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Handbook of acute toxicity of chemicals to fish and aquatic invertebrates : summaries of toxicity tests conducted at Columbia National Fisheries Research Laboratory, 1965-78

Acute toxicity is a major subject of research at Columbia National Fisheries Research Laboratory for evaluating the impact of toxic chemicals on fishery resources. The Laboratory has played a leading role in developing research technology for toxicity testing and data interpretation. In 1965-78, more than 400 chemicals were tested against a variety of invertebrates and fish species representative of both cold- and warm-water climates. The use of acute toxicity tests for assessing the potential hazard of chemical contaminants to aquatic organisms is well documented (Boyd 1957; Henderson et al. 1960; Sanders and Cope 1966; Macek and McAllister 1970). Static acute toxicity tests provide rapid and (within limits) reproducible concentration-response curves for estimating toxic effects of chemicals on aquatic organisms. These tests provide a database for determining relative toxicity of a large number of chemicals to a variety of species and for estimating acute effects of chemical spills on natural aquatic systems; they also assist in determining priority and design of additional toxicity studies. Acute toxicity tests usually provide estimates of the exposure concentration causing 50% mortality (LC50) to test organisms during a specified period of time. For certain invertebrates, the effective concentration is based on immobilization, or some other identifiable endpoint, rather than on lethality. The application of the LC50 has gained acceptance among toxicologists and is generally the most highly rated test for assessing potential adverse effects of chemical contaminants to aquatic life (Brungs and Mount 1978; American Institute for Biological Sciences 1978 a ). The literature contains numerous papers dealing with the acute toxicity of chemicals to freshwater organisms. However, there is a tremendous need for a concise compendium of toxicity data covering a large variety of chemicals and test species. This Handbook is a compilation of a large volume of acute toxicity data from the Columbia Laboratory and its field laboratories. It presents definitive acute toxicity data on 271 chemicals tested against a variety of freshwater invertebrates and fishes. The chemicals represent all major groups of pesticides, as well as numerous industrial chemicals. This compilation should serve as a useful database for the many agencies and organizations dealing with research and management programs concerned with the impact of chemicals on aquatic resources. The Columbia Laboratory has played a major role in developing currently used standard methodology for static acute toxicity testing. The use of standardized methodology greatly reduces variation in results. The data presented here have been carefully scrutinized to eliminate tests that failed to follow acceptable procedures. Handling of test organisms and procedures for static toxicity tests followed those described by Lennon and Walker (1964) and Macek and McAllister (1970), and conform well with those recommended by Brauhn and Schoettger (1975) and the Committee on Methods for Toxicity Tests with Aquatic Organisms (1975). The species of fish and invertebrates that were tested are listed in phylogenetic order in Tables 1 and 2. Fish were obtained from Federal and State hatcheries as either eggs or fry. Original stocks of invertebrates were collected and cultured from wild populations with no known source of contamination; these populations were replenished regularly. The invertebrates were cultured in the Laboratory by methods similar to those described by Sanders and Cope (1966). Test chemicals usually consisted of technical or analytical grade samples of known purity. Formulations of the chemicals were also tested when available. When purity of test chemicals was known, all calculated concentrations were based on percent active ingredients. Stock solutions were prepared immediately before each test, with commercial grade acetone as the carrier solvent. Occasionally, ethanol or dimethyl-formamide was substituted. Solvent concentrations did not exceed 0.5 mL/L in final dilution water. Test water (dilution water) was reconstituted from deionized water of at least 10 6 ohms resistivity by the addition of appropriate reagent grade chemicals (Marking 1969). Water was buffered to maintain a pH of 7.2 to 7.5, an alkalinity of 30 to 35 mg/L, and a hardness of 40 to 50 mg/L as CaCO 3 . Test water was mixed thoroughly and aerated before transfer into test chambers. Fish were acclimated to dilution water by gradually changing the water in acclimated tanks from 100% well water to 100% reconstituted water over a 1- to 3-day period at the desired testing temperature. Invertebrates were acclimated from well water to dilution water over a 4- to 6-h period. Toxicity tests were conducted under static conditions without aeration, and the organisms were not fed during acclimation or testing. Temperature of test solutions was maintained within ± 1°C of that required for a given test. Toxicity tests with fish were conducted in 18.9-liter (5-gal) wide-mouthed jars containing 15 liters of test solution. Fingerling fish weighing 0.2 to 1.5 g were tested at each concentration. Caution was taken not to exceed 0.8 g of test organisms per liter of solution. Duplicate test chambers were used to accommodate larger fish. Test chambers varied in size for invertebrates, depending on the species used; volume of test solution ranged from 0.25 to 4 liters. At least 10 organisms were exposed to each concentration for all definitive tests. At least six concentrations were used per toxicity test. The tests began upon initial exposure to the toxicant and continued for 96 h. Immobilization tests with invertebrates were conducted for only 48 h. The number of dead or affected organisms in each test chamber were recorded and the dead organisms were removed every 24 h; general observations on the condition of test organisms were also recorded at these times. Toxicity data were analyzed by a statistical method described by Litchfield and Wilcoxon (1949) to determine LC50 (theoretical estimate of the concentration lethal to 50% of the test animals) and 95% confidence intervals. This method is recommended by the American Public Health Association (1971) and by Sprague (1969) for determining median lethal concentrations. The procedure is easily modified for computing a single LC50 when replicate tests are performed.

Resource Publication

Using advanced population genomics to better understand the relationship between offshore and spawning habitat use for Atlantic Sturgeon

Atlantic Sturgeon ( Acipenser oxyrinchus oxyrinchus ) are a large-bodied anadromous fish that historically supported important fisheries along the east coast of the United States. Following years of overharvest and habitat degradation, populations experienced severe declines. In 2012, the National Marine Fisheries Service listed Atlantic Sturgeon under the Endangered Species Act (ESA; 61 FR 4722). Their listing named five Distinct Population Segments (DPSs), predicated on genetic groups composed of geographically proximate populations. Federal management of Atlantic Sturgeon presents challenges, as sturgeon from each of the five DPSs mix extensively in coastal and marine habitats yet take and recovery progress must be evaluated separately for each unit. Genetic assignment testing based on mitochondrial and microsatellite markers allows individuals to be assigned back to their natal river and DPS. However, this approach is not perfect and some individuals may be incorrectly assigned. Recent advances in genomics offer the potential of a higher resolution approach to genetic assignment testing, and thus may reduce uncertainty associated with assignment testing. In addition, genomics allows a greater number of markers to be examined from across a broader portion of the sturgeon genome, thus may provide an enhanced perspective of population structure for the species, and potentially allow other previously intractable questions to be addressed (Bernatchez et al. 2017, Supple and Shapiro 2018). We used next-generation sequencing to develop a draft genome for Atlantic Sturgeon and identify single nucleotide polymorphisms (SNPs) that could be used to resolve the natal river and DPS of individual Atlantic Sturgeon. We identified 1,210 candidate SNPs within the nuclear genome as well as 49 SNPs within the mitochondrial genome. After filtering and review, we selected 161 nuclear SNPs and 39 mitochondrial SNPs for further testing and evaluation. We used genotyping-in-thousands by sequencing (GT-seq) to simultaneously sequence nuclear SNP loci, mitochondrial SNP loci, and the existing panel of twelve microsatellite loci. This effort required a pilot sequencing run on a single sturgeon sample to test marker amplification and refine primer strengths, followed by a series of sequencing runs to generate baseline data for 288 individuals representing nine populations of Atlantic Sturgeon in four DPSs. Using baseline data from the nine populations, we ran a series of genomic analyses to characterize diversity within and among populations, providing a benchmark for this species using the new SNP markers. Allelic richness was similar for all populations, although there was a general trend of more northern population containing greater levels of allelic richness. Interestingly, we observed linkage disequilibrium among many pairs of loci within many populations. This might be the result of physical linkage but could also suggest these populations are recovering from genetic bottlenecks and/or are effectively small, leading to specific haplotypes to be favored by chance. Pairwise differentiation among populations varied among the populations ( F ST range: 0.010-0.098) and was significantly correlated ( r = 0.771; P < 0.001) to pairwise F ST observed using microsatellite markers). Population clustering and ordination techniques using the new genomic data both support an overall population structure that is similar to the current DPS management units (which were developed primarily based on microsatellite genetic data). Overall, this suggests that existing microsatellite markers and the panel of SNP markers developed in this study provide similar information about the populations structure and ecology of Atlantic Sturgeon. Given the observed differences in allele frequencies among populations, our genomic baseline supports previous assertations that Atlantic Sturgeon show natal homing, despite mixing extensively in marine waters during non-breeding periods. Lower levels of differentiation between populations in the South Atlantic DPS suggest that populations in this region may have greater levels of gene flow relative to their more northerly conspecifics, which has also previously been suggested based on microsatellite data. The observed differentiation among populations provides the necessary foundation for determining the natal river and DPS of Atlantic Sturgeon using assignment testing. We tested the utility of our new genomic baseline for resolving the population and DPS of Atlantic Sturgeon. Our nuclear SNP markers showed utility for identifying the origin of unknown Atlantic Sturgeon samples, as 86.5% were assigned to the correct DPS and 66.3% were assigned to the correct natal river. However, since this study was funded the Conservation Genetics and Genomics Laboratory at Leetown Science Center has made significant improvements to their microsatellite genetic baseline, which now performs more effectively than our new genomic approach (the genetic baseline includes 12 populations and 5 DPSs, and correctly assigns 95.8% of individuals to DPS and 84.9% of individuals to their natal population using 12 microsatellite loci). We conducted an ad hoc exploration of how additional microsatellite or nuclear SNP loci may further improve the accuracy of assignment testing. We found that additional microsatellite markers are likely to result in greater improvements in assignment efficiency than additional nuclear SNPs. However, a much larger number of SNP loci (which if identified could be sequenced using other methods that are now available; e.g., the RAD-capture approach published by Ali et al. 2016) could produce assignment efficiencies that are greater than what is currently feasible using microsatellites. In the absence of further research and development of additional SNP markers for Atlantic Sturgeon (possibly using an approach other than GT-seq), the existing microsatellite loci are the most effective means available to determine the natal river and DPS of Atlantic Sturgeon encountered in offshore waters. Because our new genomic markers were less effective than the existing panel of 12 microsatellite markers, we chose to use the existing microsatellite markers to assign Atlantic Sturgeon captured in another BOEM-funded study (cooperative agreement M16AC00003; Monitoring endangered Atlantic Sturgeon and commercial finfish habitat use offshore New York) following consultation with our project officer. Using this approach, we genotyped and assigned 186 Atlantic Sturgeon captured in coastal waters off the Rockaway Peninsula, New York. The vast majority of these sturgeon were assigned to the New York Bight DPS (94.62%), and most appear to belong to the Hudson River population (87.10%) with smaller contributions from the Delaware River population (7.53%). Smaller contributions (2.15%) were observed from six other populations, including those from the James, York, Kennebec, Ogeechee, and Edisto rivers. Although most of the fish we assigned were assigned to the nearest spawning rivers (Hudson and Delaware), the contributions from distant rivers is consistent with the propensity of this species to move long distances and form mixed stock aggregations along the continental shelf. This finding indicates that spawning populations (and their corresponding DPS) from distant locations may potentially be impacted by offshore activities. In fact, activities in this region of the New York Bight could negatively impact Atlantic Sturgeon population from at least four different DPSs. Genetic or genomic assignment testing remains an essential tool to characterize potential impacts to Atlantic Sturgeon populations and should be applied more broadly to better characterize potential impacts of activities in other locations.

Atlantic Coast

Davis Pond freshwater prediversion biomonitoring study: freshwater fisheries and eagles

In January 2001, the construction of the Davis Pond freshwater diversion structure was completed by the U.S. Army Corps of Engineers. The diversion of freshwater from the Mississippi River is intended to mitigate saltwater intrusion from the Gulf of Mexico and to lessen the concomitant loss of wetland areas. In addition to the freshwater inflow, Barataria Bay basin would receive nutrients, increased flows of sediments, and water-borne and sediment-bound compounds. The purpose of this biomonitoring study was, therefore, to serve as a baseline for prediversion concentrations of selected contaminants in bald eagle ( Haliaeetus leucocephalus ) nestlings (hereafter referred to as eaglets), representative freshwater fish, and bivalves. Samples were collected from January through June 2001. Two similarly designed postdiversion studies, as described in the biological monitoring program, are planned. Active bald eagle nests targeted for sampling eaglet blood (n = 6) were generally located southwest and south of the diversion structure. The designated sites for aquatic animal sampling were at Lake Salvador, at Lake Cataouatche, at Bayou Couba, and along the Mississippi River. Aquatic animals representative of eagle prey were collected. Fish were from three different trophic levels and have varying feeding strategies and life histories. These included herbivorous striped mullet ( Mugil cephalus ), omnivorous blue catfish ( Ictalurus furcatus ), and carnivorous largemouth bass ( Micropterus salmoides ). Three individuals per species were collected at each of the four sampling sites. Freshwater Atlantic rangia clams ( Rangia cuneata ) were collected at the downstream marsh sites, and zebra mussels ( Dreissena spp.) were collected on the Mississippi River. The U.S. Geological Survey (USGS) Biomonitoring of Environmental Status and Trends (BEST) protocols served as guides for fish sampling and health assessments. Fish are useful for monitoring aquatic ecosystems because they accumulate pesticides and other contaminants. Biomarker data on individual fish, generated at the USGS National Wetlands Research Center (Lafayette, La.), included percent white blood cells in whole blood, spleen weight to body weight ratio, liver weight to body weight ratio, condition factor, splenic macrophage aggregates, and liver microsomal 7-ethoxyresorufin-o-deethylase (EROD) activity. Fish age was estimated by comparing total lengths with values from the same species in the Southeast United States as determined from the literature. Contaminant analyses were coordinated by the U.S. Fish and Wildlife Service (USFWS) Analytical Control Facility (Laurel, Md.), where residues of organochlorine (OC) pesticides, total polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), aliphatic hydrocarbons (AHs), and trace elements were determined. The organic contaminant data were generated at the Mississippi State University Chemical Lab (Mississippi State, Miss.), and the inorganic contaminant data were generated by the Texas A&M University Geochemical and Environmental Research Group (College Station, Tex.). Statistical tests were performed to assess relationships among contaminants, fish age, fish species, and collection sites. Trends in interspecific differences among fish in concentrations of contaminants were noted. Striped mullet (hereafter mullet) frequently displayed the highest chemical concentrations. Levels of contaminants were generally higher in samples obtained from the Mississippi River than in those collected from the diversion area and were higher in mussels and clams (hereafter bivalves) than in fish. Because the Mississippi River sampling site for mullet and largemouth bass was downriver of the structure and south of New Orleans and the catfish site was upriver, the downriver data may not be directly reflective of the results from the receiving waters at the Davis Pond structure. Compared to the Caernarvon freshwater prediversion study in 1990 that assessed possible influx of contaminants with the freshwater diversion, contaminant levels in fishes and bivalves in this study were generally lower, yet three nontoxic inorganic elements in Davis Pond fish samples exhibited ranges of concentrations that were more than two times higher than did those from Caernarvon. Levels in bivalves were different between diversions but about equal in the numbers of trace elements showing high levels per location. Contaminant values were compared to those listed in various literature and agency sources, both regional and national, including the National Contaminant Biomonitoring Program (NCBP), in which the 85th percentile and above represents what is considered to be an elevated contaminant concentration and cause for concern. Generally, bivalves were at the high end of their ranges for both organic and inorganic contaminants. In this study, OCs were detectable in 67 percent of fish from the Mississippi River site, ranging from 0.15 to 1.09 μg/g wet weight (ww) or fresh weight (fw), and in 11 percent of the fish from the marsh sites, ranging from 0.06 to 0.612 μg/g ww. Bivalves from the Mississippi River had OC levels of 0.096 μg/g ww, whereas none were detectable in bivalves at the marsh sites. In this study, p,p ’-dichlorodiphenyldichloroethylene ( p,p’ -DDE) (a biodegradation product of DDT [dichlorodiphenyl trichloroethane]) and total PCBs were the most frequently detected OCs and were primarily from the Mississippi River. For total OC content, using adjusted least squares means, some significant interactions were noted between fish species and sites. PAHs were detected in aquatic animals at all sites (range of 0.017–17.534 μg/g ww), as were AHs (range of 0.423–4.549 μg/g ww); the highest levels of PAHs and AHs were found in bivalves from the Mississippi River. When analysis of variance (α = 0.05) was performed with data from aquatic animals, there were only two significant relationships between PAHs, AHs, and OCs between species, site, and age or the interaction among these variables. There was an interaction between fish species and n-decane (an AH) in that mullet and largemouth bass had significantly higher levels than did catfish ( P = 0.0175). When general linear means were used to investigate associations of inorganic contaminants among fish species, site, and age or any interactions among these variables, no significant results were noted for arsenic, cadmium, lead, beryllium, boron, molybdenum, or nickel. The range of mercury in fish in this study was 0.04–0.14 μg/g ww (0.14– 0.48 μg/g dry weight [dw]), with the most elevated levels detected in predatory largemouth bass at the sampling point farthest downstream from the structure and within the marsh area. Mercury was positively correlated with fish age ( P = 0.0152), where levels were estimated to increase 0.0253 parts per million (ppm) dw per year. In the Mississippi River, catfish showed significantly higher levels of mercury than did mullet or largemouth bass ( P = 0.00167). Among fish species, mullet displayed the highest levels in fish of aluminum, barium, manganese, and iron, all considered to have low toxicity in hydrologic systems. An interaction between fish and site was seen with aluminum ( P = 0.0031), where concentrations in mullet were significantly higher in the Mississippi River than at the other sites, as was also seen with barium ( P = 0.0009), chromium ( P = <0.0001), manganese ( P = 0.0004), strontium ( P = 0.0074), vanadium ( P = 0.0156), and zinc ( P = 0.0059). For iron ( P = 0.0.0001), mullet and largemouth bass at both the Mississippi River and Lake Salvador showed higher levels than did catfish, and these two species showed higher levels at two of the four sites. An interaction between fish and site was also seen with chromium ( P = <0.0001) in that concentrations in mullet were significantly higher in the Mississippi River than at the other sites, as was also seen with strontium ( P = 0.0074), vanadium ( P = 0.0156), and zinc ( P = 0.0059), metals for which deleterious effects have been demonstrated in other ecosystems. The NCBP program lists the 85th percentile for zinc at 34.2 μg/g fw (117.9 μg/g dw). In the Davis Pond prediversion biomonitoring study (hereafter the current study), one fish (MUL31RIVER, fish ID 8) showed values higher than that (125.4 μg/g dw or 37.54 μg/g ww), and the Mississippi River bivalve sample (MUSSRIVER) had a value of 140 μg/g dw (41.2 μg/g ww). In the current study, approximately 86 percent of the fish had measurable selenium levels, yet none reached the 85th percentile. The 85th percentile for selenium from the NCBP was 0.73 μg/g ww. Significantly higher levels of selenium were seen in mullet than in largemouth bass and catfish ( P = 0.0023). The NCBP 85th percentile for lead is 0.22 μg/g ww (0.76 μg/g dw). In the current study, the range of concentrations of lead was as much as 18.3 ppm dw (MUL31RIVER, fish ID 8), with the three most elevated values (range of 3.46–5.31 μg/g ww) coming from mullet from the Mississippi River. Biomarker data are measurable and directly reflect the condition of the animal, and measuring more than one biomarker in an individual increases confidence in health assessments. In the current study, biomarkers included macrophage aggregates (MAs), liver (hepatosomatic index [HSI]) and spleen (splenosomatic index [SSI]) weight to body weight ratios, percent white blood cells (WBCs) in whole blood, and condition factor. Few significant differences were noted with any of the biomarkers between sites, and there were no relationships between species and sites. For improved use of biomarker assessments, an increase in fish sample size would be useful for postdiversion sampling, as would comparisons of fish of the same sex and reproductive condition. During the current study, success for eagle nests in the diversion area and reference sites was similar as determined by numbers of nestlings fledged. When temperatures were below average during winter 2000, nests in both regions similarly failed. At each nest, the primary evidence of food items was small mammals. Eaglets (n = 6) generally appeared healthy, and whole blood concentrations of organic contaminants exceeded detection limits with three incidences of p,p’ -DDE (0.002–0.006 μg/L ww) and one incidence of oxychlordane (0.002 μg/L ww). The levels of p,p’ -DDE were well below those that have been inversely correlated with productivity and success rates of nesting bald eagles on a regional scale. The low values found in the whole blood samples for OC pesticides and PCBs were even lower when corrected for plasma volume. Aluminum values were 3.66 and 5.75 μg/L in two samples, zinc ranged from 5.21 to 6.77 μg/L ww in six samples, and silicon ranged from 1.7 to 4.6 μg/L in four samples. Selenium was detectable in each bird with the range at 0.332–0.566 μg/L ww, and strontium ranged from 0.0581 to 0.0975 μg/L ww. Mercury was detectable in blood samples from each bird and ranged from 0.0254 to 0.0845 μg/L ww, whereas lead was detectable in four samples and ranged from 0.0042 to 0.0136 μg/L ww. Although no detectable levels of total PCBs were found (also correlated with decreased reproductive productivity), 70 percent of the aquatic animals from the Mississippi River contained total PCBs (range 0.13–0.79 μg/L), whereas only about 7 percent of the aquatic animals sampled from the marsh area contained PCBs. Suggestions for postdiversion sampling include lowering the analytical detection limit for some metals, sampling aquatic animals over the course of a single season, obtaining a higher sample number of mature fish of one species (for example, blue catfish) within a range of total lengths for biomarker analyses, obtaining otoliths for estimating fish ages, assessing dioxins in eaglet blood, examining triazines in water, and obtaining all Mississippi River fish samples as close to the Davis Pond structure intake as possible. Because contaminants found in blood of eaglets reflect their prey species and because of the contaminant levels found in fish in the current study, eaglets may not be consuming primarily these species; therefore, obtaining juvenile nutria ( Myocastor coypus ) or turtle species for contaminant analyses might be considered, as well as collecting greater blood volume and using plasma to measure OCs and PCBs. Data obtained postdiversion will be compared with prediversion data to monitor changes.

Louisiana