Failure to transmit avian vacuolar myelinopathy to mallard ducks

Avian vacuolar myelinopathy (AVM) is a neurologic disease that has been diagnosed in free-ranging birds in the southeastern United States. Bald eagles (Haliaeetus leuocephalus), American coots (Fulica americana), and mallards (Anas platyrhynchos) have been affected. Previous investigations have not determined the etiology of this disease. In November and December 2002, we attempted to induce AVM in game-farmed mallards through four, 7-day exposure trials. Mallards were housed in six groups of eight, with two of these groups serving as controls. One group was housed with AVM-affected coots; one group was tube fed daily with water from the lake where affected coots were captured; one group was tube fed daily with aquatic vegetation (Hydrilla verticillata) from the same lake; and another group was tube fed daily with sediment from the lake. No ducks exhibited clinical neurologic abnormalities consistent with AVM and no evidence of AVM was present at histopathologic examination of brain tissue. Although limitations in sample size, quantity of individual doses, frequency of dose administration, duration of exposure, and timing of these trials restrict the interpretation of the findings, AVM was not readily transmitted by direct contact, water, hydrilla, or sediment in this investigation.     

Attempts to reproduce vacuolar myelinopathy in domestic swine and chickens

Avian vacuolar myelinopathy (AVM) was first recognized as a cause of bald eagle (Haliaeetus leucocephalus) mortality in 1994 in Arkansas (USA) and has since caused over 90 bald eagle and numerous American coot (Fulica americana) mortalities in five southeastern states. The cause of AVM remains undetermined but is suspected to be a biotoxin. Naturally occurring AVM has been limited to wild waterbirds, raptors, and one species of shorebird, and has been reproduced experimentally in red-tailed hawks (Buteo jamaicensis). In this study, chickens and swine were evaluated for susceptibility to vacuolar myelinopathy with the intent of developing animal models for research and to identify specific tissues in affected coots that contain the causative agent. Additionally, submerged, aquatic vegetation, primarily hydrilla (Hydrilla verticillata), and associated material collected from a reservoir during an AVM outbreak was fed to chickens in an effort to reproduce the disease. In two separate experiments, six 4-wk-old leghorn chickens and ten 5-wk-old leghorn chickens were fed coot tissues. In a third experiment, five 3-mo-old domestic swine and one red-tailed hawk, serving as a positive control, were fed coot tissues. In these experiments, treatment animals received tissues (brain, fat, intestinal tract, kidney, liver, and/or muscle) from coots with AVM lesions collected at a lake during an AVM outbreak. Negative control chickens and one pig received tissues from coots without AVM lesions that had been collected at a lake where AVM has never been documented. In a fourth experiment, eight 3-wk-old leghorn chickens were fed aquatic vegetation material. Four chickens received material from the same lake from which coots with AVM lesions were collected for the previous experiments, and four control chickens were fed material from the lake where AVM has never been documented. Blood was collected and physical and neurologic exams were conducted on animals before and once per week during the trials. All animals were sacrificed and necropsies were performed on Day 29 of feeding, with the exception of one treated chicken that was sacrificed and necropsied on Day 15 of feeding. Microscopic lesions of vacuolar myelinopathy were present in the red-tailed hawk and five chickens that received a mixture of all tissues and two chickens that received only gastrointestinal tissues of coots with AVM lesions. Three of four treated chickens in the aquatic vegetation trial developed vacuolar lesions. None of four treatment pigs or any of the negative control animals developed vacuolar lesions. Chickens are susceptible to AVM and may serve as a useful animal model for future studies. Swine may be refractory to AVM or not affected by AVM at the same dose as are chickens and red-tailed hawks. The causative agent of AVM in affected coots is associated with the gastrointestinal tissues. Furthermore, AVM can be reproduced in chickens via ingestion of aquatic vegetation and associated materials collected from a lake during an AVM outbreak. The cause of AVM is most likely present in the materials associated with submerged vegetation because the vegetation itself (hydrilla) was the same at our AVM-positive and AVM-negative sites.     

Avian vacuolar myelinopathy outbreaks at a southeastern reservoir

Avian vacuolar myelinopathy (AVM) is a neurologic disease of unknown etiology that affects bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), and several species of waterfowl. An unidentified neurotoxin is suspected as the cause of AVM, which has been documented at several reservoirs in the southeastern United States. We conducted diagnostic and epidemiologic studies annually during October-March from 1998-2004 at Clarks Hill/Strom Thurmond Lake on the Georgia/South Carolina border to better understand the disease. Avian vacuolar myelinopathy was confirmed or suspected as the cause of morbidity and mortality of 28 bald eagles, 16 Canada geese (Branta canadensis), six American coots, two great-horned owls (Bubo virginianus), and one killdeer (Charadrius vociferus). Active surveillance during the outbreaks yielded annual average prevalence of vacuolar lesions in 17-94% of coots, but not in 10 beavers (Castor canadensis), four raccoons (Procyon lotor), and one gray fox (Urocyon cinereoargenteus) collected for the study. Brain lesions were not apparent in 30 Canada geese collected and examined in June 2002. The outbreaks at this location from 1998-2004 represent the most significant AVM-related bald eagle mortality since the Arkansas epornitics of 1994-95 and 1996-97, as well as the first confirmation of the disease in members of Strigiformes and Charadriiformes.     

Experimental vacuolar myelinopathy in red-tailed hawks

Avian vacuolar myelinopathy (AVM) was recognized in 1994 as a cause of wild bird mortality when 29 bald eagles (Haliaeetus leucocephalus) succumbed to the disease at DeGray Lake, Arkansas (USA). The cause of AVM and its source remain undetermined despite extensive diagnostic and research investigations. Two years later, when AVM killed 26 eagles in the same area in Arkansas, it became apparent that American coots (Fulica americana) had identical neurologic signs and lesions, and it was hypothesized that eagles acquired AVM via ingestion of affected coots. In order to test this hypothesis, we fed coot tissues (brain, liver, kidney, muscle, fat, and intestinal tract) to rehabilitated, non-releasable red-tailed hawks (Buteo jamaicensis). Five hawks received tissues from coots with AVM lesions, and one hawk received tissues from coots without brain lesions that had been collected at a site where AVM never has been documented. All hawks received 12-70 g/day (mean = 38 g) of coot tissues for 28 days. All six hawks remained clinically normal during the study. The birds were euthanatized on day 29 and microscopic lesions of AVM were found in all hawks that received tissues from affected coots, but not in the hawk that received tissues from unaffected coots. This marks the first time that AVM has been produced in birds under laboratory conditions and proves that birds of prey can acquire AVM via ingestion of tissues from affected coots.     

Epizootiologic studies of avian vacuolar myelinopathy in waterbirds

Epizootic avian vacuolar myelinopathy (AVM) was first recognized as a neurologic disease in bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana) in Arkansas, USA in 1994 and 1996, respectively, but attempts to identify the etiology of the disease have been unsuccessful to date. Between 1998 and 2001, wing clipped sentinel birds (wild American coots and game farm mallards [Anas platyrhynchos]) were released at Lake Surf, North Carolina, a lake with recurrent outbreaks of AVM, in order to gain a better understanding of the epizootiology of the disease. As early as 5-7 days post-release, sentinel coots and mallards showed neurologic signs of disease and were confirmed with AVM upon histologic examination of their brains. Serial releases of sentinel mallards during the summer, fall, and winter of 2000-01 demonstrated that exposure to the causative agent at a threshold sufficient to manifest disease was seasonal and occurred over about a 2 mo period, during November and December. Our findings that disease onset can be very rapid (5-7 days) and that exposure to the causative agent of AVM is site-specific, seasonal (late fall to early winter), and occurs over a relatively short duration (several months) supports the hypothesis that the disease is caused by a chemical substance, most likely of natural origin.     

Investigation of the link between avian vacuolar myelinopathy and a novel species of cyanobacteria through laboratory feeding trials

Avian vacuolar myelinopathy (AVM) is a neurologic disease affecting Bald Eagles (Haliaeetus leucocephalus), American Coots (Fulica americana), and other birds in the southeastern United States. The cause of the disease has not yet been determined, although it is generally thought to be a natural toxin. Previous studies have linked AVM to aquatic vegetation, and the current working hypothesis is that a species of cyanobacteria growing epiphytically on that vegetation is producing a toxin that causes AVM. Surveys of epiphytic communities have identified a novel species of cyanobacteria in the order Stigonematales as the most likely suspect. The purpose of this study was to further examine the relationship between the suspect Stigonematales species and induction of AVM, by using animal feeding trials. Adult Mallards and domestic chickens were fed aquatic vegetation from two study sites containing the suspect cyanobacterial epiphyte, as well as a control site that did not contain the Stigonematales species. Two trials were conducted. The first trial used vegetation collected during mid-October 2003, and the second trial used vegetation collected during November and December 2003. Neither treatment nor control birds in the first trial developed AVM lesions. Ten of 12 treatment Mallards in the second trial were diagnosed with AVM, and control birds were not affected. This study provides further evidence that the novel Stigonematales species may be involved with AVM induction, or at the least it is a good predictor of AVM toxin presence in a system. The results also demonstrate the seasonal nature of AVM events.     

Attempts to identify the source of avian vacuolar myelinopathy for waterbirds

Attempts were made to reproduce avian vacuolar myelinopathy (AVM) in a number of test animals in order to determine the source of the causative agent for birds and to find a suitable animal model for future studies. Submerged vegetation, plankton, invertebrates, forage fish, and sediments were collected from three lakes with ongoing outbreaks of AVM and fed to American coots (Fulica americana), mallard ducks and ducklings (Anas platyrhynchos), quail (Coturnix japonica), and laboratory mice either via gavage or ad libitum. Tissues from AVM-affected coots with brain lesions were fed to ducklings, kestrels (Falco sparverius), and American crows (Corvus brachyrhynchos). Two mallards that ingested one sample of Hydrilla verticillata along with any biotic or abiotic material associated with its external surface developed brain lesions consistent with AVM, although neither of the ducks had clinical signs of disease. Ingestion of numerous other samples of Hydrilla from the AVM affected lakes and a lake with no prior history of AVM, other materials (sediments, algae, fish, invertebrates, and water from affected lakes), or tissues from AVM-affected birds did not produce either clinical signs or brain lesions in any of the other test animals in our studies. These results suggest that waterbirds are most likely exposed to the causative agent of AVM while feeding on aquatic vegetation, but we do not believe the vegetation itself is the agent. We hypothesize that the causative agent of AVM might either be accumulated by aquatic vegetation, such as Hydrilla, or associated with biotic or abiotic material on its external surfaces. In support of that hypothesis, two coots that ingested Hydrilla sampled from a lake with an ongoing AVM outbreak in wild birds developed neurologic signs within 9 days (ataxia, limb weakness, and incoordination), and one of two coots that ingested Hydrilla collected from the same site 13 days later became sick and died within 38 days. None of these three sick coots had definitive brain lesions consistent with AVM by light microscopy, but they had no gross or histologic lesions in other tissues. It is unclear if these birds died of AVM. Perhaps they did not ingest a dose sufficient to produce brain lesions or the lesions were ultrastructural. Alternatively, it is possible that a separate neurotoxic agent is responsible for the morbidity and mortality observed in these coots.     

Predicting Bald Eagle Collision at Wind Energy Facilities

Bald eagles (Haliaeetus leucocephalus) are currently protected in the United States under the Bald and Golden Eagle Protection Act of 1940 and Migratory Bird Treaty Act of 1918. Given these protections and the increasing development of wind energy throughout the United States, it is important for regulators and the wind industry to understand the risk of bald eagle collisions with wind turbines. Prior probability distributions for eagle exposure rates and collision rates have been developed for golden eagles (Aquila chrysaetos) by the United States Fish and Wildlife Service (USFWS). Given similar information has not been available for bald eagles, the current recommendation by the USFWS is to use the prior probability distributions developed using data collected on golden eagles to predict take for bald eagles. But some evidence suggests that bald and golden eagles may be at different risk for collision with wind turbines and the prior probability distributions developed for golden eagles may not be appropriate for bald eagles. We developed prior probability distributions using data collected at MidAmerican Energy Company's operating wind energy facilities in Iowa, USA, from December 2014 to March 2017 for bald eagle exposure rates and collision rates. The prior probability distribution for collision rate developed for bald eagles has a lower mean collision rate and less variability relative to that developed for golden eagles. We determined that the prior probability distributions specific to bald eagles from these operating facilities are a better starting point for predicting take for bald eagles at operating wind energy facilities in an agricultural landscape than those developed for golden eagles. © 2021 The Wildlife Society.     

Use of Upland and Riparian Areas by Wintering Bald Eagles and Implications for Wind Energy

Weather can shape movements of animals and alter their exposure to anthropogenic threats. Bald eagles (Haliaeetus leucocephalus) are increasingly at risk from collision with turbines used in onshore wind energy generation. In the midwestern United States, development of this energy source typically occurs in upland areas that bald eagles use only intermittently. Our objective was to determine the factors that cause wintering bald eagles to occupy riparian areas and riskier, upland areas. We tracked 20 bald eagles using telemetry in the Upper Midwest (MN, IA, MO, WI, IL, USA) during winter 2014–2015 and 2015–2016 and evaluated habitat use by eagles in response to variation in weather and time of year. Eagles used riparian areas more when wind speed and atmospheric pressure were low. Exclusive use of uplands was more frequent during weather systems with low pressure and high humidity and after long periods of cold weather. There was a non-linear response to time of year (measured by days before migration) in the frequency of exclusive use of uplands or riparian areas. Probability of exclusive use of either landscape was generally constant within 95 days prior to migration. The probability of use of riparian areas, however, was markedly less during dates >100 days before migration. Our results suggest that eagles are most likely to be exposed to wind energy developments located in upland areas during low pressure systems, after long periods of cold weather, and several months before the onset of spring migration. This information helps to better understand the factors influencing bald eagle habitat use in winter and will be useful to managers and developers wishing to establish effective strategies to avoid, minimize, and mitigate take, and to survey for mortalities at wind energy developments. © 2020 The Wildlife Society.     

Lead Exposure in Bald Eagles from Big Game Hunting,the Continental Implications and Successful Mitigation Efforts

Studies suggest hunter discarded viscera of big game animals (i.e., offal) is a source of lead available to scavengers. We investigated the incidence of lead exposure in bald eagles in Wyoming during the big game hunting season, the influx of eagles into our study area during the hunt, the geographic origins of eagles exposed to lead, and the efficacy of using non-lead rifle ammunition to reduce lead in eagles. We tested 81 blood samples from bald eagles before, during and after the big game hunting seasons in 2005–2010, excluding 2008, and found eagles had significantly higher lead levels during the hunt. We found 24% of eagles tested had levels indicating at least clinical exposure (>60 ug/dL) during the hunt while no birds did during the non-hunting seasons. We performed driving surveys from 2009–2010 to measure eagle abundance and found evidence to suggest that eagles are attracted to the study area during the hunt. We fitted 10 eagles with satellite transmitters captured during the hunt and all migrated south after the cessation of the hunt. One returned to our study area while the remaining nine traveled north to summer/breed in Canada. The following fall, 80% returned to our study area for the hunting season, indicating that offal provides a seasonal attractant for eagles. We fitted three local breeding eagles with satellite transmitters and none left their breeding territories to feed on offal during the hunt, indicating that lead ingestion may be affecting migrants to a greater degree. During the 2009 and 2010 hunting seasons we provided non-lead rifle ammunition to local hunters and recorded that 24% and 31% of successful hunters used non-lead ammunition, respectively. We found the use of non-lead ammunition significantly reduced lead exposure in eagles, suggesting this is a viable solution to reduce lead exposure in eagles.     

Hemograms for and nutritional condition of migrant bald eagles tested for exposure to lead

Plasma proteins, hematocrit, differential blood counts were examined and nutritional condition was estimated for bald eagles (Haliaeetus leucocephalus) trapped (n = 66) during antumn migration, 1994-95 at Galloway Bay (Saskatchewan, Canada), for the purposes of estimating prevalence of exposure to lead. Sex and age differences in hematocrit and plasma proteins were not observed; however, female eagles exhibited larger median absolute heterophil counts than males. Hematologic values were similar to those previously reported from eagles in captivity. Departures from expected hematological values from a healthy population of eagles were not observed in birds with elevated levels of blood lead (> or =0.200 microg/ml). Similarly, nutritional condition was not related to blood-lead concentrations. Therefore, it appears that lead exposure in this population was below a threshold required to indicate toxicological alteration in the hematological values and index of nutritional condition that we measured.     

Identification of the sex chromosomes in the bald eagle.

Karyotypes of five American bald eagles (Haliaeetus leucocephalus and H. alacanus) are compared. All had 2n=66 chromosomes which fell into 3 size groups: A, 20 pairs of biarmed chromosomes; B, 9 pairs of acrocentric chromosomes and C, 4 pairs of microchromosomes. C-banding was done in two eagles and a heterochromatic W chromosome was identified in a presumptive female. The ZZ and ZW chromosomes could be identified in the karyotypes.     

Modeling climate change impacts on overwintering bald eagles

Bald eagles (Haliaeetus leucocephalus) are recovering from severe population declines, and are exerting pressure on food resources in some areas. Thousands of bald eagles overwinter near Puget Sound, primarily to feed on chum salmon (Oncorhynchus keta) carcasses. We used modeling techniques to examine how anticipated climate changes will affect energetic demands of overwintering bald eagles. We applied a regional downscaling method to two global climate change models to obtain hourly temperature, precipitation, wind, and longwave radiation estimates at the mouths of three Puget Sound tributaries (the Skagit, Hamma Hamma, and Nisqually rivers) in two decades, the 1970s and the 2050s. Climate data were used to drive bald eagle bioenergetics models from December to February for each river, year, and decade. Bald eagle bioenergetics were insensitive to climate change: despite warmer winters in the 2050s, particularly near the Nisqually River, bald eagle food requirements declined only slightly (<1%). However, the warming climate caused salmon carcasses to decompose more rapidly, resulting in 11% to 14% less annual carcass biomass available to eagles in the 2050s. That estimate is likely conservative, as it does not account for decreased availability of carcasses due to anticipated increases in winter stream flow. Future climate-driven declines in winter food availability, coupled with a growing bald eagle population, may force eagles to seek alternate prey in the Puget Sound area or in more remote ecosystems.     

Sources of Mortality in Bald Eagles in Michigan, 1986–2017

As bald eagle populations recover, defining major sources of mortality provides managers important information to develop management plans and mitigation efforts. We obtained data from necropsies on 1,490 dead bald eagles (Haliaeetus leucocephalus) collected in Michigan, USA, conducted from 1986 to 2017 to determine causes of death (COD). Trauma and poisoning were the most common primary COD categories, followed by disease. Within trauma and poisoning, vehicular trauma (n = 532) and lead poisoning (n = 176) were the leading COD subcategories, respectively. Females comprised a greater number of carcasses for most COD diagnoses. The proportion of trauma and poisoning CODs significantly increased in the last few years of the study in comparison to a select few years at the beginning. Trauma CODs were greater in autumn months during whitetail deer (Odocoileus virginianus) breeding and hunting seasons and in February, when aquatic foraging is unavailable and eagles are likely forced to scavenge along roadsides. Poisoning CODs were greatest in late winter and early spring months, when deer carcasses containing lead ammunition, which are preserved by the cold weather, also become a supplemental food source. The major infectious disease CODs, West Nile virus and botulism (Clostridium botulinum type E), were more prevalent during summer months. We recommend moving road-killed carcasses, especially white-tailed deer, from the main thoroughfare to the back of the right-of-way, and the transition from lead ammunition and fishing tackle to non-toxic alternatives to decrease these main anthropogenic sources of mortality for bald eagles, and other scavenger species. © 2020 The Wildlife Society.     

Eagle visitation rates to carrion in a winter scavenging guild

Understanding the behavioral ecology of species of conservation concern can help to inform better management. During winters 2011 through 2017, we placed camera traps at stations baited with carrion to investigate characteristics of winter scavenging by golden eagles (Aquila chrysaetos) and bald eagles (Haliaeetus leucocephalus) in eastern Washington and Oregon, USA. Our objectives were to better understand exposure risk of individual eagles to lead contaminants and evaluate factors that affect eagle visitation to carrion to inform measures that reduce lead exposure. We studied photo sequences from 108 traps ( = 2,725 ± 306 [SE] images/trap) and used plumage and physical characteristics to track visitation of 183 individual golden eagles and 90 bald eagles at deer (Odocoileus spp.) carrion until it was totally consumed. At least 1 eagle visited 76% of traps ( = 2.5 ± 0.3 unique eagles/trap). On average, an eagle visited a trap 3.4 ± 0.2 times (range = 1–19 visits) over 1.9 ± 0.1 days (range = 1–9 days). We used general linear mixed models to identify influences on number of eagle visits and pooled visit duration. Individual golden eagles visited carrion about 25% more often and 50% longer than bald eagles, and individual juvenile eagles visited carrion more often and longer than immature and adult eagles. On average, an eagle made an additional visit to carrion for every golden eagle that came to the same trap. Eagles spent less time at offal ( = 26.2 ± 6.4 min) than at a whole carcass ( = 92.9 ± 7.5 min), and understory vegetation immediately surrounding carrion was associated with a 30% reduction in visitation time. In the Pacific Northwest during winter, adult and juvenile golden eagles, by virtue of their abundance and visitation to carrion compared to the immature age class and bald eagles of all ages, have the highest potential for exposure to anthropogenic effects from carrion visitation. Concealment of offal piles in vegetation may reduce, but not eliminate, eagle use because of competing scavengers that expose carrion locations. We found no evidence that carrion proximity to nearest known nests, topography, or snow cover affect visitation by eagles. Thus, short of using alternative ammunition to lead, we recommend burial or removal of offal from hunter-killed ungulates. © 2019 The Wildlife Society.     

Secondary poisoning of eagles following intentional poisoning of coyotes with anticholinesterase pesticides in western Canada

Records of eagles, coyotes (Canis latrans), and red foxes (Vulpes vulpes) necropsied at the Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada, between 1967 and 2002 were reviewed for cases suggestive of anticholinesterase poisoning. From 1993 to 2002, 54 putative poisoning incidents involving 70 bald eagles (Haliaeetus leucocephalus) and 10 golden eagles (Aquila chrysaetus) were identified. Of these, 50 incidents occurred in Saskatchewan, two were in Manitoba, and one occurred in each of Alberta and the Northwest Territories. The diagnosis was confirmed in eight instances by demonstration of pesticide in ingesta from eagles or known use of pesticide at the site together with brain cholinesterase (AChE) reduction of >50% in at least one animal. A presnmptive diagnosis of poisoning was made in 33 incidents based on brain AChE reduction of >50% in at least one animal; 13 incidents were considered suspicious because of circumstantial evidence of the death of eagles in association with other species and limited AChE reduction. Other wild species were found dead in 85% of the incidents involving eagles. Coyotes, foxes, black-billed magpies (Pica pica), and striped skunks (Mephitis mephitis) were associated with 34, six, six, and three incidents, respectively. There were eight additional incidents that did not involve eagles in which poisoning was diagnosed in coyotes. Carbofuran was identified in nine incidents. Carbamate poisoning was indicated on the basis of reactivation of brain AChE activity in two additional incidents. Brain AChE activity was not reduced from normal in eagles in four of seven incidents in which carbofuran was identified. The organophosplorous insecticide terbufos was found together with carbofuran in one incident. Brain AChE activity was measured in wild canids and in eagles in 15 incidents; in all of these incidents, brain AChE was redulced by >50% in at least one mammal, whereas this level of reduction occrred in eagles in only four incidents. Use of anticholinesterase pesticides to poison coyotes is illegal, but the practice continues and secondary poisoning of eagles is a problem of unknown proportions in western North America.     

Native flora and fauna response to removal of the weed Hydrilla verticillata (L.f.) Royle in Lake Tutira

Hydrilla has been in New Zealand since the 1960s and formed major infestations in four lakes in the Hawkes Bay region. Challenges to controlling hydrilla in New Zealand have included a lack of tools and the changing responsibilities of local management agencies. A grass carp field trial was initiated in 1988 in the smallest of the hydrilla infested lakes to assess the feasibility of eradicating hydrilla. After the main hydrilla beds were consumed, regrowth from tubers still occurred for a further 12 years. In 2008, grass carp were released into the remaining three hydrilla-infested lakes in a central government led response to eradicate hydrilla. This paper describes the changes in the flora and fauna in the largest of these lakes, Lake Tutira, following the introduction of grass carp and the removal of the hydrilla weed beds. Annual surveys of aquatic vegetation and macroinvertebrates in the lake from 2008 to 2012 have shown that, following the removal of the hydrilla weed beds by 2010, there was a shift in grass carp grazing to marginal emergent plants, and a general increase in the distribution of the native plant vegetation, although there was some evidence of a decline in charophyte abundance. Macroinvertebrate diversity was maintained although there were changes in the relative abundance of taxa linked to changes in the littoral vegetation.     

EVALUATION OF PIGMENT EXTRACTION METHODS AND A RECOMMENDED PROTOCOL FOR PERIPHYTON CHLOROPHYLL a DETERMINATION AND CHEMOTAXONOMIC ASSESSMENT1

Many methods have been proposed to extract and quantify algal pigments. Comparative studies have found that pigment extraction efficiency varies among solvent and mechanical disruption protocols due to differential cellular resistance, thereby, leading to potential misinterpretation of pigment data. When the type or resistance of algae are unknown, a method is required that efficiently extract pigments from all taxonomic groups. The objective of this study was to develop a simple and efficient one stage periphyton pigment extraction protocol by comparing the extractability of four solvents (acetone, methanol, methanol/acetone, and methanol/acetone/N,N‐dimethylformamide), the effects of grinding, and the effects of freeze‐drying. The best overall extraction was obtained using freeze‐dried samples extracted with methanol/acetone/DMF/water (MAD). Eighty‐six percent more chlorophyll was extracted when the sample was freeze‐dried relative to fresh/frozen samples extracted with 90% acetone. Freeze‐drying greatly improved the extraction of both polar and non‐polar (lipophilic/hydrophobic) pigments while MAD increased the extractability of polar pigments and improved peak resolution of all pigments. Chemotaxonomic assessment differed between samples that were fresh/frozen or freeze‐dried before extraction. The relative abundance of cyanobacteria was greater for freeze‐dried material compared with fresh/frozen due to the improved extractability of cyanobacterial pigments. Based on the results of this study, the traditional approach of 90% acetone as a solvent is not recommended for periphyton samples containing cyanobacteria or when the composition of the mat is unknown. The combination of freeze‐drying and MAD was sufficient for the extraction of pigments from a periphyton mat containing filamentous cyanobacteria, green algae, and diatoms.