THE EFFECT OF INTERTIDAL EXPOSURE ON THE SURVIVAL AND EMBRYONIC DEVELOPMENT OF PACIFIC HERRING SPAM by BARRY CYRIL JONES B.Sc, University of British Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1971 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ZOOLOGY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada November 26, 1971. ABSTRACT Eggs of P a c i f i c herr ing were exposed to a i r for d i f f e r e n t periods of time i n s imulat ion of t i d a l e f fects on spawn deposits at varying beach heights . The maximum exposure range was 2/3 of a 2k hour day corresponding roughly to the exposure of eggs at k meters above mean low t ide on the B r i t i s h Columbia coast. Egg s i ze , spawning f i s h length , and egg clump s ize were examined as secondary factors modifying the effect of exposure. Incubation time dropped from 19 to 18 days with only two 2-hour periods of exposure per day and thereaf ter f e l l s lowly. It i s suggested that oxygen depr ivat ion tr iggered a hatching response for the i n i t i a l drop, whereas the gradual decrease was due to a higher a i r temperature increas ing metabolism. Hatching m o r t a l i t y rose s t ead i ly from an unexposed 1J% to yi% at maximum exposure time, with s i g n i f i c a n t l y higher contr ibut ions from eggs of smaller f i s h and smaller egg clumps. Larva l length at hatching for the unexposed eggs was 7*7 mm.5 lengths for a l l degrees of exposure were s i m i l a r (7% l e s s than for no exposure). Larva l weight (body plus yolk) remained r e l a t i v e l y constant (0.099 mg.) u n t i l the longest exposure period when i t dropped to O.O87 mg. This decrease coincided with s i m i l a r sharp trends i n incubation time and hatching m o r t a l i t y , and suggests a " c r i t i c a l point" near the upper experimental range of exposure, above which eggs stand l i t t l e chance of normal development or s u r v i v a l . Beach surveys to note poss ible egg s ize s t r a t i f i c a t i o n , although suggesting the deposi t ion of l arger eggs at the top l e v e l s , proved inconc lus ive , but point up the p o s s i b i l i t y that a heavy f i s h i n g pressure which reduces mean f i s h s ize might detr imenta l ly a f fec t po tent ia l stock recruitment v i a the i n t e r t i d a l exposure effect on the spawn. TABLE OF CONTENTS i i i Page ABSTRACT i LIST OF TABLES iv LIST OF FIGURES v ACKNOWLEDGEMENTS • v i i INTRODUCTION 1 MATERIALS AND METHODS 3 Spawner Characteristics Analyses.- 3 Exposure Laboratory Experiment 5 Egg Size Distribution Beach Surveys 9 RESULTS 10 Effects of Exposure 10 Incubation Time 12 Hatching Mortality 12 Larval Length 15 Larval Weight 15 Beach Stratification 18 DISCUSSION 18 LITERATURE CITED 26 APPENDICES 29 A - Apparatus Design 30 B - Raw Data 33 C - Spawner Correlations ^2 D - Computations Summary E - Statistical Analyses 52 iv LIST OF TABLES Table Page I Summary of experimental conditions 6 II Group means and standard deviations in the analyses 11 Appendix Table IA Spawner data l i s t 36 IIA Incubator data l i s t • 37 IIIA Computations for incubation time (days) 7^ IVA Computations for hatching mortality {%) ^8 VA Computations for larval length (mm.) ^9 VIA Computations for larval weight (mg;..) 50 VILA. Computations for beach stratification of egg weight (mg.), showing beach height (m. ) 50 VIIIA Significance of differences within the total data 53 IXA Significance of differences between groups 5^ XA Significance of interaction 55 XIA Significance of differences between beach levels 56 LIST OF FIGURES v Figure Page 1 Relationship of beach height to exposure time. Data for Vancouver, B.C., (March, 1970) meaned from Straits Towing calendar 6 2 Relationship of incubation time to exposure time for total data 13 3 Relationship of hatching mortality to exposure time for total data 13 k Fish length effects in the relationship between hatching mortality and exposure time Ik 5 Clump size effects in the relationship between hatching mortality and exposure time Ik 6 Relationship of larval length to exposure time for total data 16 7 Egg size effects in the relationship between larval length and exposure time..... 16 8 Relationship of larval weight to exposure time for total data 17 9 Egg size effects in the relationship between larval weight and exposure time 17 10 Relationship of egg size to beach height at spawning, Bedwell Bay, April 20, 1970 19 11 Relationship of egg size to beach height at mid-incubation (8'days), Nanoose Bay, March 27, 1970 19 12 Relationship of egg size to beach height just after spawning (k days) and at hatching (16 days) for the same egg mass. The latter is for larvae as the eggs hatched en route to the lab. These samples taken at Icarus Point, March 17 and 29, 1971 20 LIST OF FIGURES (CONT.) Appendix Figure Page 1 A Tank set-up for each exposure time 32 2 A Cross-section of incubator in tank 32 3A Relationship of egg size to spawner length 4 3 4A Relationship of egg size to spawner weight with gonads removed 43 5A Relationship of egg size to spawner age 6 A Relationship of spawner length to age kk 7A Relationship of spawner weight with gonads removed to age 4 5 8A Relationship of spawner weight with gonads removed to length 4 5 ACKNOWLEDGEMENTS v i i I would l i k e to thank Dr. P .A. L a r k i n , my supervisor , of the Department of Zoology, Un ivers i ty of B r i t i s h Columbia, who gave me the opportunity , support from h i s Nat ional Research Council grant, and guidance i n th i s work over the past two years. The ass is tance of Dr. F . H . C . Taylor and the other members of h i s Herring Invest igat ion group of the F i s h e r i e s Research Board of Canada's B i o l o g i c a l S ta t ion , Nanaimo, i s a lso g r a t e f u l l y appreciated. They supplied me with the f a c i l i t i e s , the f i s h , and checked, cer ta in of my data. Thanks also go to Dr. N. G i l b e r t for h i s help in the set-up and use of h i s non-orthogonal ana lys i s of variance computer program and to Dr. D . J . Randall for h i s h e l p f u l c r i t i c i s m s of the manu-s c r i p t . Drs. G i l b e r t and Randall are of the Department of Zoology, Un ivers i ty of B . C . THE EFFECT OF INTERTIDAL EXPOSURE ON THE SURVIVAL AND EMBRYONIC DEVELOPMENT OF PACIFIC HERRING SPAWN INTRODUCTION The eggs of the P a c i f i c herr ing (Clupea p a l l a s i i V a l . ) are spawned i n and below the i n t e r t i d a l zone. Due to t h e i r adhesive nature, they become attached to c e r t a i n forms-of seaweed and are e s s e n t i a l l y immobile. For th i s reason most of them are subjected to regular periods of exposure and sub-mergence. Such condit ions cause considerable f l u c t u a t i o n i n the environment of the eggs and may a f fec t t h e i r s u r v i v a l and development. The ef fect of thi>s f l u c t u a t i o n i s os tens ib ly d i r e c t l y r e l a t e d to the height up the beach that the eggs are l a i d , and thus, the amount of time they are exposed. Within the spawning zone a var ie ty of egg s izes can be expected because each spawner produces a range of egg s izes (for example, for A t l a n t i c h e r r i n g , Clupea harengus, Hempel and Blaxter , 1 9 6 7 ) . In a d d i t i o n , every reproductive stock comprises a var ie ty of i n d i v i d u a l s d i f f e r i n g i n length, weight, and age, and several studies (Rannak, 1 9 5 8 ; B laxter and Hempel, 19^3) have shown that mean egg s ize i s a funct ion of s i ze and maturity . The adhesiveness of herr ing eggs also causes the formation of clumps when exposed to sea water. Such clumps are of d i f f e r i n g thickness and vary i n egg s ize and number. Hence, egg s i ze , f i s h s i ze , and clump s ize a l l 2 have some bearing on the poss ib le ef fects of environmental f l u c t u a t i o n r e s u l t i n g from exposure. The c h a r a c t e r i s t i c s most notably af fected are incubation time, hatching m o r t a l i t y , and l a r v a l length and weight at hatching. In th i s regard, Blaxter and Hempel (1963) noted that egg s i ze d id not a f fec t incubation time, whereas hatching m o r t a l i t y was found by other studies (Runnstrom, 19^1; McMynn and Hoar, 1953) to be d i r e c t l y re la ted to egg number. The larvae have been shown to be af fected by both egg and f i s h s i zes . For instance, Toom (1958) has demonstrated that l a r v a l s i ze i s d i r e c t l y r e l a t e d to egg s i ze , and Cushing and Bridger (I966) have noted that larvae from f i r s t spawners are l e ss v iab le than those from l a r g e r f i s h . In a d d i t i o n , i t has also been shown (Nagasaki, 1958) that fecundity i s d i r e c t l y r e l a t e d to spawner s i ze . Because f i s h i n g i n t e n s i t y reduced the mean s i z e , age, and numbers of spawners of B r i t i s h Columbia stocks of herr ing (Taylor , 1963) and North Sea h e r r i n g , Clupea harengus (Cushing and Br idger , 1966), then i t must fol low that mean egg s i ze a lso decreased. There would be fewer, smaller eggs produced than i n former years, and with a l e s ser chance of l a r v a l s u r v i v a l . The s u r v i v a l advantage accruing to a f i s h stock due to the presence of l a r g e r eggs and larvae has been pointed out by Marshall (1953)- If environmental fac tors operating i n the spawning zone are more detrimental to smaller eggs or the eggs from smaller f i s h , then there could be serious repercussions on recruitment p o t e n t i a l , 3 i . e . the number of immature f i s h a v a i l a b l e to enter the reproductive populat ion. Previous work on herr ing egg development has been concerned with condit ions for submerged eggs. This study-sought to examine incubation time, hatching m o r t a l i t y , and l a r v a l length and weight at hatching i n r e l a t i o n to varying degrees of exposure. The laboratory experiment was conducted and analyzed using as a d d i t i o n a l var iab les the effects of egg s i z e , f i s h s i ze , and clump s i z e . A beach survey was a l so undertaken to note poss ible egg s ize s t r a t i f i c a t i o n . MATERIALS AND METHODS The eggs used i n t h i s study were taken from spawning P a c i f i c herr ing of the Lower East Coast stock (inner southern Vancouver Is land region) of B r i t i s h Columbia, and the l abora -tory experiment was done at the F i s h e r i e s Research Board of Canada's B i o l o g i c a l Stat ion i n Nanaimo, B . C . Spawner C h a r a c t e r i s t i c s Analyses Forty female spawners were used to determine i f egg s i ze was r e l a t e d to f i s h s ize and maturity . The f i r s t 29 were taken by beach seine and held a l i v e i n l a r g e , we l l - f lushed hold ing tanks for one week p r i o r to use. The other 11 were obtained dead from l o c a l trawlers within 6 hours of capture and used i:mmedlately. A f t e r s t r i p p i n g the experimental eggs, the spawners were measured for standard length ( t ip of snout to end of ver tebra l column) and three or more scales plus 4 both o t o l i t h s were taken for age determinations. The gonads were then removed and the spawner wet weight recorded. The f i s h were then tagged and preserved i n 5$ formal in for poss ib le future reference. The age of each spawner was determined by reading the scales from the areas above and below the l a t e r a l l i n e between the rear of the g i l l cover and the front of the dorsal f i n (Tester, 1937). These were cleaned, dyed, and mounted on a glass s l i d e . The 11 trawl caught f i s h had very few scales , and hence, any scale was used. These ages were checked with the o t o l i t h s which had been cleaned and preserved i n 5$ forma-l i n . Samples of each spawners' gonads were immediately pre-served i n S% formalin when removed. This succeeded i n hardening and separating the eggs from each other and the ovarian t i s sue so that they could be e a s i l y counted. Sub-sequently, the gonad samples were broken up to re lease the eggs which were then thoroughly washed i n fresh water. F ive samples of 100 eggs were taken from each of two f i s h and put i n a drying oven for 24 hours at 50° Centrigrade^. Several p r i o r tests confirmed that there were no effects of pos i t ion of samples i n the dryer , the dryer handling capaci ty , the est imation 'Of residue weight, and the length of dry ing time. The samples were i n d i v i d u a l l y removed from the oven, weighed on a Cenco e l e c t r i c a l balance to the nearest 0.1 mg., weighed 1 These condit ions are the same as those used by Blaxter and Hempel (1963 ). again as a check, and then discarded. 5 Exposure Laboratory Experiment Five tanks (see Appendix A) simulated condit ions at d i f f erent beach l e v e l s (Figure 1) ranging from the contro l (0) which was continuously submerged, through 2, 4, 6, and 8 hours of exposure twice per day. These exposure times simulate a f ixed t i d a l cycle of roughly 2 meters amplitude (not found i n th i s area , but necessary as an experimental feature) . Each tank contained for ty incubators , and a l l were kept i n a small temperature-control led room under regulated condit ions (Table 1). From every female spawner approximately 100 eggs were s tr ipped into each of f i ve separate incubators . In th i s operat ion, clumping of the eggs was unavoidable, but an <$ attempt was made to produce the same clump form i n a l l incubators . The incubators were then simultaneously placed into a glass f e r t i l i z a t i o n tray containing a sperm so lu t ion and allowed to stand for 60 seconds. The sperm so lu t ion was prepared using 500 ml. of sea water and s u f f i c i e n t sperm from 2 or 3 males (to ensure v i a b l e sperm) to turn the water opaque. The incubators were transferred to another tray and gently f lushed with fresh sea water to prevent polyspermy and remove any excess organic matter which might decay i n the tanks. They were then transferred to t h e i r respect ive exposure tanks and kept submerged for 12 hours before the The small s ize and adhesiveness of the eggs prevented counting. In f a c t , i t was found that the mean was 132 eggs; standard deviat ion t kj,. 6 Exposure time twice per day (hr . ) Figure 1: Re lat ionship of beach height to exposure time. Data for Vancouver, B . C . , (March, 1970) meaned from S t r a i t s Towing calendar. Table 1: Summary of experimental condi t ions . Factor Mean Standard Dev.(SD) (1) Light (a) (b) • • Day length Intens i ty 13 hours 60 watt bulb at cm. above each 75 tank — (2) A i r : —Ta) (b) Temperature Relat ive humidity 1 1 . 7 ° C 65% to. 6° t5% (3) Sea Water* (a) Temperature (b) Oxygen (c) Flow rate (d) Depth 7.8° C 6.5 m l ./ I . 55ml. per min. per incubator 5 cm. +0.4° ±3 ml. 7 experimental condit ions were i n i t i a t e d . The a r t i f i c i a l environment (summarized i n Table I) was s i m i l a r to that recorded on the beach surveys during the experimental incubation per iod. An attempt was made to main-t a i n the laboratory temperature at 12° C. A maximum-minimum thermometer checked d a i l y gave a mean of 11.7° G; SD * 0.6°. The mean r e l a t i v e humidity determined by s l i n g psychrometer was 65%', SD * 5%> The day length was regulated by time clock and set at th i r t een hours (9 am to 10 pm) so that one exposure period was i n the l i g h t and the other i n darkness. The l i g h t source was a s ingle 60-watt incandescent bulb per tank. Each bulb had a white porce la in rear r e f l e c t o r and was suspended 75 cm. above the l e v e l of the eggs i n the center of the tank. The sea water or ig inated from the bottom of the l o c a l bay and ran continuously through the tanks at a mean rate of 55 m l • per minute per incubator; SD * 3 nil. When the tanks were f u l l , a l l the eggs were suspended at an equal depth of 5 cm. Several oxygen determinations were c a r r i e d out on the i n l e t and out le t waters by the Improved Winkler Method and a l l came to approximately 6.5 ml. per 1. This would suggest that with p l e n t i f u l oxygen i n the i n l e t waters and the open c i r c u l a t o r y system, oxygen was not a l i m i t i n g factor-^. Regular water temperature measurements y ie lded a mean of 7•8° C; SD - 0.4°. This resu l ted i n an a i r /water temperature d i f f e r e n t i a l of 4° C. 3 This was v e r i f i e d by a tank pos i t ion analys i s of the r e s u l t s using Dr. N. G i l b e r t ' s program. However, because the system was open and appropriate water sampling proved d i f f i c u l t , I would question the v a l i d i t y of these deter-minations, although not the conclusions drawn. 8 After 15 days the larvae "began to hatch. Throughout the hatching period collection was done immediately prior to exposure (10 am and 10 pm) of the eggs . Upon removal by large-mouth pipette, they were immobilized in a 1:50,000 solution of MS222 (Tricaine Methanesulfonate). This treatment caused the larvae to straighten out and stiffen. They were then preserved in 5% formalin. When larval emergence ceased, the incubators were cleaned out and the dead eggs counted-'. Prom this data the incubation time (from f e r t i l i z a t i o n to 50$ hatch) and mortality were determined. At convenient times during and after the experiment the larvae were counted and the lengths (from t i p of snout to end of t a i l ) of a l l measur-able larvae were determined by graduated microscopic eyepiece. This work took some 3 months, during which time a companion shrinkage test was run. When the measuring was completed, the test was terminated and a table of daily shrinkage correction values was computed and used to correct the mean larval length obtained for each incubator. The shrinkage was found to be only U-,2% over the entire three month measuring period. Once the larvae from each incubator had been counted and measured, they were a l l put into one v i a l . When a l l the incubators had been processed in this way, ten vials (incubators) at a time were taken, the larvae recounted, washed thoroughly in fresh water, and dried and weighed in Larvae did not hatch out during the exposure periods. The dead larvae were in many stages of development. 6 7 the same manner as for the spawner egg weights ' . Egg Size D i s t r i b u t i o n Beach Surveys A number of recent spawning s i t e s were examined during daytime low t i d e s . For purposes of comparison, the deter-mination of beach height was based on the datum establ ished by the sea l e v e l at the exact time of low t ide (as ind icated i n the Canadian Tide and Current Tables - #5, using Point Atkinson as a reference) . The sea l e v e l at t h i s time was used as sample area M, the middle region of f i v e beach l eve l s sampled on each survey. The bottom sample (B) was then taken i n as great a depth as p r a c t i c a l (about 1 meter), and another sample (L-low) taken halfway between these two (about 50 cm.). The ac tua l sample depths were determined with a graduated s t a f f . Two further samples were taken above M — T (top), as high as the spawn was deposited, and H (high), halfway between T and M. The heights of these were determined by clinometer and tape measure. The samples, taken i n 500 ml. j a r s , included as many eggs and the seaweed they adhered to as poss ib le . Environmental condit ions were also recorded at the spawning s i t e s . Among these were the a i r and sea water F i x a t i o n i n formalin over a three month period was shown to have n e g l i g i b l e ef fects on l a r v a l weight (-0.4$) and egg weight {-0.2%) by Blaxter and Hempel ( 1 9 6 6 ) . Larva l weight i n t h i s experiment means the t o t a l weight of the body and the yolk sac. 10 temperature, and r e l a t i v e humidity as determined by s l i n g psychrometer. These data were used as a guide for the exper i -mental regime. Upon re turning to the l a b , the age of the spawn was estimated (Outram, 1955)« the samples were preserved i n 5% formal in , and the beach l e v e l for each sample r e l a t i v e to mean low t i d e was ca l cu la ted . Later , the eggs were separated from the seaweed by trans fer to a one normal KOH so lu t ion which was then heated to 30° C. and allowed to stand for 2 h o u r s ® . The eggs and seaweed were then transferred to a 5% formalin so lu t ion again to harden for 24 hours before the seaweed was removed and discarded. This treatment not only loosened the eggs from the seaweed, but a l so from each other. The eggs were then thoroughly washed i n fresh water, and ten 100-egg samples were taken from each beach l e v e l for drying and weighing as per the spawner egg weight determinations. RESULTS Ef fec t s of Exposure Eggs from s ix of the trawl caught f i s h had 100^ morta l i ty i n a l l tanks. The data from these incubators was discarded on the grounds that the eggs were probably already d i s i n t e g r a t i n g when used. Data for one spawner from the beach seine group was discarded for the same reason. The net Q Procedure by word-of-mouth from herr ing researchers at the B i o l o g i c a l S ta t ion , Nanaimo, but s l i g h t l y a l t e r e d . 11 r e s u l t was data from 3 3 spawners. On cons iderat ion , the experimental data was d iv ided into three groups — noted as smal l , medium, and large . in the analyses (see Appendix D). The data were i n i t i a l l y analysed i n t o t a l to note the general trend of each c h a r a c t e r i s t i c i n r e l a t i o n to increased exposure time. They were then treated separately according to t h e i r groupings as noted above. Egg s i ze as determined from the preserved gonads was f i r s t examined for poss ible d i f ferences between groups. It was found of s ign i f i cance only i n l a r v a l length and weight (see Appendix E ) . The second analys i s examined the ef fects of f i s h length. Here hatching morta l i ty and l a r v a l weight were shown to be a f fec ted . Since f i s h length and weight are so h igh ly corre la ted (see Appendix C ) , the ana lys i s was not repeated for weight. The effect of age was not examined as the spawners were predominantly 3 -year o ld f i s h , with only a few 4 and 5 -year o lds . Because the egg number (clump s ize) was d i f f e r e n t for each incubator, a t h i r d test was run to see i f t h i s had any e f fec t . It proved n e g l i g i b l e for a l l c h a r a c t e r i s t i c s but hatching m o r t a l i t y . The mean group values for these three analyses are given i n Table I I . Table I I : Group means and standard deviat ions i n the analyses. Grouping Small Medium Large ( 1 ) Egg s ize (mg.) ( 2 ) P i sh length (mm.) ( 3 ) Clump Size (no.) 0 . 2 4 3 * 0 . 0 1 5 1 9 9 * 5 8 9 * 1 7 0 . 2 7 1 * 0 . 0 0 5 2 1 1±4 1 3 0 ± 1 2 0 . 3 0 0 * 0 . 0 1 5 223±6 1 7 5 * 2 9 12 Another analysis was performed to determine i f there was any interaction among egg size, fish length, and clump size. This was found to be non-significant in most cases for a l l factors and hence w i l l not be referred to further. These various analyses are discussed together for each of the variables examined. Incubation Time The relationship of incubation time to exposure time is shown in Figure 2. The control or unexposed incubators had a slightly greater than 19-day incubation period. The f i r s t exposure period (2 hours) showed an abrupt decrease of close to one f u l l day (p < . 0 1 ) . Thereafter, there i s only a gradual decrease through the remaining exposure periods, but the total decrease (from 2 to 8 hours) of 0.4 days is significant (p = . 0 1 - . 0 5 ) . Hatching Mortality As expected, the hatching mortality showed a continuous increase with increasing exposure time (Figure 3 ) . rising from 13$ in the control to 31$ in the 8-hour exposure period. For the total data, this is significant (p < .01)9 . Eggs from smaller f i s h had a higher mortality (Figure 4), but the effect was not s t a t i s t i c a l l y significant (p = . 0 5 - . 1 0 ) . Analysis of this small fis h data did not indicate which egg 9 A l l hatching mortality s t a t i s t i c a l tests were done on arcsin transformation of the percentage data. 13 19-5 • 17. 0 • 0 2 4 6 8 Exposure time twice per day (hr.) Figure 2 : Relationship of incubation time to exposure time for t o t a l data. 53%T 0 2 4 6 8 Exposure time twice per day (hr.) Figure 3 8 Relationship of hatching mortality to exposure time f o r t o t a l data. 40 14 > 5 •P •H H 03 P o g to •H -C o -p 03 m 30 20 10 • medium f i s h Figure 4: 8 ) 2 4 Exposure time twice per day (hr . ) F i s h length effects i n the r e l a t i o n s h i p between hatching morta l i ty and exposure time. - P o3 -p u o g faD fl •H .c o p 03 K 40 30 20 10 small clumps large clumps 6 2 5 6" 8" Exposure time twice per day (hr . ) Figure 5« Clump s ize effects i n the r e l a t i o n s h i p between hatching morta l i ty and exposure time. sizes within the group might be suffering greater mortality. Smaller egg clumps also had a significantly higher mortality (p < .01 for several exposure periods) than larger egg clumps (Figure 5 ) . Larval Length Larval length at hatching in relation to the exposure time (Figure 6) follows closely the pattern of incubation time. The i n i t i a l drop between the control and the 2-hour exposure periods from 7.7 mm. to 7-2 mm. is significant (p < . 0 1 ) . From exposure periods of 2 to 8 hours there was no further, decrease. Larvae were shorter (Figure 7) from smaller eggs, but this difference was not significant (p = . 0 5 - . 1 0 ) . Larval Weight The relationship of larval weight to exposure time (Figure 8) follows a concave curve, rising from 0.092 mg. to a high of 0.099 mg. at the 4-hour period, and f a l l i n g back to 0.087 mg. by the 8-hour period. None of the differences was s t a t i s t i c a l l y significant. For egg size groups (Figure 9) there was a pronounced (p < .01) relationship to larval weight. Fish length had similar effects (not shown), except that they were not sig-nificant (p = . 0 5 - . 1 0 ) . 8 . 5 1 6 Xi •p faO fl) I - H iH f-l 5 8 . 0 7 . 5 7 . 0 6 . 5 F i g u r e 6 : ) 2 4" 6 8 Exposure t i m e t w i c e p e r day ( h r . ) R e l a t i o n s h i p o f l a r v a l l e n g t h t o exposure t i m e f o r t o t a l d a t a . xi •p bD C 0 r-n rH cd l - H 8 . 5 8 . 0 7 - 5 7 . 0 6 . 5 s m a l l eggs-0 F i g u r e 7 * Exposure t i m e t w i c e p e r day ( h r . ) Egg s i z e e f f e c t s i n t h e r e l a t i o n s h i p between l a r v a l l e n g t h and exposure t i m e . 17 s .p s: bO •H