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<br />111"
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<br />102
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<br />D.W. BEYERS ET AL.
<br />
<br />from excess reproduction were available for experimental
<br />purposes. Fertilized eggs were allowed to water harden for
<br />48 h before being packaged and transported to laboratory
<br />culture facilities at Colorado State University (Fort Collins,
<br />CO). Upon arrival, fertilized eggs were acclimated to culture-
<br />facility water temperature (190C) and transferred to a "vertical-
<br />tray" incubator. Hatching occurred approximately 4 and 5 d
<br />after fertilization for Colorado squawfish and bony tail, re-
<br />spectively. After hatching, larvae were transferred to flow-
<br />through troughs where they were maintained until selected
<br />for a toxicity test. After onset of first feeding (approximately
<br />11 d after fertilization), larvae were fed live:::; 24-h-old brine
<br />shrimp nauplii (Aquarium Products, Glen Burnie, MD) two
<br />or three times a day.
<br />
<br />Toxicity test overview
<br />
<br />Because we had no previous knowledge of the sensitivity
<br />of Colorado squawfish or bony tail to the toxicants, 24-h
<br />range-finding tests were conducted. The lowest concentration
<br />lethal to all test organisms in 24 h was the highest test con-
<br />centration in 4-d renewal-acute tests. Results of 4-d renewal-
<br />acute tests were used to select a toxicant concentration range
<br />for 32-d ELS tests. The lowest toxicant concentration in
<br />renewal-acute tests that caused abnormal behavior (erratic
<br />swimming, lethargy, or loss of equilibrium) was the highest
<br />toxicant concentration in ELS tests.
<br />Toxicity tests were conducted with Colorado squawfish
<br />and bony tail during 1989 and 1990, respectively. Because
<br />young Colorado squawfish and bony tail were available only
<br />on an annual basis, toxicity tests were conducted sequentially.
<br />Unfortunately, by the time ELS tests were initiated, consid-
<br />erable ontogenetic development had occurred, and embryos
<br />and proto larvae [12] were not present during the exposure
<br />period. Mesolarval, metalarval, and juvenile life stages were
<br />present during the exposure period.
<br />
<br />Exposure systems
<br />
<br />Range-finding and 4-d renewal-acute tests were conducted
<br />using 1-L glass beakers containing 0.75 L of toxicant solu-
<br />tions. Contents of each beaker were renewed every 24 h. The
<br />dilution factor was 0.75.
<br />ELS tests were conducted using a continuous-flow mini-
<br />diluter exposure system [13]. The diluter maintained a 0.5
<br />dilution factor and provided a volume of 0.055 L/min to
<br />replicate aquaria. Aquaria were 10 x 20 x 15 cm high, and
<br />depth of test solutions was 12 cm.
<br />In both exposure systems, treatments were assigned to two
<br />replicate exposure chambers by a randomized block design.
<br />Test animals were randomized to one of seven treatment
<br />groups: five toxicant concentrations, a solvent control, and
<br />a dilution-water control. Cool-white fluorescent lamps were
<br />the only source of illumination, and a 16:8-h light:dark pho-
<br />toperiod was maintained.
<br />
<br />Physical and chemical conditions
<br />
<br />Dilution water for all toxicity tests was supplied by a well
<br />on the Colorado State University campus and was vigorously
<br />aerated for approximately 24 h while being heated to a test
<br />
<br />temperature of 22:t IOC. In renewal-acute tests, alkalinity,
<br />hardness, and specific conductance were measured at the be-
<br />ginning and end of the exposure period. Dissolved oxygen
<br />and pH were monitored daily, and water temperature was
<br />measured continuously. For 32-d ELS tests, alkalinity, hard-
<br />ness, pH, and specific conductance were measured weekly.
<br />Dissolved oxygen was measured daily, and water tempera-
<br />ture was measured continuously. Dilution water character-
<br />istics for all tests, except Colorado squawfish renewal-acute
<br />tests, had the following ranges: dissolved oxygen, 6.1 to 7.0
<br />mg/L; pH, 7.9 to 8.2; temperature, 21.2 to 22,70C; alkalin-
<br />ity, 237 to 259 mg/L as CaC03; hardness, 344 to 378 mg/L
<br />as CaC03; and specific conductance, 720 to 780 ILS/cm. Di-
<br />lution water used in Colorado squawfish renewal-acute tests
<br />inadvertently underwent a different aging process; its char-
<br />acteristics were dissolved oxygen, 7.1 to 7.2 mg/L; pH, 8.5
<br />to 8.6; temperature, 22.0 to 22.80C; alkalinity, 104 to 110
<br />mg/L as CaC03; hardness, 212 to 216 mg/L as CaC03; and
<br />specific conductance, 600 ILS/cm.
<br />
<br />Toxicant solutions
<br />
<br />Technical carbaryl (I-naphthyl methylcarbamate, 99070)
<br />and Sevin-4-0il (a formulation containing 49% carbaryl and
<br />petroleum distillates) were obtained from Rhone-Poulenc
<br />(Research Triangle Park, NC). Technical malathion (diethyl
<br />mercaptosuccinate, S-ester with 0, O-dimethyl phosphoro-
<br />dithioate, 93%) was obtained from American Cyanamid Co.
<br />(Princeton, NJ). Technical carbaryl and technical malathion
<br />are henceforth referred to as "carbaryl" and "malathion" re-
<br />spectively. Stock solutions were prepared by dissolving each
<br />toxicant in pesticide-grade acetone or acetone-dilution-water
<br />mixtures. Renewal-acute exposure concentrations were pre-
<br />pared by pipetting the desired amount of toxicant stock into
<br />beakers containing 0.75 L dilution water. Test solutions were
<br />stirred and transferred to exposure chambers within 30 min
<br />of preparation. In ELS tests, toxicant stock solutions were de-
<br />livered to the diluter via peristaltic pump. The amount of ace-
<br />tone in any exposure concentration never exceeded 0.5 ml/L.
<br />
<br />Analytical procedures
<br />Toxicant concentrations in renewal-acute tests were mea-
<br />sured twice during the exposure period. Toxicant concentra-
<br />tions in ELS tests were measured weekly (four occasions).
<br />Samples for analysis were taken from alternate replicate ex-
<br />posure chambers. In renewal-acute tests, toxicant concentra-
<br />tions nearest the median lethal concentration were also
<br />measured 24 h after renewal to estimate the amount of toxi-
<br />cant breakdown during the 24 h between renewals. Toxicants
<br />were extracted with solid-phase extraction and analyzed with
<br />GC [14]. Extracted samples were stored at -40C until they
<br />could be analyzed.
<br />
<br />Exposure conditions
<br />
<br />The 4-d renewal-acute tests with Colorado squawfish and
<br />bonytail were initiated with 26- and 6-d-old (postfertilization)
<br />larvae, respectively. Mean wet weight and total length, re-
<br />spectively, at start of renewal-acute tests were 4 mg and
<br />9.4 mm for Colorado squawfish and 2 mg and 6,8 mm for
<br />
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