STRUHSAKER: EFFECTS OF BENZENE ON SPAWNING HERRING 



ovaries prepared for radiometric or gas 

 chromatograph analyses. Methods of preparation 

 for radiometric and chromatograph measure- 

 ments are described elsewhere (Benville and Korn 

 1974; Korn, Hirsch, and Struhsaker 1976, see 

 footnote 2). It should be emphasized that the 

 radiometric technique measures total radioactiv- 

 ity and concentrations calculated may include 

 metabolites of benzene as well as benzene itself. 



Ovaries were examined under a dissecting 

 microscope for developmental stage [Hjort's stage 

 (Bowers and Holliday 1961)] and the presence of 

 opaque dead or dying eggs, and the gross ap- 

 pearance (color and degree of deliquescence) was 

 ranked. The maximum diameters of 10 eggs from 

 the ovary of each female were measured and the 

 eggs examined for abnormal development. 



On day 3, after cessation of exposure, pieces of 

 clean plastic screening were placed around the 

 standpipe in the center of the 800 ppb and 100 ppb 

 exposure and control tanks to provide substrate 

 for spawned eggs. Males were placed with females 

 in the 100 ppb tank. After spawning occurred, the 

 screens were removed and eggs examined for 

 developmental stage and mortality. Pieces of 

 screen with 75 eggs on each (most in 4-cell stage) 

 were cut apart. Pieces of screen were selected with 

 a single layer of relatively separated eggs because 

 previous experience showed reduced survival in 

 dense egg clusters. Two pieces of screen with 75 

 eggs each were placed in each 8-liter rearing 

 container (total of 150 eggs). There were five rep- 

 licate containers for each treatment and control 

 (total of 15 containers). Temperature during 

 development was 11.0°-12.0°C, and salinity, 

 22.0%o. Other rearing conditions were as pre- 

 viously described (Struhsaker et al. 1974). 

 Hatching occurred 10 days after fertilization, and 

 percent survival at hatching was determined from 

 three replicate counts of swimming larvae in each 

 container and by counting the number of dead and 

 abnormal embryos left on the screen. The screens 

 were removed and surviving larvae fed the rotifer, 

 Brachionus plicatilis, through the remainder of 

 the experiment (past yolk absorption to larval day 

 7). Surviving larvae were counted and the percent 

 survival through yolk absorption determined from 

 the original egg number. 



Data were analyzed, depending upon variables, 

 with the methods of analysis of variance and 

 covariance using University of California 

 Biomedical programs, BMD 01V, 02V, and 03V 

 (Dixon 1970). 



RESULTS 



No adult mortalities occurred during the 6 days 

 of the experiment. Stress behavior was noted in 

 exposed fish, particularly at the constant 800 ppb 

 exposure. Definite distress was observed by the 

 end of the first day, although oxygen levels were 

 above saturation. Milling was disrupted, fish were 

 gaping at the surface, and many exhibited dis- 

 equilibrium. Even after cessation of exposure, 

 stress behavior continued for the duration of the 

 experiment. Control fish may also have been 

 stressed by the capture conditions and the short 

 acclimation period, but they exhibited none of the 

 stress symptoms of exposed fish and milled 

 normally. 



Although behavior was abnormal in exposed 

 fish, spawning occurred in the tanks. In fact, the 

 stress from benzene exposure appeared to pre- 

 maturely induce spawning. This is illustrated in 

 Table 1 by the percentage of exposed fish which 

 were spent (Stage VII) compared with control fish. 

 At the end of the 6-day experimental period, 73% 

 (100 ppb) and 70% (800 ppb) of the exposed fish 

 were spent, compared with only 25% of the con- 

 trols. The higher percentage of spent females in 

 the 100 ppb static treatment than in the 800 ppb 

 open flow treatment during the first 4 days may be 

 a result of additional stress imposed by static 

 conditions. At all treatments, most unspent 

 ovaries were ripe (Stage VI); only 7-10% were 

 immature (Stages III-V) (Table 1). 



No changes in growth (as indicated by wet 

 weight and length) were expected in females over 

 the short experimental period. However, these 

 measurements were taken to determine the 

 similarity of fish between the treatments and to 

 adjust effect of size on the differences in weights of 

 ovaries between the treatments. Ovary length and 

 weight and egg diameters were measured to 

 determine if benzene uptake affected the growth 

 or resorption of ovaries or eggs and to determine 

 the ripeness or proximity to spawning. Data are 

 summarized in Table 2. Egg diameter did not 

 correlate with any other measurement variable. 

 Analysis of variance revealed no significant 

 difference (P>0.25) in egg diameter between and 

 800 ppb benzene treatments. Since the size range 

 of females varied somewhat between the two 

 treatments (Table 2), analysis of covariance was 

 used to compare the weights of females and 

 ovaries between concentrations and days after 

 adjustment for the effect of lengths (Table 3). No 



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