Sarrm~uth, N.S. B2Y $A2. FOWLER, G. M., AND S. J. S~IITM. 1983. Length changes in silver hake (Merluccius bilinearis) larvae: effects of formalin, ethanol, and ...
Length Changes in Silver Hake (Merluccius bilinearis) Larvae: Effects of Formalin, Ethanol, and Freezing G . h4. FOWLER AND S.
Department of Fisheries and Oceans, Marine Fish Division, Bedford Insrirute of Ocesratography, P.O. Box 1006, S a r r m ~ u t hN.S. , B2Y $A2 FOWLER,G. M., AND S. J . S~IITM.1983. Length changes in silver hake (Merluccius bilinearis) larvae: effects of formalin, ethanol, and freezing. Can. J . Fish. Aquat. Sci. 40: 866-870. Silver hake (Mer6ucciu.~biainearis) larvae preserved in either formalin or ethanol decreased in length over time, most shrinkage being achieved in the first 15 d. The magnitude and variability of the length reduction was greater for smaller larvae. Rate and magnitude of shrinkage for ethanol-preserved larvae exceeded those of formalin-preserved larvae. Frozen specimens were extremely variable in length change response, and we noted a transition from shrinkage to expansion with increasing larval length. FOWLER,G . M., AND S. J. SMITH. 1983. Length changes in silver hake (Mevluccius bilineuris) larvae: effects of formalin, ethanol, and freezing. Can. J. Fish. Aquat . Sci. 40: 866-870. Ees larves Be merlu argent6 (Merkucckus hilinearis) conservees soit dans la formaline, soit dans B'Cthanol. rktrtcissent avec le temps, Ba plus grande partie du r6trCcissement se prsduisant durant les premiers 15 jours. L'ampleur et la variabilit6 de cette diminution de longueur ssnt plus prononcees chez les petites lames. Le taux et I'ampleur du r6tr6cissement des lames consew6es dans 196thanolsont plus importants que ceux des Iarves conserv6es dans la formaline. Le changement de longueur de sujets congelCs est extr6mement variable, et on observe une transition du r6tr6cissement i?~ l'expansion h mesure qu'augmente la longueur larvaire. Received December 20, 1982 Accepted April 7, 1983
R e p le 28 d6cembre 1982 Accept6 le 7 avril 1983
FIXATIONand preservation techniques are known to cause changes in the length, weight, and quality of larval samples for a number of fish species, necessitating the use of adjustments to convert from preserved to fresh measuren~ents.Most of the work to date has concentrated on the effects of various fomalin solutions in relation to preservation time and larval size (Blaxter 197 1; May 1% I , 1982; Lockwood and Daly 1975; Parker 1963: Wosenthal et al. 1978; Schnach aqd Rosenthal 1978; Theilacker 1980). These studies demonstrated a reduction in the lengths of the larvae. the magnitude of which was dependent upon the duration of storage and the initial lengths of the specimens. The preservation effects of ethanol have received less attention than formalin (Radtke and W a i w ~ o d 1980; Theilacker 1980). Radtke and Waiwood found that for Atlantic cod (Gadus marhraa), preservation in ethanol resulted in age-dependent reduction in larval lengths whereas Theilacker concluded that there was no effect at all for northern anchovy (Engruulis nzordax). Freezing has not been investigated previously with respect to its effects on larval morphometrics, although its effects on adults have been studied (Engel 8974; Halliday and Roscoe B 967). Our objective was to study the effects of the three pres-
ervation treatments on the lengths saf larval silver hake (MevCucciusbilinecrris) specimens. This information is necessary if larval lengths obtained from preserved specimens are used to provide age and growth parameters for stock assessment purposes.
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Methods Silver hake larvae were obtained from 10- to 20-min mesh) during the Scotian Shelf oblique bongo tows (333-~FII Ichthyoplanktsn Program cruise AR02 (Argus, Sept. 3-29. 1980). Standard length (tip of upper jaw to end of notochord) of each larva was measured by a precalibrated ocular micrometer to 0.1 mm, within 10-15 man of capture. Subsequent measurements (postpreservation) were accurate to 0.05 mm, the greater precision achieved in the absence of vessel movements. Larvae were randomly allotted to three preservation treatments: 4% neutral formalin- seawater (3 1- 32°/00) solution, 95% ethanol, and freezing in petri dishes on wetted filters. Identification of individual specimens was maintained by the use of numbered vials and filter papers. Sample sizes of eighty-three individuals were subjected to each treatment. Size ranges were 3.7- 14.4 mm (formalin), 3.2- 16.8 mm (ethanol), and 4.6-41.0 mm (frozen), with most of the speci-
FOWLER AND SMITH: LENGTH CHANGES IN SILVER HAKE
TABLE1. Average percent shrinkage of silver hake lanae with time. Values in parentheses denote mean lengths (mm) of treatment groups. Range of initial lengths (mm)
15 d (T,)
30 d ( Tz)
3.7-14.4(8.50) 3.2-16.8 (9.78) 4.6-41.0 (9.48)
3.4(8.21) 4.8 69.30) -
3.7(8.B#) 5.6 (9.25)
4.3(8.14) 7.0 (9 16) 1.4 (9.44)
Formalin Ethanol Freezing
83 83 83
"T4= 337 d (formalin), 205 d (ethanol), and 338 d (freezing).
TABLE2. Statistical analysis of percent shrinkage results obtained from formalin and ethanol treatments of silver hake larvae. Hypothesis tested
Test statistic (observed F )
Degrees of freedom
I ) Parallelism: formalin vs. ethanol" 2) Equality treatment means 3) Quality of effects over time:
a) Formalin Contrasts To- T , Tt-T2 TZ-Tq Ti- T4
Contrasts T o - T I 7'1-T2 T2-T ? Ti-T,
"Tested for comparable times (T,,, T,. T z , T I ) . "Because parallelism was rejected at cr = 0.001, a test of this hypothesis is meaningless (see text).
mens in the 4- to 15-mm range for a11 treatments. Ethanol samples were remeasured at 15, 30, 50, and 205 d foIlowing fixation. One or two drops of distilled water were applied ts specimens on the slide to counteract thc dehydrating effect of rapid evaporation of ethanol. Formalin samples were remeasured at 15, 30, 50, and 337 d. No compensation for dehydration was believed necessary for formalin-preserved larvae. In both cases 20-30 s elapsed bctwcen removal and return of larvae to their vials. The frozen samples were maintained at a temperature of about - 15°C for a period of 338 d, thawed with distilled water, and measured. Repeated thawing and refreezing would damage the specimens, rendering observatiolras at discrete time intervals useless. The length measurements were expressed in terms of percent shrinkage, defined as
length at time T' x 100 initial length
for each specimen. Results from the formalin and ethanol treatments were amalyzcd by a repeated measures approach which incorporates temporal correlation between measurement intervals into the test statistics. Details of the test statistics are discussed by Morrison (1976, sections 4.5 and 4.6).
Results The average percent shrinkage results obtained from the preservation experiment are presented in Table 1 . Hw Figures 1, 2, and 3, box and whisker plots are used to present final percent shrinkage results relative to the initial lengths of the larvae (arbitrarily divided into 2-mm groups). The upper and Bower edges of the boxes in the figures mark the positions of the upper and lower quartiles, respectively, with the position of the median indicated by an asterix. The dotted lines (whiskers) join the upper and lower extreme values to the boxes. For further information on this type sf plot see Tukcy (1 977) and Reckhow (1980). We followed the analysis hierarchy described in Morrison (1976; p. 153- 154); the results of the statistical test are presented in Table 2. Because the null hypothesis of parallelism between ethanol and fomalin treatments was rejected, we caw conclude that the two preservatives do not affect larval shrinkage in a similar manner over time. As a further test of equality of treatment means would be meaningless, the results were analyzed separately for each treatment. For larvae preserved in formalin an average percent shrimkage of 4.3 (range = 0.0- 14.$%) was observed after 337 d. The most significant reduction in length occumed during the first 15 d (Table 2) with the mean larva1 length continuing to
CAN. J . FISH. AQUAT. SCI., VOL. 40. 1083
INITIAL LENGTH jrnrn)
FIG. I .
Box and whisker plot of percent shrinkage results of formalin-preserved silver hake larvae at T4 (337 d) relative to initial larval lengths. ( * = position of median. See text for explanation.)
7-9 9-11 11-13 INITIAL LENGTH ( m m )
PIG. 2. Box and whisker plot of percent shrinkage results of ethanol-preserved silver hake larvae at Tl(205 d) relative to initial larval length. (* = position of median, x = position of observations when three or fewer points were available. See text for explanation.)
FIG. 3. Box and whisker plot of percent shrinkage results of frozen silver hake larvae at 338 d relative to initial larval length. C* = position of median, x = position of observations when three or fewer points were available. See text for explanation.)
INlPiAL LENGTH (mm)
FOWLER AND S M I T H : LENGTH C H A N G E S IN S I L V E R H A K E
shrink for the duration of the experiment, but at a slower rate. The increase in significance of the test statistic for the final contrast (T,-T,) relative to the previous contrast reflects the longer time interval involved rather than a sudden increase in shrinkage. In addition to the temporal effects there was also a relationship between treatment response and initial larval length. Figure 1 indicates that the median percent shrinkages for larvae greater than 9 mm initial length were less than those for smaller larvae. Larvae greater than 7 mrn exhibited less variability than smaller larvae, this variability being measured by comparing the lengths of the boxes bi.e. interquartile range). The largest interquartile range and range of extreme values occurred for larvae in the 5- to 7-mm group. Ethanol-preserved larvae had a mean shrinkage of 7.0% (range = 0.0-20.0%) after 205 d. Though the greater part of this reduction occurred in the first 15 d, shrinkage from day 50 to day 2665 was highly significant (Table 2) despite the declining rate of shrinkage over time. With respect to initial Iength Fig. 2 demonstrates that there was larger variation associated with the response of the smaller larvae (less than 7 mm) and a gradual decrease in shrinkage with increasing larval size. The highest median percent shrinkage and variability was exhibited by larvae in the 5- to 7-mrn group. The estimate of 1.4% shrinkage for frozen samples at 338 d was derived from length changes which ranged from -29.9% (expansion) to +26.5% (reduction). The highly variable nature of length response to freezing is depicted in Fig. 3. Larvae which were greater than 19 mm are not included in this figure because no change in length was observed. A transition in median response from shrinkage to expansion as larval size increases is also evident in this figure.
Discussion The observation that larval silver hake shrink over time when preserved in fomalin with the greater part of this shrinkage occurring in the first few days is consistent with findings reported for other species (Blaxter 197 1; Lockwood and Baly 1975; Schnack and Rosenttaal 1978; Hay 1981, 1982). In addition the tendency for the magnitude of shrinkage to decrease with increasing initial larval length has also been observed by Lockwood and Daly (1975), Schnack and Rosenthal 61978), and Hay ( 1981, 1982). However, Theilacker (1980), in noting for laboratoryreared northern anchovy larvae that capt~are/handlingstress (including death) was chiefly responsible for larval shrinkage, further concluded that size-dependent shrinkage was a consequence of handling alone and not preservation. This conclusion was based on a comparison of ratios of posttreatment lengths to pretreatment lengths of larvae after ( 1 ) preservation in formalin, (2) simulated net-capture, and (3) preservation after net-capture. The inconsistency between Theilacker's results and those of other studies may be explained by the shorter duration time of preservation in her experiment. As Schnack and Rosenthal6 1978) and Hay (1982) have noted for Pacific herring (Clupeer hareragus pcallasi), size-dependent shrinkage becomes more pronounced the longer the larvae are stored in formalin. The difference may also be due to a species-specific response, as kockwood and Daly (1975)
found that larval dabs (Limanda limundu) and plaice (Pleuronecbtesplates~a)react differently to storage in formalin. The observation with silver hake larvae of an increase in variability of the shrinkage response with decreasing larval size has not been mentioned abs occurring in previous investigations of preservation shrinkage, possibly because of the limited range of lengths treated in other studies. The only exception is Theilacker's study, which covered as wide a size spectrum as ours did but only reported averaged results for smaller larvae such that the degree of variation present could not be evaluated.
Significant reduction in length and decreasing magnitude of shrinkage with increasing initial length for larvae preserved in ethanol was found both in our experiment with silver hake and for cod (posthandling lengths vs. postpreservation lengths, data pooled by us) in Radtke and Waiwood (1980). The change in variability of response with size could not be compared for cod data because that study concentrated on very small larvae (4.27 -5.62 mm). Theilacker ( 1980) examined the effects of ethanol on simulated nct-caught and laboratoryreared northern anchovy larvae in the same manner as she did for formalin. She concluded that ethanol did not cause larvae to shrink. However, it as not possible for us to evaluate these findings because no data were presented for net-treated specimens. Neither the cod nor the anchovy studies looked at duration effects of ethanol. Our observations then, that larvae preserved in ethanol undergo increasing magnitude but decreasing rate of shrinkage over time cannot be compared with these investigations.
For silver hake larvae both treatments exhibited decreasing rates of shrinkage over time with most of the length reduction occurring in the first 15 d and decreasing percent shrinkage and variability of response with increasing size of larvae. Size-dependence in this relationship was likely a function of increased rigidity of larger specimens due to ossification, muck as Theilacker (1980) suggested to explain shrinkage differences resulting from capture alone. The greater rate of shrinkage noted for the first 15 d relative to the later periods we believe attribuvdble to continuing autolysis prior to tissue saturation with preservative, and to tissue degradation during the more pronounced osmotic changes of newly preserved samples. The greater rate and magnitude of shrinkage with the ethanol-preserved samples constitutes the major difference between the two treatments. It was also noted that larvae preserved in ethanol were firmer than those in formalin and more faded in coloration (most notably the color of the stomach).
Larvae demonstrated extreme variability in length change response to freezing, being easily stretched or con~pressed without visible structural damage. Thus it is our opinion that
CAN. J. FISH. AQUAT. SCI.. VOL. 40. 1983
freezing is an unsuitable method for preserving fragile larval samples for length-related analyses.
Length measurements froin preserved larvae have been used to obtain mortality and recruitment estimates for commercial fish stocks (e.g. Laugh et al. 19801, but given the results of this and other studies such practices may have significant ramifications. Where measurements are equated to predetermined length at age estimates and applied to growth/mortality formulae, underestilnation of age due to larval shrinkage could result in overestimation of mortality, especially if no allowance is made for capture-induced shrinkage. The lower recruitment estimates derived from length data without adjusting for shrinkage may thus bias predictions sf s t ~ e kabundance and hence influence potential yield determinations. This bias might explain intances of stock abundance estimates from larval surveys falling short of virtual population assessment estimates, as noted in Louph et al. (1980).
PRACTICAL CCPNSIDERP~TIONS AND SUGGESTIOKS The use of preserved saniples to derive larval length data is statistically risky because of the extent and variability of length changes resulting from stress and preservation. Nor would measurements of freshly caught larvae alleviate the problem as many authors report that the influence of capture and handling alone is of greater magnitude than any subsequent changes during preservation. But whcre rough estimates of larval lengths of wild populations are desired, and measuring feasibility and precision under land-based laboratory conditions is significantly superior to shipboard attempts, it becomes necessary to develop the best methods possible to obtain and analyze preserved larval samples. We believe freezing to be a very poor preservation technique where larval length estimates are required due to the unpredictability of individual length changes. In choosing between formalin and ethanol. it might be argued that forrnalin is the better option by virtue of the lesser rate of shrinkage and greater abundance of previous data in the literature available for comparison. With respect to measurement. however, the firmer specimens obtained by preservation in ethanol might be more desirable provided a 95% concentration of the solution is consistently achieved to avoid the destructive lower pH levels of less concentrated so%utionsBK. Waiwoc~d. Fisheries and Oceans, Biological Station, St. Andrews, N. B., personal communication). It is also the best choice when correlations between larval length and the growth rings of otoliths are required, as formalin dissolves calcium. Should ethanol be preferred it would be advisable to generate a background of experimental data to provide consistent estilnates of shrinkage with the preservative. In either case the nature of the preservation responses are essentially the same. Larvae undergo most of their shrinkage within a few days, after which shrinkage continues but at a much lesser rate. Magnitude of shrinkage increases as the initial size of larvae decreases, likely a function of greater rigidity of body structure with maturity (Radtke and Waiwood 1980). Coupled with this relationship we expect an increase in variability of length change response to preservation with decreasing larval size.
Where length data is to be obtained from preserved samples, we propose a waiting period of at least 50 d between collection and measurement to avoid the more dynamic shrinkage trends of recently stored samples. Furthermore, the problem of response variability observed with small larvae must be approached cautiously. If possible. the appropriate size range should be dealt with separately, as a single class, while applying a different method to larger specimens. Any loss in continuity must be weighed against the loss in precision of length estimates over the larger size range.
We thank Y. IPeLafontaine (McGill University) for his contributions at the beginning of this study and I%. N. O'Bc~yle,W. T. Stobo. and K. G. Waiwood, all of the Marine Fish Division, for reviewing and commienting on earlier drafts. In addition we thank D. E. Hay (DFO, Nanaimo, B.C.) and J. 5 . Colton (NMFS. Narragansett) for reviewing the final draft. BI~AXTER, J . H. S. 197 1. Feeding and condition of Clyde herring Larvae. Rapp. P.-V. Reun. Cons. Int. Explor. Mer 160: 128- 136. ENGEI,,S. 1974. Effects of forrnalial and freezing on length, weight and condit~onfactor of cibco and yellow perch. Trans. Am. Fish. SOC. 103: 136- 138. HALLIDAY, R. G., AND B. I P ( W ~ E . 1969. The effects of icing and freezing osn the length and weight of groundfish species. ICNAF Wes. Doc. 69/2. HAY,I).E. 198 l . Effects of capture and fixation on gut contents and body size of Pacific herring larvae. Wapp. P.-V. Reun. Cons. Int. Explor. Mer 178: 395-400. 1982. Fixation shrinkage of herring larvae: effects of salinity, formalin concentration, and other factors. Can. J. Fish. Aquat. Ssi. 39: 1 138- 1 143. LC~CKWOOD, S . J . , AND C. DE B . DAI-Y.1975. Further observations on the effects of preservation in 4% neutral formalin on the length and weight of 0-group flatfish. J. Cons. Int. Expior. Mer 36(2): 170- 175. LOC'GH,R . 6., 6. W. RC~LZ,M. I%. PENNINGTON, AND h'f. D. GROSSLEIN. 1980. PLbundance and mortality estimates for sea herring (Ciltpeu hirrcngus L.) larvae spawned In the Georges Bank-Nantucket Shoals area, 197 B - 1878 seasons, in relation to spawning stock and recruitment. NAFO SCR Doc. 8Q/IX/ 129. MORRISON, D. F. B 974. Muitivariate statistical methods. 2nd ed. McGraw-Hill. New York. 4L5 p. PARKER, R. W. 1963. Effects of formalin on length and weight of fishes. J. Fish Res. Board Can. 20: 1441-1455, RADTKE,R. L., AND W. G . WAIWOOD. 1980. Otolith formation and h d y shrinkage due to fixatioia in Barval cod (Gadas morhuu). Can. Tech. Rep. Fish. Aquat. Sci. 929: 10 p. RECKHOW, K . H. 1980. Techniques for exploring and presenting data applied to lake phosphorous concentration. Can. J. Fish. Aquat. Sci. 37: 290-294. ROSENI'HAL, H., D. KIIHEMANN, AND 0. FUKL'I-IAWA. 1978. Shrinkage of newly hatched larvae of the Red Sea bream iChrysophrp malor, Ternmink Wr Schlegel) preserved in fornaalin. Arch. Fischereiwiss. 29: 59-63. SCHWACK, D., AND H. ROSENTHAL. 1978. Shrinkage of Pacific herring larvae due to formalin fixation and preservation. Ber. Dtsck. Wiss. Komm. Meeresforsch. 26: 222-226. THLII-ACKER, G. H. 1980. Changes in body measurements of larval northern anchovy, Engraulis rnordar, and other fishes due to handling and preservation. Fish. Bull. 78i3):685-692. TUKEY,J. W . 1977. Exploratory data analysis. Addison-Wesley, Reading, MA. 688 p.