Non-lethal Sampling Research Articles

Non-Lethal Sampling Methods for Assessing Environmental Impacts Using a Small-Bodied Sentinel Fish Species

Abstract Under the Canadian Fisheries Act, pulp and paper mills and metal mines must conduct a cyclical monitoring program for potential environmental effects that includes a fish survey. Study designs for the fish survey have been evolving over the past few years, and there has been increased emphasis on the use of small-bodied fish species. Increasing concerns about the potential impacts of sampling programs on the fish populations in smaller receiving waters have led us to develop non-lethal sampling methodologies that will satisfy the information requirements for the environmental effects monitoring program. This manuscript outlines the use of a non-lethal sampling program to collect information on age distributions, growth rates, reproductive performance and fish condition in populations of slimy sculpin inhabiting forested and agricultural sections of a small New Brunswick river.

Overview of Freshwater Fish Studies from the Pulp and Paper Environmental Effects Monitoring Program

Abstract The second cycle of the pulp and paper environmental effects monitoring (EEM) program reported its results on April 1, 2000, and preliminary analysis of the fish survey results have been conducted. The EEM program is a cyclical evaluation of the receiving water impacts associated with the discharge of effluent, consisting of evaluations of fish populations, benthic invertebrate communities, effluent toxicity and other components. This paper represents a summary of the preliminary evaluations of 114 EEM surveys for the fish survey results, as reported by the consulting companies to the mills. Less than 10% of the cases failed to find a statistical difference in the key measurements of gonad size, liver size and condition factor. When effects were seen in a sex of one species, 67% of the time, a similar effect was seen in the opposite sex and when a difference was seen in one sex of one species, 54% of the time, the same difference was seen in the same direction in a second species sampled at that site. This reflects that differences were often consistent between sexes and consistent between species. Results, problems encountered, and lessons learned will be compared for cycle 1 and 2 data, and general response patterns and national trends in reported data will be presented. A summary of research needs is presented, as are recommendations for cycle 3 pulp and paper. New developments in the program reflect some of the adaptations developed for metal mining EEM, including a) setting alpha and beta equal, and its consequences for study design and interpretation, b) discussion of the role of effect size in power analysis and study design, and c) non-lethal sampling protocols for EEM.

Anesthesia and Liver Biopsy Techniques for Pigeon Guillemots (Cepphus columba) Suspected of Exposure to Crude Oil in Marine Environments

This paper reports on the anesthesia and liver biopsy techniques used in adult and nestling pigeon guillemots (Cepphus columba) to test for continued exposure to residual crude oil in the marine environment. Populations of pigeon guillemots have declined significantly in Prince William Sound, Alaska, USA, possibly because of residual effects of crude oil in the environment after the Exxon Valdez oil spill in March 1989. Measurement of hepatic cytochrome P450 1A (CYP1A) is currently the best way to assess crude oil exposure from food sources; however, lethal sampling to obtain adequate liver tissue was not desirable in this declining population of birds. As part of a larger study to identify factors limiting the recovery of pigeon guillemots and other seabird populations, we surgically collected liver samples from adult and nestling guillemots to provide samples for measurement of hepatic CYP1A concentrations. Results from the larger study were reported elsewhere. Liver samples were taken from 26 nestling (1998) and 24 adult (1999) guillemots from a previously oiled site (Naked Island; 12 chicks, 13 adults) and from a nonoiled site (Jackpot Island/Icy Bay; 14 chicks, 11 adults). The birds were anesthetized with isoflurane. No surgical complications occurred with any of the birds and all adult and nestling birds survived after surgery to the point of release or return to the nest. Thirteen out of 14 chicks from the Jackpot Island/Icy Bay and 8 out of 12 chicks from Naked Island fledged. Four chicks at Naked Island were depredated before fledging. All adults abandoned their nests after surgery, so the study sites were revisited the following summer (2000) in an attempt to assess overwinter survival of the adults. All but 1 adult biopsied bird at the nonoiled site (Icy Bay) was found renesting, whereas only 2 birds at the previously oiled site (Naked Island) were similarly observed. The percent of 1999 breeders at Naked Island that returned to their nest sites to breed again in 2000 was low at nests of biopsied birds and nonbiopsied birds alike, suggesting that factors other than the surgical procedure were responsible for the low return rate among this group. These survival results provide strong support for using experienced veterinarians for nonlethal invasive sample collection from birds to document exposure to crude oil in the marine environment.

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.

Induction of cytochrome P450 1A1 expression in captive river otters fed Prudhoe Bay crude oil: evaluation by immunohistochemistry and quantitative RT-PCR

Numerous studies have explored the relationships between exposure to a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons, and induction of cytochrome P450 1A (CYP1A) in different vertebrates. Few controlled studies, however, simulated chronic long-term exposure with repeated non-lethal sampling of the same individuals, which should better represent repeated exposure incidents in animals inhabiting polluted areas. In this study, we investigated the effects of chronic exposure to crude oil on levels of CYP1A1 in endothelial cells of skin biopsies and peripheral mononuclear blood cells in captive river otters (Lontracanadensis) using repeated sampling of the same individuals. We hypothesized that ingestion of oil would result in an increase in levels of CYP1A1 in both targets, and predicted that the relationship between prolonged exposure and expression of CYP1A1 would reach a plateau indicative of continuous detoxification of hydrocarbons. Fifteen wild-caught male otters were acclimated to captivity, and then fed diets containing no oil (control) or diets containing weathered crude oil at 5 mg day-1 kg-1 body weight (low-dose) and 50 mg day-1 kg-1 body weight (high-dose), at the Alaska Sealife Center in Seward, Alaska, USA. Expression of CYP1A1 was assessed with immunohistochemical analysis of CYP1A1 protein in skin biopsies and by quantitative RT-PCR analysis of CYP1A1 mRNA in mononuclear blood cells. Both assays revealed a decrease between capture and the transfer to captivity, indicating that the enclosure at the Alaska Sealife Center, and the food we offered to the otters were free of potential inducers of CYP1A1. During the exposure period, increases in CYP1A1 expression were registered by both techniques, followed by a decline in CYP1A1 after oil administration ended. Levels of endothelial CYP1A1 in the high-dose group were comparable to those recorded for wild river otters in PWS in 1996 and 1997. Levels of CPY1A1 mRNA in mononuclear blood cells, however, were well below levels recorded for river otters in Prince William Sound, and no correlation was detected between values obtained from the two methods. Thus, our results from this longitudinal study with repeated sampling of the same individuals provide support for the use of cytochrome P450 1A1 as a biomarker for hydrocarbon exposure. Nonetheless, our results also suggest that the induction process of CYP1A1 may be complicated and interacting with other processes in vivo. Such interactions may obscure our ability to describe specific, quantitative, predictable, dose-response relationships between exposure to hydrocarbons and induction of CYP1A1, which are required of reliable biomarkers. Evaluations of such interactions based on theoretical physiological models in live-animals merit further investigation.

Strategies for the Diagnosis ofCeratomyxa shastaUsing the Polymerase Chain Reaction: Comparison of Lethal and Nonlethal Sampling with Microscopic Examination

A single-round and a nested polymerase chain reaction (PCR) assay were compared with microscopic examination for their ability to detect infections caused by the myxozoan parasite Ceratomyxa shasta. Tissues for the comparison were periodically sampled from susceptible rainbow trout Oncorhynchus mykiss naturally exposed to the parasite. Spores of the parasite were first detected in the intestine by microscopic examination at 41 d postexposure. Parasite DNA was detected in the intestine at 20 d postexposure by single-round PCR and at 7 d using the nested PCR assay. Using a nonlethal sampling method, parasite DNA was detected from intestinal swabs at 27 d postexposure by single-round PCR and at 13 d using the nested reaction. Although PCR diagnosis using the nonlethal sampling method was less effective in detecting C. shasta than diagnosis using lethally collected tissue, it was a more reliable assay than microscopic examination.

Application of molecular techniques to non-lethal tissue samples of endangered butterfly populations ( Parnassius apollo L.) in Norway for conservation management

Tissue from the wing tips of the endangered Apollo butterfly ( Parnassius apollo L.) were collected at the end of a breeding season from two sites in Norway: Bandak and Gjendetunga/Bukkelægret about 250 km apart. We investigated the suitability of such non-lethal samples as a source of DNA for studying these populations. Universal mitochondrial DNA (mtDNA) primers were used to amplify DNA from the wing tip extractions. Primers spanning the 12s rRNA region produced 0.35 kb fragments that proved to be invariable in the selected samples when tested for restriction length polymorphisms (RFLPs). These sequences had high homology with analogous mtDNA from other insect sources, e.g. lepidoptera [ Spodoptera litura (Fabricius)], and served to authenticate the DNA. Random amplified polymorphic DNA (RAPDs) technology was then used to screen for genetic variability. Of several primers tested, Primer-01 (Operon Kit F) produced a profile that differentiated the population with 88% ( P<0.001) efficiency based on multivariate logistic regression analysis of banding profiles. RAPDs were also adapted to develop different molecular marker approaches to screen the samples quickly and cheaply. This work indicates that reliable ‘molecular tags’ can be designed to track demographic populations to study their distribution without endangering the butterfly and highlights the suitability of these markers for use in future population studies.

Cadmium and lead exposure associated with reduced growth rates, poorer fledging success of little blue heron chicks (Egretta caerulea) in south Louisiana wetlands.

Persistent contaminants in ecosystems are often monitored via organisms that bioaccumulate the pollutant of interest. Several published studies have used colonial wading birds to assess contamination by heavy metals in aquatic ecosystems. Using a nonlethal sampling regimen, this study identified chick feathers and guano as tissues that best reflect local contamination. Both cadmium and lead were detected in food samples (0.3-4.3 microg/g Cd, 0.8-9.3 microg/g Pb), guano (0.2-3.2 microg/g Cd, 1.0-9.5 microg/g Pb), and feathers (0.6-25.4 microg/g Cd, 1.2-16.9 microg/g Pb) from little blue heron chicks at levels similar to other studies. Chicks exposed to cadmium had significantly slower growth rates than nonexposed chicks, and exposure to lead was correlated with increased nestling mortality. The appropriate scale of comparison necessary to demonstrate these effects and the usefulness of wading birds as bioindicators are discussed.

Allozyme Studies of Pacific Salmonids with Nonlethal Sampling of Fin Tissue

We evaluated the use of nonlethal sampling of bony fin tissue to obtain allozyme data for Pacific salmonids Oncorhynchus spp. Enzyme activity was detected for 53 loci, including many polymorphic loci, in caudal fin tissue sampled from chinook salmon O. tshawytscha, chum salmon O. keta, coho salmon O. kisutch, sockeye salmon O. nerka, and steelhead O. mykiss. This represents 61% of all the loci (N = 87) routinely analyzed using eye, heart, liver, and muscle tissue. Anal, dorsal, caudal, pelvic, and pectoral fin tissues all expressed the same loci with approximately the same strength of activity. Fin tissue sampled from adult salmonids yielded a slightly greater number of loci than did juvenile samples. Sufficient allozyme activity was observed for 9 mg of juvenile chinook salmon and steelhead fin tissue for some loci; however, the minimal amount of tissue needed to obtain all detectable loci was 54 mg. We performed computer simulations to test the feasibility of using allozyme data from fin tissue in mixed-stock analyses. The simulations showed that using the loci expressed in fin tissue samples, we were able to accurately estimate the stock composition of adult steelhead migrating up the Columbia River. Fin tissue sampling will be useful for collecting allozyme data when other tissues are not available or when lethal sampling is not desirable and will complement mitochondrial and nuclear DNA techniques that also use nonlethal sampling.

Effects of Invasive Tissue Collection on Rainbow Trout, Razorback Sucker, and Bonytail Chub

Abstract Histological examination and biochemical assays of fish tissues are used for disease detection, genetic characterizations, contaminant analyses, and the detection of stable isotopes. However, tissue sampling usually requires invasive sampling procedures that may harm or kill an organism under investigation. For this reason, invasive procedures are rarely used with threatened, endangered, or other sensitive species. If invasive tissue sampling could be performed with little harm, a wide array of sophisticated techniques could be employed to assist in protection and management of species in decline. We subjected rainbow trout Oncorhynchus mykiss to nonlethal invasive sampling of fin, muscle, and liver tissue. Techniques first developed for trout were applied to two endangered fishes, razorback sucker Xyrauchen texanus and bonytail chub Gila elegans. Acute (mortality) and chronic (suppression of growth rate) effects of tissue removal were evaluated in a hatchery for control and treatment groups of e...

Use of Biopsy Samples of Humpback Whale (Megaptera novaeangliae) Skin for Stable Isotope (δ13C) Determination

Previous work has shown that stable isotope indicators taken from the muscle tissue of dead, stranded cetaceans can be used to assess diet. Recent advances in remote biopsy techniques have provided a means to collect skin and blubber tissues from live animals. This study examines the potential of biopsy samples taken from humpback whales (Megaptera novaeangliae) for isotopic assessments of diet by 1) determining if isotopic differences exist between the two tissue types obtained in a biopsy (skin and blubber) and the traditional source for isotopic analysis, muscle tissue, 2) examining the effects of two different lipid extraction techniques on the removal of the preservative dimethyl sulfphoxide (DMSO) from tissues, and 3) assessing procedural reproducibility for automated isotopic analysis of skin derived from biopsy samples. Results demonstrate that carbon isotopic values ( δ 13C) of muscle were not significantly different from those of skin (Scheffe, p = 0.4985; δ 13C = -19.1‰ ± 0.7 and -19.5‰ ± 0.5 for muscle and skin respectively; mean ± SD). The values for blubber ( δ 13C = -23.7‰ ± 0.2) were significantly lower than those of muscle or skin (Scheffe, p = 0.0001). This result was consistent with previous studies indicating that the δ 13C of lipids is typically lighter than those tissues with which it is associated. The analysis also indicates that samples preserved in DMSO have significantly lower δ 13C than unpreserved samples (paired t-test, p = 0.010). Two methods of lipid extraction, sonication and Soxhlet ext rac t ion, were successful in removing DMSO from samples . The procedural reproducibility for δ 13C was 0.1‰. In summary, skin tissue yielded from biopsy samples may be used in isotopic assessments of diet. The use of biopsies as a source allows the technique to be used in longitudinal, non-lethal sampling.

Effects of different protein and fiber levels on growth, protein digestibility and carcass composition of carp ( Cyprinus carpio L.)

Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1β1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and β-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment.

Survival and pesticide exposure of northern bobwhites (Colinus virginianus) and eastern cottontails (sylvilagus floridanus) on agricultural fields treated with counter® 15G

During 1988 and 1989 northern bobwhites (Colinus virginianus) and eastern cottontails (Sylvilagus flondanus) were radiotagged and their daily survival monitored on six study sites treated with the organophosphorus insecticide-nematicide Counter®15G (terbufos), at the rate of 1.45 kg a.i. per hectare, and on three reference sites. Nonlethal blood samples were collected at the time of capture and during subsequent recaptures to determine exposure to terbufos through blood ChE analysis. Survival rates of radiotagged eastern cottontails and blood plasma ChE activities were not different between treated and reference sites in either year. Gastrointestinal (GI) tracts of two cottontail carcasses found in 1988 contained traces of terbufos in concentrations <0.046 ppm. No terbufos was detected in GI tracts in 1989. There were no differences in survival rates of radiotagged northern bobwhites among treatments. Mean blood plasma ChE activities of northern bobwhites were significantly lower (P < 0.05) on treated sites than on reference sites both years, including sublethal exposure to the test chemical. No GI tract residues were detected in bobwhites in 1988, and GI tract residues were detected in trace amounts (<0.046 ppm) in only one bobwhite carcass in 1989.

Glucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout ( Oncorhynchus mykiss)

Elevations in the non-specific defence mechanisms were noted after trout were injected or bathed in glucan solutions or in solutions containing the glucan combined with a bacterin, Y. ruckeri O-antigen. Periodic non-lethal blood samples showed that neutrophil activity as evaluated by the ability of the cells to stick to glass and produce oxidative radicals as detected by the nitroblue tetrazolium (NBT) assay, rose after treatments within 2 days. Elevated phagocytosis, assessed by increased uptake of glutaraldehyde-treated sheep red blood cells, also confirmed these kinetics. The numbers of circulatory glass-adherent cells from fish given the glucans by injection or bath was twice the level of the sham-treated controls; likewise the phagocytic ratio also was two-fold higher. In following the kinetics of the non-specific defensive responses, the injection of the glucan caused an immediate, slight reduction of NBT staining cells and numbers of leukocytes before the 2–3 day rise; this was not obvious in the fish given the glucan by bath. The glucans could play an important role in the prevention of diseases in fish culture.

Toxicity of complex waste mixtures: a comparison of observed and predicted lethality.

The ability to predict the biological effect of complex waste mixtures from chemical characterization data was examined by comparing observed mortality to that predicted by a mathematical additivity model with literature LD50 values for the chemicals identified in the mixtures. Male F344 rats were exposed by gavage to 1 of 10 samples of complex industrial waste. Seven of the 10 waste samples caused death within 24 h of administration at dosages ranging from 1 to 5 ml/kg body weight. Two of the 7 lethal waste samples produced 100% mortality at a dosage of 2.5 ml/kg; another 2 waste samples produced 100% mortality at 5 ml/kg. The partial chemical analysis, although providing more extensive information on chemical composition than might normally be available for most complex waste mixtures, was not sufficient to distinguish lethal from nonlethal waste samples or to indicate lethal potency. Possible explanations for the apparent inability to predict readily lethality from the chemical characterization data include the possible inappropriateness of an additivity model due to the presence of interactions, such as synergism or antagonism; the kinetics of chemical absorption, distribution, and elimination, which may be affected by administration of the chemical in a complex matrix; and the presence of unidentified chemicals in the mixture that may have contributed to the observed toxicity.

EXPERIMENTAL ESCHERICHIA COLI MENINGITIS IN NEWBORN SOS GUINEA PIGS

Newborn albino guinea pigs (GP's) were used as an animal model for Gram negative neonatal meningitis (NM). Twenty-four GP's 4-5 days old were inoculated intranasally with 108-9 E. coli K1 organisms (MIC 0.1 ug/ml gentamicin) originally isolated from human NM. Bacteremia occurred in 50% of GP's within 48h, and meningitis (defined by positive CSF culture) was detected in 80% of bacteremic GP's within 5 days. Seven of 8 (88%) GP's with meningitis died within 7 days. All animals were sacrificed at 7 days post-inoculation and brains were examined histologically. There was 100% agreement between positive or negative CSF culture and presence or absence of inflammatory meningeal reaction. The majority (88%) of GP's with meningitis also had ventriculitis. Two further experiments revealed that the results (rate of bacteremia, positive CSF, mortality) were reproducible. A group of animals was also treated with gentamicin (2.5 mg/kg IM q12h) begun after diagnostic cisternal puncture at 16h post inoculation. Mean serum and CSF gentamicin levels were 3.6 and 0.16 ug/ml, respectively. Mortality in meningitic GP's was reduced by about 60% with gentamicin therapy. These results suggest that the newborn GP may be a useful model to study pathogenesis and efficacy of therapeutic agents in E. coli NM, since a) meningitis can be induced in a predictable and reproducible manner and b) the non-lethal sampling of CSF permits assessment of therapeutic responses and measurement of antibiotic levels.

Protecting garment for aviators : Anon. ( Sci. Amer., cxi, No. 12, 223)

In this report, we investigated the accumulation of heavy metals in the lizard Microlophus atacamensis, in three coastal areas of the Atacama Desert, northern Chile. We captured reptiles in a non-intervened area (Parque Nacional Pan de Azúcar, PAZ), an area of mining impact (Caleta Palitos, PAL) and an active industrial zone (Puerto de Caldera, CAL). Our methods included a non-lethal sampling of reptiles’ tails obtained by autotomy and a few sacrificed animals to perform a stomach contents analysis. The concentrations of lead, copper, nickel, zinc and cadmium were measured by atomic absorption spectrophotometry in both soil and prey and compared to those recorded in the lizards’ tails. Data obtained from lizard tails captured in PAL showed significantly high concentrations of Pb, Cu, Ni, and Zn compared to the other two sites PAZ and CAL. We did not find statistically significant differences among PAZ, PAL and CAL soils, probably due to the similar geological composition of the sites. However, the regional background values for Pb indicate contamination or at least metal enrichment in soils of the three sites, for Cu the global background values indicate contamination for the three sites, and for Cd both the regional and global backgroud values show high values. The analysis of the stomach content showed differences in the food sources of the lizards among the sites studied. The concentration of heavy metal in lizard tissues versus prey delivered values of the Trophic Transfer Factor higher than one (1), suggesting that food may be a primary source of metals in the tissues of M. atacamensis. Calculations of the Bioaccumulation Factor (BAF) and the Ecological Risk (IR) resulted in values higher than one (1) indicating the relevance of this process in the sites studied. In this article, we report relationships between environmental contaminants, mainly putative preys, and concentrations found in lizard tails, which is more substantial in areas with historical heavy metal contamination such as PAL where the non-lethal technique developed in this research suggests a process of metal bioaccumulation in M. atacamensis.