity to both herbicides. The green algae, diatoms, and duckweed (all eukaryotes) were more sensitive to hexazinone than were cyanobacteria (prokaryotes). Mean concentrations at which 50% inhibition occurred in these groups were 0.01 (green algae), 0.05 (diatoms), 0.07 (duckweed), and 0.6 (cyanobacteria) mg hexazinone per litre, respectively. In contrast, green algae were comparatively tolerant of diquat (EC 50 values approximately 0.6 mg l −1 ), while the cyanobacteria and diatoms were much more sensitive (mean EC 50 values of 0.074 and 0.079 mg diquat per litre). Duckweed showed the greatest sensitivity to diquat, with 50% inhibition of growth occurring at 0.004 mg l −1 . That some non-target aquatic plants are susceptible to diquat toxicity at less than 0.01 mg l −1 is significant, as most methods of diquat analysis have minimum detection limits of 0.01 to 0.05 mg l −1 . Therefore, algae and vascular aquatic plants may suffer phytotoxic effects in a contaminated water body even when diquat is undetectable chemically. Differential sensitivity to herbicide contaminants among taxonomic groups of plants may have ecological consequences. Cyanobacteria may produce neuro- and hepato-toxins and constitute a poor food resource for higher trophic levels relative to the other organisms tested. Contamination of surface water with hexazinone may degrade water quality for wildlife, both by causing food reduction and habitat loss through inhibition of green algae, diatoms and macrophytes, and by allowing the proliferation of cyanobacteria. Varying responses among the different test algae to the two herbicides under study indicated that a wide taxonomic range of test species is necessary in evaluating the impacts of contaminants in aquatic systems.

IMMUNOTOXICITY OF THE COMMERCIAL POLYBROMINATED DIPHENYL ETHER MIXTURE DE-71 IN RANCH MINK (MUSTELA VISON)

Polybrominated diphenyl ethers (PBDEs) are persistent, bioaccumulative, organohalogen compounds that are increasing exponentially in the Great Lakes (Canada/USA) biota. The present study was undertaken to examine the immunological effects of a commercial PBDE mixture in ranch mink (Mustela vison). Twenty-week-old mink (n = 10 mink/group) were exposed to 0, 1, 5, or 10 ppm of DE-71 through their diet for eight weeks. The phytohemagglutinin-induced cutaneous reaction, and antibodies specific to keyhole limpet hemocyanin conjugated to dinitrophenol were measured. Liver microsomal ethoxyresorufin-O-deethylase (EROD) activity also was measured. Organs were weighed and spleens were examined histologically. No differences were found in the PHA-induced skin response in exposed mink; mink in the two highest treatments exhibited significant increases in antibody production over control mink. Systemic toxicity was apparent; significant body weight reductions were found in mink exposed to 5 and 10 ppm of DE-71. Exposed mink had significantly larger relative spleen, adrenal, and liver masses than control mink. Spleens of mink exposed to 10 ppm of DE-71 had significantly increased germinal center development and incidence of B-cell hyperplasia. The activity of EROD was induced in all treated mink relative to controls and was positively associated with the liver somatic index. Hematocrit in mink from the two highest exposure groups was significantly lower than control mink. Percentage neutrophils increased and percentage lymphocytes decreased significantly in mink from the higher two dosage groups. Our findings have direct relevance to wild mink in the Great Lakes ecosystem, because mink are top predators of the aquatic food web, providing evidence for the vulnerability of this species to the effects of environmental PBDE mixtures.

Perfluoroalkyl carboxylates and sulfonates and precursors in relation to dietary source tracers in the eggs of four species of gulls (Larids) from breeding sites spanning Atlantic to Pacific Canada

In the present study, we identified and examined the spatial trends, sources and dietary relationships of bioaccumulative perfluorinated sulfonate (PFSA; C(6), C(8), and C(10) chain lengths) and carboxylate (PFCA; C(6) to C(15) chain lengths) contaminants, as well as precursor compounds including several perfluorinated sulfonamides, and fluorotelomer acids and alcohols, in individual eggs (collected in 2008) from four gull species [glaucous-winged (Larus glaucescens), California (Larus californicus), ring-billed (Larus delawarensis) and herring (Larus argentatus) gulls] from 15 marine and freshwater colony sites in provinces across Canada. The pattern of PFSAs was dominated by perfluorooctane sulfonate (PFOS; >89% of ΣPFSA concentration) regardless of egg collection location. The highest ΣPFSA concentrations were found in the eggs collected in the urbanized areas in the Great Lakes and the St. Lawrence River area [Big Chicken Island 308 ng/g ww, Toronto Harbour 486 ng/g ww, and Ile Deslauriers (HG) 299 ng/g ww]. Also, eggs from all freshwater colony sites had higher ΣPFSA concentrations, which were significant (p<0.05) in many cases, compared to the marine sites with the exception of the Sable Island colony in Atlantic Canada off the coast of Nova Scotia. C(6) to C(15) chain length PFCAs were detected in the eggs, although the pattern was variable among the 15 sites, where PFUnA and PFTrA dominated the pattern for most colonies. Like the ΣPFSA, the highest concentrations of ΣPFCA were found in the eggs from Big Chicken Island, Toronto Harbour, Ile Deslauriers (HG), and Sable Island, although not all freshwater sites had higher concentrations compared to marine sites. Dietary tracers [δ(15)N and δ(13)C stable isotopes (SIs)] revealed that PFSA and PFCA exposure is colony dependent. SI signatures suggested that gulls from most marine colony sites were exposed to PFCs via marine prey. The exception was the Mandarte Island colony in Pacific British Columbia, where PFSA and PFCA exposure appeared to be via terrestrial and/or freshwater prey consumption. The same was true for the freshwater sites where egg SIs suggested both aquatic and terrestrial prey consumption as the source for PFC exposure depending on the colony. Both aquatic (marine and freshwater) and terrestrial prey are likely sources of PFC exposure to gulls but exposure scenarios are colony-specific.

Reproductive and Developmental Toxicity of a Pentabrominated Diphenyl Ether Mixture, DE-71, to Ranch Mink (Mustela vison) and Hazard Assessment for Wild Mink in the Great Lakes Region

Polybrominated diphenyl ethers (PBDEs) are widespread persistent and bioaccumulative environmental contaminants. Recent scientific attention has focused on the developmental toxicity of PBDE commercial mixtures following perinatal exposure of rodents; however, these studies do not necessarily predict toxicity to highly exposed top predators, such as mink (Mustela vison). Here we assessed the effects of environmentally relevant doses (0, 0.1, 0.5, and 2.5 ppm [wt/wt] in feed) of a technical pentabrominated diphenyl ether mixture, DE-71, on reproductive performance of mink and on development of offspring exposed perinatally and post-weaning until 33 weeks. A dietary concentration that causes no effects on reproduction in rodents, 2.5-ppm DE-71, resulted in complete reproductive failure in these mink, while whelping rates were not affected at all lower does. Developmental effects in offspring were evident in 33-week-old juveniles, which were more sensitive to effects than their respective dams. Juvenile thyroid hormone homeostasis was also much more sensitive compared to rodents, and at 0.5-ppm DE-71, total triiodothyronine (T3) was significantly decreased in all males and females, even despite a compensatory increase of total thyroxine (T4) in females. T4-outer-ring deiodinase activity, mainly contributed by type II deiodinase, was not affected at any dose for any life stage, but thyroid follicular epithelium cell height was elevated in the 0.5-ppm-treated juveniles (p = 0.057). Ethoxyresorufin O-deethylase activity was significantly induced in all offspring at 33 weeks, most likely as a consequence of polybrominated dioxin, furan, or biphenyl impurities in DE-71. Biomonitoring of wild mink in the Great Lakes region indicated that most populations had lower concentrations than what are expected to affect thyroid hormone homeostasis, but margins of safety are small and mink around Hamilton Harbour exceeded the no observed adverse effect level for T3 disruption.

EFFECTS OF ENVIRONMENTALLY RELEVANT CONCENTRATIONS OF ATRAZINE ON GONADAL DEVELOPMENT OF SNAPPING TURTLES ( CHELYDRA SERPENTINA )

The herbicide atrazine has been suspected of affecting sexual development by inducing aromatase, resulting in the increased conversion of androgens to estrogens. We used snapping turtles (Chelydra serpentina), a species in which sex is dependent on the production of estrogen through aromatase activity in a temperature-dependent manner, to investigate if environmentally relevant exposures to atrazine affected gonadal development. Eggs were incubated in soil to which atrazine was applied at a typical field application rate (3.1 L/ha), 10-fold this rate (31 L/ha), and a control rate (no atrazine) for the duration of embryonic development. The incubation temperature (25 degrees C) was selected to produce only males. Although some males with testicular oocytes and females were produced in the atrazine-treated groups (3.3-3.7%) but not in the control group, no statistical differences were found among treatments. Furthermore, snapping turtle eggs collected from natural nests in a corn field were incubated at the pivotal temperature (27.5 degrees C) at which both males and females normally would be produced, and some males had oocytes in the testes (15.4%). The presence of low numbers of males with oocytes may be a natural phenomenon, and we have limited evidence to suggest that the presence of normal males with oocytes may represent a feminizing effect of atrazine. Histological examination of the thyroid gland revealed no effect on thyroid morphology.

European Starlings (Sturnus vulgaris) Suggest That Landfills Are an Important Source of Bioaccumulative Flame Retardants to Canadian Terrestrial Ecosystems

Landfills are used as the primary means for the disposal of municipal solid waste in Canada. In the present study, polybrominated diphenyl ethers (PBDEs) and other flame retardants (FRs) were determined in fresh European starling ( Sturnus vulgaris ) eggs collected in 2009, 2010, and 2011 from nest boxes established within, adjacent to, and 10 and 40 km distant to five major urban centers across Canada, i.e., Vancouver, British Columbia (BC); Calgary, Alberta (AB); Hamilton, Ontario (ON); Montréal, Québec (QC); and Halifax, Nova Scotia (NS). Nest boxes were located in several land use types: urban industrial areas (districts of industrial activity within city limits), landfill sites (adjacent to cities), and rural (agricultural) sites located 10 and 40 km distant from the major urban centers, as well as a national reference site. Of the 14 PBDE congeners and 16 non-PBDE FR substances determined in the starling eggs, BDE-17, -28, -47, -49, -66, -85, -99, -100, -138, -153, -154, -183, and -209, Dechlorane Plus isomers (anti and syn), and bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (BEHTBP) were most frequently quantifiable. The data revealed orders of magnitude greater PBDE concentrations in eggs from starlings nesting in landfill sites (median: 28-280 ng/g wet weight) relative to those from urban industrial and rural environments. However, the percent fractional composition of the PBDE congener patterns did not vary significantly between the types of land uses or between years. Additionally, the median ∑PBDE concentration in eggs from landfill sites and the human population density of the metropolitan region that the landfill serves were highly correlated (r(2) = 0.998, p < 0.001). As the first transcontinental effort in assessing FR contamination in Canadian terrestrial ecosystems, the present study strongly suggest that landfills are an important FR source to starlings nesting nearby and that other terrestrial organisms could also be similarly exposed.

Spatial and temporal comparisons of legacy and emerging flame retardants in herring gull eggs from colonies spanning the Laurentian Great Lakes of Canada and United States

In the Laurentian Great Lakes basin of North America, an increasing number of chemicals of emerging concern (CECs) are being investigated, including legacy and replacement flame retardants (FRs). In the present study, 14 polybrominated diphenyl ethers (PBDEs), 23 non-PBDEs halogenated FRs (NPHFRs) and 16 organophosphate ester FRs (OPE-FRs) were analyzed in 100 individual eggs collected in 2012 and 2013 and in 15 egg pools of herring gulls collected in 2012 from 20 colonies across the entire Laurentian Great Lakes basin. For CEC-FRs in eggs from all colonies, 14 PBDEs, 12 NPHFRs and 9 OPE-FRs were quantifiable in at least one of the 115 analyzed samples. The mean sum PBDE (Σ14PBDE) concentrations ranged from 244 to 657 ng/g wet weight (ww), and on average were 1-2 orders of magnitude greater than the Σ12NPHFR concentrations (13.8-35.6 ng/g ww), and 2-3 orders of magnitude greater than Σ9OPE-FR concentrations (0.31-2.14 ng/g ww). Mean Σ14PBDE and sum of syn- and anti-Dechlorane Plus isomer (Σ2DDC-CO) concentrations in eggs from colonies within Laurentian Great Lakes Areas of Concern (AOCs) were in most cases greater than in eggs from nearby colonies outside of AOCs. Comparing CEC-FR concentrations in eggs collected in 2012-2013 to those previously measured in eggs collected approximately 7 years earlier (2006 and 2008) showed that Σ7PBDE (BDE-28, -47, -100, -99, -154,-153 and -183) mean concentrations in eggs from 6 colonies were approximately 30% less than they were in eggs from the same colonies from the earlier time period, whereas 3 current-use FR (BDE-209, HBCDD and Σ2DDC-CO) concentrations were significantly greater (p<0.05) than previously measured. Between 2006 and 2013 there were significant changes in individual PBDE patterns for BDE-71, -138, -153, -203, -206 and -207. Among all of the examined CEC-FRs, concentrations of Σ4PBDE (BDE-47, -99, -100 and -153) and HBCDD in gull eggs from all colonies were greater than or comparable to their lowest observed effect concentrations (LOECs) based on in ovo egg injection studies. Overall, the current profiles of a broad suite of FRs in Laurentian Great Lakes herring gull eggs highlights the need to better understand e.g., exposure-effect implications and metabolism of FRs, i.e. OPE-FRs, and emphasizes the importance of continued monitoring of CEC-FRs whose concentrations appear to be increasing, including BDE-209, HBCDD and DDC-COs.