Improving the design and conduct of aquatic toxicity studies with oils based on 20 years of CROSERF experience.

Laboratory toxicity testing is a key tool used in oil spill science, spill effects assessment, and mitigation strategy decisions to minimize environmental impacts. A major consideration in oil toxicity testing is how to replicate real-world spill conditions, oil types, weathering states, receptor organisms, and modifying environmental factors under laboratory conditions. Oils and petroleum-derived products are comprised of thousands of compounds with different physicochemical and toxicological properties, and this leads to challenges in conducting and interpreting oil toxicity studies. Experimental methods used to mix oils with aqueous test media have been shown to influence the aqueous-phase hydrocarbon composition and concentrations, hydrocarbon phase distribution (i.e., dissolved phase versus in oil droplets), and the stability of oil:water solutions which, in turn, influence the bioavailability and toxicity of the oil containing media. Studies have shown that differences in experimental methods can lead to divergent test results. Therefore, it is imperative to standardize the methods used to prepare oil:water solutions in order to improve the realism and comparability of laboratory tests. The CROSERF methodology, originally published in 2005, was developed as a standardized method to prepare oil:water solutions for testing and evaluating dispersants and dispersed oil. However, it was found equally applicable for use in testing oil-derived petroleum substances. The goals of the current effort were to: (1) build upon two decades of experience to update existing CROSERF guidance for conducting aquatic toxicity tests and (2) to improve the design of laboratory toxicity studies for use in hazard evaluation and development of quantitative effects models that can then be applied in spill assessment. Key experimental design considerations discussed include species selection (standard vs field collected), test substance (single compound vs whole oil), exposure regime (static vs flow-through) and duration, exposure metrics, toxicity endpoints, and quality assurance and control.

Effects of Feeding Strategy, Sediment Characteristics, and Chemical Properties on Polychlorinated Biphenyl and Polybrominated Diphenyl Ether Bioaccumulation from Marine Sediments in Two Invertebrates.

Shellfish and sediment invertebrates have been widely used to assess pollution trends over space and time in coastal environments around the world. However, few studies have compared the bioaccumulation potential of different test species over a range of sediment-contaminant concentrations and profiles. The bioavailability of sediment-related contaminants was evaluated using sediments collected from sites (n = 12) throughout the Salish Sea, British Columbia, Canada. Two benthic marine invertebrates-the Baltic clam Macoma balthica and the polychaete worm Neanthes arenaceodentata-were exposed for 28 days in a controlled environment to these field-collected coastal sediments. The congener-specific uptake of legacy polychlorinated biphenyls (PCBs) and emergent polybrominated diphenyl ethers (PBDEs) was determined using high-resolution gas chromatography/mass spectrometry in sediments and in invertebrates after the experimental exposure. The polychaete Neanthes accumulated lower concentrations of PCBs but higher concentrations of PBDEs. The present study indicates that differences in bioaccumulation between these two invertebrates shape the accumulation of PCB and PBDE congeners, reflect differences in feeding strategies, and reveal the physicochemical properties of the contaminants and sediment properties. Because biota-sediment accumulation factor values are often calculated for environmental monitoring or site-specific impact assessments, our results provide insight into potentially confounding factors and the need for caution when selecting indicator species for coastal marine pollution.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles. II: agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory conditions.

Glyphosate-based herbicides are currently the most commonly used herbicides in the world. They have been shown to affect survival, growth, development and sexual differentiation of tadpoles under chronic laboratory exposures but this has not been investigated under more environmentally realistic conditions. The purpose of this study is (1) to determine if an agriculturally relevant exposure to Roundup WeatherMax®, a relatively new and understudied formulation, influences the development of wood frog tadpoles (Lithobates sylvaticus) through effects on the mRNA levels of genes involved in the control of metamorphosis; (2) to compare results to the well-studied Vision® formulation (containing the isopropylamine salt of glyphosate [IPA] and polyethoxylated tallowamine [POEA] surfactant) and to determine which ingredient(s) in the formulations are responsible for potential effects on development; and (3) to compare results to recent field studies that used a similar experimental design. In the present laboratory study, wood frog tadpoles were exposed to an agriculturally relevant application (i.e., two pulses) of Roundup WeatherMax® and Vision® herbicides as well as the active ingredient (IPA) and the POEA surfactant of Vision®. Survival, development, growth, sex ratios and mRNA levels of genes involved in tadpole metamorphosis were measured. Results show that Roundup WeatherMax® (2.89 mg acid equivalent (a.e.)/L) caused 100% mortality after the first pulse. Tadpoles treated with a lower concentration of Roundup WeatherMax® (0.21 mg a.e./L) as well as Vision® (2.89 mg a.e./L), IPA and POEA had an increased condition factor (based on length and weight measures in the tadpoles) relative to controls at Gosner stage (Gs) 36/38. At Gs42, tadpoles treated with IPA and POEA had a decreased condition factor. Also at Gs42, the effect on condition factor was dependent on the sex of tadpoles and significant treatment effects were only detected in males. In most cases, treatment reduced the normal mRNA increase of key genes controlling development in tadpoles between Gs37 and Gs42, such as genes encoding thyroid hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail. We conclude that glyphosate-based herbicides have the potential to alter mRNA profiles during metamorphosis. However, studies in natural systems have yet to replicate these negative effects, which highlight the need for more ecologically relevant studies for risk assessment.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frogs (Lithobates sylvaticus) tadpoles. I: chronic laboratory exposures to VisionMax®.

The purpose of this study was to determine if chronic exposure to the glyphosate-based herbicide VisionMax(®) affects the survival, development, growth, sex ratios and expression of specific genes involved in metamorphosis of wood frog tadpoles (Lithobates sylvaticus). We hypothesized that exposure to this herbicide will affect developmental rates by disrupting hormone pathways, sex ratios and/or gonadal morphology. Tadpoles were chronically exposed in the laboratory from Gosner developmental stage 25 to 42 to four different concentrations of VisionMax(®) (ranging from 0.021 to 2.9 mg acid equivalents/L). Chronic exposures to VisionMax(®) had direct effects on the metamorphosis of L. sylvaticus tadpoles by decreasing development rates, however, there was a decrease in survival only in the group exposed to the highest dose of VisionMax(®) (2.9 mg a.e./L; from approximately 96% in the control group to 77% in the treatment group). There was a decrease in the number of tadpoles reaching metamorphic climax, from 78% in the control group to 42% in the VisionMax(®) (2.9 mg a.e./L) group, and a 7-day delay to reach metamorphic climax in the same treatment group. No effects of exposure on sex ratios or gonadal morphology were detected in tadpoles exposed to any of the concentrations of VisionMax(®) tested. Gene expression analyses in brain and tail tissues demonstrated that exposure to VisionMax(®) alters the expression of key genes involved in development. Results showed significant interaction (two-way ANOVA, P<0.05) between developmental Gosner stage and treatment in brain corticotropin-releasing factor, deiodinase type II (dio2) and glucocorticotiroid receptor (grII) and tail dio2 and grII. This demonstrates that mRNA levels may be differently affected by treatment depending on the developmental stage at which they are assessed. At the same time there was a clear dose-response effect for VisionMax(®) to increase thyroid hormone receptor ß in tadpole brain (F(2,69)=3.475, P=0.037) and tail (F(2,69)=27.569, P<0.001), regardless of developmental stage. Interestingly, delays in development (or survival) were only observed in the group exposed to 2.9 mg a.e./L of VisionMax(®), suggesting that tadpoles need to be exposed to a "threshold" concentration of glyphosate-based herbicide to exhibit phenotypic observable effects. We suggest that the upregulation of genes that trigger metamorphosis following VisionMax(®) herbicide exposure might result from a compensatory response for the delays in development observed. Further studies are needed to determine if disruption of expression of these key genes leads to long-term effects when metamorphs reach adult stages.

Toxicity testing of freshwater sediment collected near freshwater aquaculture facilities in the Maritimes, Canada.

In the Atlantic region of Canada, there are close to 50 land-based freshwater aquaculture facilities, most of which discharge wastewater to freshwater receiving environments. This study was designed to assess the chemical and toxicological characteristics of sediments in those receiving environments. Thirty sediment samples were collected from 3 locations (upstream, outfall and downstream) at seven freshwater aquaculture facilities. Toxicity tests conducted included amphipod growth, amphipod survival and Microtox™. Sediments were also analysed for ammonia/porewater ammonia, redox and sulphide. Porewater ammonia concentration for the amphipod survival test ranged from 0.01 to 42 mg/L at the conclusion of the 14-day survival test. Ammonia concentration in sediment ranged from 0.3-202 µg/g, sulphide concentration ranged from 0.15 to 17.4 µg/g, yet redox ranged from 32 to 594 mV. Microtox™  IC50 values ranged from 108,00 to >164,000 mg/L, yet amphipod survival ranged from 0 to 100%, depending on sampling locations. Amphipod survival was significantly related (P < 0.05) to porewater ammonia (at time = 0 and 14 days) and Microtox™  IC50 was significantly related (P < 0.05) to ammonia, sulphide and redox levels. These results indicate that discharges from some the land-based aquaculture facilities are impacting sediment dwelling benthic invertebrates at the outfall but that impact largely disappears by 100 m downstream. Furthermore those impacts were rarely detected during the early winter season, when biomass production was at the lowest.

Evaluation of buffer zone effectiveness in mitigating the risks associated with agricultural runoff in Prince Edward Island.

To minimize the risk posed by runoff from row crops, Prince Edward Island introduced buffer legislation in 2000. The legislation mandates 10-m and 20-m buffers, respectively, for moderate sloped (i.e. <5%) and steep sloped (i.e. >5%) agricultural fields that border streams. Since 2001, Environment Canada has been evaluating the effectiveness of various buffer widths on operational farms in reducing toxicity and contaminant concentrations in runoff. Sample collectors, placed in 44 fields at the field edge (0m), 10m and at distances out to 30m, collected overland flow following rainfall-induced runoff events. Samples were collected within 24 hours of an event and analysed for seven pesticides (endosulfan, chlorothalonil, carbofuran, linuron, metribuzin, metalaxyl, mancozeb), water quality parameters and Daphnia magna toxicity. The 10-m buffer required for moderate sloped fields was effective at reducing contaminant concentrations but not always to less than lethal concentrations to Daphnia magna. Limited data beyond 10m for fields of both slope types precluded making recommendations on a suitable buffer width for shallow sloped fields and evaluating the effectiveness of 20-m buffers for steep sloped fields. When paired data were combined and statistically tested for all fields, the studied pesticides underwent a 52-98% and 68-100% reduction in aqueous and particulate concentrations within 10m and 30m, respectively. In addition, by 10m, soluble phosphorus, nitrate-nitrogen and total suspended solids were reduced by 34%, 38% and 64%, respectively. Results suggest buffer zones on operational farms are capable of achieving contaminant reductions comparable to those reported for controlled experiments. Inconsistent siting of sample collectors beyond 10m limited the evaluation of the effects of field slope and buffer width on buffer effectiveness on working farms. Future studies on buffer efficiency on operational farms should focus on building the data set beyond 10m and evaluating load reductions.

Dispersion and toxicity to non-target aquatic organisms of pesticides used to treat sea lice on salmon in net pen enclosures.

Pesticides are used extensively in the finfish aquaculture industry to control sea lice infestations on farmed salmon. The most prevalent method of use is to enclose a net pen with an impervious tarpaulin and mix a pesticide solution within that enclosure. After treatment for short periods (1 h) the pesticide solution is released to the environment. Concerns have been raised that there is a potential risk to non-target aquatic organisms from those releases. The fate of dispersing pesticide solutions was measured after six simulated treatments in the Lower Bay of Fundy, New Brunswick. Three simulated treatments were done with azamethiphos and three with cypermethrin. Rhodamine dye was added to all pesticide solutions in order to facilitate tracking of the dispersing plume through real-time measurements of dye concentrations by a flow-through fluorometer coupled with a differential global positioning system (DGPS). Water samples were obtained from within the plumes at various times after release and analysed for pesticide content and toxicity to a benthic amphipod Eohaustorius estuaris. Dye concentrations were detectable for time periods after release which varied from 2 to 5.5 h. Distances travelled by the dye patches ranged from 900 to 3000 m and the dye concentrations at the final sampling period were generally 1/200-1/3000 the pre-release concentrations and cypermethrin concentrations were generally 1/1000-1/2000 the pre-release concentrations. Cypermethrin concentrations in water samples were closely correlated with dye concentrations, indicating that dye analyses were an accurate surrogate for cypermethrin concentrations. Most samples taken after the releases of azamethiphos were not toxic to test organisms in 48 h exposures and none were beyond 20 min post-release. By contrast, almost all samples taken after the release of cypermethrin, even up to 5-h post-release, were toxic. Data indicate the potential to cause toxic effects over areas of hectares from a single release of cypermethrin.

The differentiation of asaccharolytic anaerobic gram-positive cocci by protein electrophoresis.

Conventional biochemical and antibiotic sensitivity tests were used to allocate 87 clinical isolates of anaerobic gram-positive cocci to currently recognised species, in comparison with type and other authentic reference strains. Whole-cell protein electrophoresis was then performed with extracts of each strain. Allowing for difficulties of standardisation, it was possible to allocate most of the organisms to species-related groups on the basis of protein patterns. Organisms identified conventionally as Peptostreptococcus anaerobius and P. micros formed homogeneous groups by protein electrophoresis. There was evidence for heterogeneity amongst strains identified as P. asaccharolyticus (two groups, including P. indolicus), P. prevotii and P. magnus. However, aberrant P. prevotii strains were allocated to the P. asaccharolyticus groups, leaving a homogeneous P. prevotii group, and if P. variabilis were re-instated as a species, the remaining P. magnus strains could be divided into two groups. Of the anaerobic gram-positive cocci in the National Collection of Type Cultures deposited by Hare, Group IV is P. magnus, Group IX is P. micros and Groups I, III and VIII appear to be related to the butyrate-producing species P. asaccharolyticus and P. prevotii, but are strongly saccharolytic.

Detection and quantification of microorganisms in a heterogeneous foodstuff by image analysis.

A relatively inexpensive image analysis system has been developed to semi-automate the detection and quantification of microbial growth in sections of food. A system based on an IBM PC compatible, with a frame store card, was programmed to scan Gram-stained sections using a motorized stage. Each field of view was thresholded after subtraction of a background image and the area between two thresholds measured. In the food studied it was found that, by using a size limit, it was possible to reduce the number of fields that needed to be examined by a microscopist to approximately 3% of those scanned. Visual examination was still required to distinguish bacterial cells from other stained objects which occasionally occur.

Characterization of Corynebacterium group JK by whole-cell protein patterns.

A total of 102 strains received as Corynebacterium 'group JK' were characterized by SDS-PAGE of their whole-cell proteins. Numerical taxonomy based on the protein pattern absorbance profiles indicated that 91 of the strains formed a cluster. Seventy strains isolated in the UK were identified as group JK, indicating the increasing detection of this group as opportunistic pathogens. Fine differences between strain patterns were visible but it was not possible to associate these with any particular clinical source.

Testosterone metabolism by isolated human axillary Corynebacterium spp.: a gas-chromatographic mass-spectrometric study.

Transformations of [4-14C]testosterone have been studied in Corynebacterium spp. isolated from the axillae of men. Metabolites have been separated by TLC and capillary gas chromatography and have been identified by gas chromatography-mass spectrometry (GC-MS). The introduction of a clean-up step using Florisil columns, prior to TLC, removed Tween-80 which co-extracted from the medium with the metabolites. This procedure greatly improved TLC resolution. Testosterone was converted enzymically to 5 alpha- and 5 beta-DHT, identification being assisted by the inclusion of [3,4-13C]testosterone in some incubations. Other metabolites formed enzymically were 4-androstene-3,17-dione, 5 beta-androstane-3,17-dione, 3 beta-hydroxy-5 beta-androstan-17-one and 5 beta-androstane-3 alpha .17 alpha-diol. Some spontaneous breakdown of [14C]testosterone occurred giving rise to 5 alpha (beta)-DHT, androstanediol and a monohydroxy-diketo-androstene, the latter being reduced enzymically to 2 monohydroxy-diketo-androstanes. Under the conditions used, no clear evidence has been obtained for the formation of 5 alpha-androst-16-en-3-one, an odorous steroid that occurs in the axillae of men; the possible reasons why we were unable to prove the biosynthesis of this compound are discussed.

Transformation of steroids by axillary coryneform bacteria.

Synopsis The contribution made by the apocrine glands to body odour has been discussed. In particular, the role of odorous steroids, such as 5alpha-androst-16-en-3-one (5alpha-A), and their formation by axillary bacteria, has been explored. Isotopically labelled testosterone was incubated (21 days at 37 degrees C) with strains of axillary bacteria, both separately and in mixtures, and the products characterized by capillary gas chromatography (GC) and GC-mass spectrometry. In many cases testosterone was converted into large yields of 5beta-dihydrotestosterone and 4-androstenedione, although 5alpha-dihydrotestosterone, 5beta-androstanedione and androstanediols were also produced. Mixing certain coryneform bacteria appeared to cause some additive and some inhibitory effects. A non-polar steroid, with polarity similar to that of 5alpha-A, was formed in some incubations, but identification could not be confirmed. Nevertheless, preliminary experiments have indicated that 5alpha-A is present in axillary hair of men, and the possible reasons why we have failed to prove the biosynthesis of this odorous steroid are discussed.

Fingerprinting Acinetobacter strains from clinical sources by numerical analysis of electrophoretic protein patterns.

A total of 57 strains of Acinetobacter calcoaceticus was fingerprinted by SDS-PAGE of cellular protein. All strains were also examined by conventional, API and N/F-Tek methods, and antibiotic sensitivity patterns were determined. In general, using the API 20E and N/F-Tek methods, it was possible to assign isolates of A. calcoaceticus to the two accepted "biotypes" or "varieties", A.c. anitratus and A.c. lwoffi, but these methods did not offer useful subdivision of the biotypes. Gel electrophoresis permitted subdivision of the strains into clusters in a manner which suggests that the technique may be valuable in typing strains isolated during outbreaks of infection in hospital.

The similarities between Pseudomonas paucimobilis and allied bacteria derived from analysis of deoxyribonucleic acids and electrophoretic protein patterns.

The chromosomal DNA was isolated and purified from 17 strains of Pseudomonas paucimobilis, and from the type or reference strains of Flavobacterium capsulatum, F. devorans, F. multivorum, 'Chromobacterium lividum', Xanthomonas campestris and seven species of Pseudomonas. The DNA base compositions (mol% G + C) of P. paucimobilis strains were between 62.2 and 68.6%, and typical strains had a mean value of 65.3 +/- 0.4 mol%, determined from thermal denaturation temperature. DNA-DNA molecular hybridization with 3H-labelled probe DNA from NCTC 11030 P. paucimobilis (the type strain) indicated that the species comprised a core of 13 closely related strains (74 to 96%), which included F. devorans NCIB 8195 (= ATCC 10829). Four P. paucimobilis strains displayed lower levels of hybridization (less than or equal to 38%). The hybridization results showed that P. paucimobilis was not closely related to allied yellow-pigmented bacteria or to other reference pseudomonads. The electrophoretic protein patterns of representative strains were analysed by computer-assisted techniques and similarity coefficients were calculated. A high degree of congruence was obtained with the DNA hybridization data, and the protein analyses indicated that the four atypical P. paucimobilis strains were heterogeneous and not a single group within the species. The generic affinities of P. paucimobilis, F. capsulatum, 'P. azotocolligans' and P. echinoides are uncertain, but available chemotaxonomic data indicate these species could provide the basis for a new genus.

Classification of Corynebacterium species from axillary skin by numerical analysis of electrophoretic protein patterns.

The genus Corynebacterium, defined by the presence of mesodiaminopimelic acid, arabinose and corynomycolic acids in the cell wall, contains a very diverse group of organisms according to the results of numerical analysis of protein patterns; 13 groups were distinguished in this study. Strains isolated from axillary skin and hair included representatives of several groups but many strains were placed in groups 1 and 6A. Neither of these groups contained any reference strains and may constitute hitherto undescribed species. The reference strains of C. diphtheriae formed a coherent group not closely related to any non-pathogenic Corynebacterium species.