Effects of methanol on the fertilisation of chum salmon (Oncorhynchus keta) ova

EFFECTS OF M E T H A N O L ON THE FERTILISATION OF C H U M SALMON (ONCORHYNCHUS KETA) OVA P. C. CRAIG Aquatic Environments Ltd, 53 Howard Avenue, Nanaimo, B.C., Canada V9R 3P9 & F. C. WITHLER • R. B. MORLEY Department o[ the Environment, Fisheries and Marine Service, Pacific Biological Station, P.O. Box 100, Nanaimo, B.C., Canada V9R 5K6

ABSTRACT

The ejJect of methanol on the jertilisation oJ chum salmon ova was investigated because methanol jrom oil and gas pipeline testing is a potential contaminant in northern watercourses. Both gametes and fertilised eggs were subjected to methanol concentrations (0.001% to 10 %)jor brief periods. An exposure time of 32 sec allowed an examination of ejJects on gametes; a 30-min exposure included possible lethal or sublethal effects oJmethanol uptake during imbibition of the ova. These exposures at methanol concentrations up to and including 1% did not significantly aJ]ect Jertilisation success, survival to hatching, hatching time, alevin size at hatching or physical deJormities among alevins. A 10 % methanol concentration was lethal in most cases.

INTRODUCTION

Large quantities of methanol may be used as an antifreeze in the hydrostatic testing of northern oil and gas pipelines. It has been suggested that salmonid sperm and ova might be extremely sensitive to low concentrations of this chemical (McMahon & Cartier, 1974). Ifthiswere true, a methanol spill could affect the spawning success of salmonids occupying streams into which methanol drained. The purpose of this study is to examine the effects of methanol on the sperm and ova of a salmonid fish during fertilisation. The salmonid used in this study was the Pacific chum salmon (Oncorhynchus keta). Although not abundant in Arctic waters, chums have been taken in several Beaufort Sea drainages (McPhail & Lindsey, 1970; Yoshihara, 1973), and there is a large run in the Porcupine River (Elson, 1975). 85 Environ. Pollut, (14) (1977)--~ Applied Science Publishers Ltd, England, 1977 Printed in Great Britain

86

P . C . CRAIG, F. C. WITHLER, R. B. MORLEY

MATERIALS A N D M E T H O D S

Experimental design Exposure times were determined on the basis of gamete behaviour during the fertilisation process of salmonids. At spawning, the time between shedding of gametes and fertilisation of ova is extremely brief. Copious quantities of milt eddying within the redd ensure that most eggs are fertilised within 30 sec. During this time and for the next half-hour or so, the eggs imbibe water and become 'waterhardened' (Morley & Withler, 1969). With respect to the potential effects of methanol, there are two important periods: (l) the briefl~eriod prior to sperm penetration when both sperm and unfertilised ova would be exposed to methanol and (2) the period after penetration when the ova imbibe water and, presumably, methanol. There is some information available concerning the movements of materials through egg membranes during the latter period. For example, the transfer of cadmium through herring egg capsules is extremely rapid during imbibition. Some of this contaminant also passes through the vitelline membrane and enters the yolk (Rosenthal & Sperling, 1974; Rosenthal & Alderdice, 19?6). These authors also found that herring eggs became impermeable to cadmium at later stages, so that most of the cadmium which entered the egg did so during water hardening. Hence, an exposure time of 32 sec was chosen to examine the effects of methanol on gametes alone, and an exposure of 30 min was chosen to examine the effects of methanol uptake during water hardening. Methanol concentrations used were 0.001 0/0, 0.01 ',)Jo,0' 1 '~'0, 1.0"J~oand 10"/jo by volume.

Methods Eight chum salmon (four males and four females) were collected alive by seine and dipnet from spawning grounds in Nanoose and Bonell creeks on Vancouver Island, British Columbia, Canada. They were held overnight and tests were carried out the following day (29 October 1975). The experiment was replicated twice, using different parents for each replicate. Within replicates, the ova from two females were pooled, as was the milt from two males. Within 0.5-1-5 h of stripping, ova were fertilised in plastic buckets containing 1 litre of either plain water (control) ot one of five methanol solutions. The solutions were drawn in a randomised sequence, but the first and last tests in each series were controls, to permit detection of any decrease in viability of ova and/or sperm with time. As in previous tests (McMahon & Cartier, 1974), a single batch of reagent grade methanol was used. In order to dissipate heat of reaction all test solutionswere stored in a 9-5°C water bath for at least 1.5 h prior to testing. The procedure was to add simultaneously 2 ml of milt and approximately 50 ml of ova to water (ifa control) or to a methanol solution and to stir briefly. Rinsing of the eggs was initiated after 15 sec in the short-exposure tests and after 30 rain in the longexposure tests. The time involved in thorough rinsing and subsequent flooding with

EFFECTS OF METHANOL ON FERTILISATION OF SALMON OVA

87

fresh water was 17 sec (in each of three timings). Whilst the time involved in rinsing is a negligible addition to the 30-rain exposures, it doubles the exposure time in the short-exposure tests. The latter is therefore referred to as a 32-sec exposure time. After rinsing, the eggs from each treatment were placed in randomised compartments of two trays of a Heath vertical stack incubator. They were incubated at 9-3°C (SD +_ 0-33°C) for the duration of the experiment. Temperatures were recorded with a Taylor seven-day continuous recorder. Daily temperature was calculated by averaging temperatures recorded at 0600, 1200, 1800 and 2400h. Time-temperature units are expressed as 'Celsius degree days' (C ° days) of incubation. At the 300 C ° day stage, when the eggs were eyed, they were shocked with a fine spray of water to reveal dead or unfertilised eggs (the yolk material of these eggs coagulates and turns white). These were examined for the presence or absence of embryos after being cleared with Stockard's solution. Once hatching began, alevins were counted daily and removed from trays. Every third or fourth alevin (for a total of thirty for each treatment) was measured to the nearest 0-5 mm. All alevins were preserved in 10 ~o formalin for later examination for physical deformities.

RESULTS

Fertilisation success

Methanol concentrations up to and including 1 ~o did not significantly affect fertilisation. For an exposure of 32 sec, 96 ~ or more of ova in combined replicates were fertilised (Table 1). Slightly fewer ova were fertilised when the exposure period was extended to 30 min, but even then the fertilisation rate was at least 90 ~0 (Table 2). Within the 0.001 ~o-1.0% range of methanol concentrations, the highest concentration (i.e. 'worst case' treatment) was compared statistically with the control data. For both exposure periods there was no significant difference between the results obtained from controls and the 1 ~o methanol treatments (32-sec series: Z2 = 0.3, P = 0.9; 30-min series: ~(2 = 0.2, P = 0.9). Methanol concentrations of 10 ~o proved lethal. Fertilisation was impaired for nearly all ova subjected to this concentration for 30 min. Embryos were present in only four (1.5 ~o) of the 267 eggs tested. Even when the gametes were exposed to a 10 'Yomethanol concentration for just 32 sec, only 28 ~o (range 10-49 ~o) of the eggs were fertilised. It is possible that somewhat more eggs were fertilised than shown above. The criterion for successful fertilisation was the presence of an embryo within the egg, but embryos are not large enough to be detected by eye during the first ten days after fertilisation. Thus some embryos may have been present but not counted. However, for our purposes it is not important whether the gametes or the newly formed zygotes were killed by the 10 ~o concerttration.

88

P.c. CRAIG, F. C. WITHLER, R. B. MORLEY

TABLE 1 FERTILISATION RATES, SURVIVALS AND TIMES TO HATCHING. AND LENGTH OF ALEVINS FOR CHUM SALMON GAMETES EXPOSED TO VARIOUS CONCENTRATIONS OF METHANOL FOR 32 SEC. LENGTH MEASUREMENTS ARE BASED ON A SAMPLE SIZE OF 3 0 (EXCEPT IN ONE CASE. REPLICATE 2-10 ~o. WHERE n = 12)

Ova

Alevins Length (ram)

Replicate 1

2

1 and 2 combined

')o Methanol Control" Control b 0.001 0.01 0.1 1-0 lif0 Control ~ Control ~' 0.001 0.01 0.1 1.0 10.0 Control a Control b 0-001 0.01 0.1 1.0 10.0

N

~o Fertilised

% Survival to hatching

99 150 128 142 109 122 117 141 146 148 130 150 123 135 240 296 276 272 259 245 252

96 99 100 99 97 100 49 100 97 97 94 98 94 10 98 98 99 96 98 97 28

96 99 99 98 97 100 49 99 97 95 94 97 93 09 98 98 97 96 97 96 27

Mean hatching (C ° days) Mean 539-8 539.4 539-6 539-4 539.7 539-8 539-8 531.4 535.5 531.4 537.0 534-3 533.4 536-7 534.8 537.4 535.3 538.3 536.6 536.5 539.3

24.3 24.0 24.2 24.0 24.2 24.1 24.4 24.4 24.2 24.1 24.5 24.1 24.2 24.2 24.4 24.1 24.2 24.2 24.2 24.1 24.3

Range

SD

(23.0-25.0) (21.5-25.5) (23.0-25.0) (21.0-25.5) (23.5-25.5) (23.0-25.0) (23-5-25.5) (23.0-25.5) (23.0-25.5) (21.0-25.5) (23.5-25.5) (23.0-25-0) (22.0-25.0) (23.5-25.0) (23.0-25.5) (21.5-25.5) (21.5-25.5) (21-0-25.5) (23.0-25.5) (22.0-25-5) (23.5-25.5)

0-4 0.9 0.5 0.9 0.5 0.6 0.5 0.6 0.7 0.9 0.5 0.6 0.7 0.4 0.5 0.8 0.7 0-7 0.6 0.6 0-4

" Control test at beginning of series. b Control test at end of series.

Survival to hatching B e c a u s e few e m b r y o s d i e d d u r i n g i n c u b a t i o n , n u m b e r s o f eggs w h i c h survived m e t h a n o l t r e a t m e n t s a n d successfully h a t c h e d are similar to t h o s e d e s c r i b e d a b o v e . T h e results are a l m o s t i d e n t i c a l for p r o p o r t i o n s fertilised a n d s u b s e q u e n t survival to h a t c h i n g in the 32 sec series, i n d i c a t i n g t h a t there was n o d e l a y e d effect w h i c h c a u s e d m o r t a l i t y a m o n g d e v e l o p i n g e m b r y o s . D a t a in the 3 0 - m i n series w e r e m o r e v a r i a b l e , p a r t i c u l a r l y in r e p l i c a t e 2 ( T a b l e 2), b u t t h e r e was n o c o n s i s t e n t trend. E x p e r i m e n t a l e r r o r c o u l d a c c o u n t for an i n d i c a t e d g r e a t e r survival rate a f t e r a ! ~o m e t h a n o l t r e a t m e n t t h a n after a 0.1 ~0 t r e a t m e n t , H a t c h i n g time H a t c h i n g t i m e was e x a m i n e d b e c a u s e a d e l a y in d e v e l o p m e n t m i g h t s e r i o u s l y i n t e r f e r e w i t h later survival a n d g r o w t h o f the fish. E a c h r e p l i c a t e in this e x p e r i m e n t was d e r i v e d f r o m a different set o f p a r e n t s a n d m i n o r differences b e t w e e n r e p l i c a t e s were n o t e d , i n c l u d i n g t i m e to h a t c h i n g . A l e v i n s in r e p l i c a t e 1 h a t c h e d significantly later t h a n t h o s e in r e p l i c a t e 2 (t = 8.5, P < 0.005).

EFFECTS OF METHANOL

89

ON FERTILISATION OF SALMON OVA

TABLE 2 FERTILISATION RATES, SURVIVALS AND TIMES TO HATCHING, AND LENGTH OF ALEVINS FOR CHUM SALMON GAM ETES EXPOSED TO VARIOUS CONCENTRATIONS OF METHANOL FOR 30 MIN. LENGTH MEASUREMENTS ARE BASED ON A SAMPLE SIZE OF 30 (EXCEPT IN ONE CASE, REPLICATE 1 - 1 0 % , WHERE n = I )

Ova

AIcvins Length (rnm)

%

%

Replicate

Methanol

,N

Fertilised

1

Control ° Control b 0.001 O.Ol 0-1 1.0 10.0 Control ~ Control b 0.001 0.01 0-1 1.0 10.0 Control" Control b 0.001 0-01 0.1 1-0 10-0

104 135 115 130 112 126 124 139 146 158 154 153 139 143 243 281 273 284 265 265 267

100 99 I00 98 94 94 1.6 91 93 96 86 87 98 1.4 95 96 97 91 90 96 1.5

2

1 and 2 combined

Survi~:al Mean hatching to hatching (C ° days) %

100 96 IO0 94 83 88 0.8 84 90 88 79 65 95 0 91 93 93 86 72 92 0.4

539.1 537.8 539.6 539-7 539.0 538-3 540-0 535.6 532.0 531.6 532.4 534-6 537-4 . . 537-2 534.9 535.2 536-1 536.7 537-8 540.0

Mean

24.4 24-3 24.0 24.0 24.5 24.4 24.0 24-3 23.4 24-1 24.0 23-8 24.4 . . 24.4 23-8 24.0 24-0 24.2 24.4 24-0

Range

SD

(22.5-26-0) (23.5-25.5) (23.0-25.0) (22.5-25.0) (23-5-25.5) (23.0-25.0) -(23-5 25.0) (21-5 25-0) (22.5 25.0) (23.0-25.0) (22.0-25-0) (21.5-25-0) . . (22.5 26-0) (21.5-25.5) (22.5-25.0) (22.5 25-0) (22.0-25-5) (21.5-25.0) --

0.7 0-6 0.6 0-5 0.5 0-5 -0.5 0.7 0-6 0.6 0-8 0.7 0.6 0-6 0.6 0.6 0.6 0.6 --

" Control test at beginning of series. b Control test at end of series. H o w e v e r , the difference b e t w e e n m e a n s was small, a m o u n t i n g to o n l y 5-3 C ° days, o r 1/2 d a y o f i n c u b a t i o n , B e c a u s e o f this difference, r e p l i c a t e s are t r e a t e d s e p a r a t e l y in this s e c t i o n . M e t h a n o l t r e a t m e n t s c a u s e d n o d e t e c t a b l e c h a n g e in h a t c h i n g times. In r e p l i c a t e 1 h a t c h i n g t i m e was e x t r e m e l y precise a n d u n i f o r m b o t h w i t h i n a n d b e t w e e n t r e a t m e n t s . T h e m e a n t i m e to h a t c h f o r all c o n t r o l s a n d t r e a t m e n t s was 539.4 C ° d a y s a n d the r a n g e o f v a l u e s p e r t r e a t m e n t was small. O v e r 92 ~o o f the eggs in e a c h t r e a t m e n t h a t c h e d at 540 + 4.6 C ° d a y s in the 32-sec series. T h e r e was slightly m o r e v a r i a b i l i t y in the 3 0 - m i n series o f r e p l i c a t e 1, b u t there, t o o , m o s t eggs ( 7 8 - 1 0 0 °/o) h a t c h e d at 540 + 4.6 C ° days. In r e p l i c a t e 2 t h e eggs h a t c h e d o v e r a slightly l o n g e r p e r i o d t h a n t h e y did in r e p l i c a t e 1. O t h e r m i n o r differences i n c l u d e the e a r l i e r average hatching mentioned previously and more variation between treatments. O t h e r w i s e , o v e r a l l results b e t w e e n r e p l i c a t e s are similar. T h e r e is n o significant difference a s s o c i a t e d w i t h e x p o s u r e times, i.e. m e a n h a t c h i n g t i m e s w e r e s i m i l a r in b o t h t h e 32-sec a n d 3 0 - m i n e x p o s u r e s ( r e p l i c a t e 1 : t = 1-86, P = 0.09; r e p l i c a t e 2: t = 0-24, P = 0.8).

90

P . c . C R A I G , F. C. W I T H L E R , R. B. M O R L E Y

Alevin length at hatching

Regardless of the methanol treatment, average lengths of newly hatched alevins showed little variation (Tables 1 and 2). The range of mean lengths was only 24.0-24.5mm in the 32-sec series and 23.4-24-5mm in the 30-rain series. Comparisons of'worst case' treatments in each replicate (i.e. the 10 ~o concentration in the 32-segseries and the I ?/o concentration in the 30-min series due to insufficient numbers surviving in the higher concentration) with controls showed no significant differences (t = 0.2-1.1, P = 0.25-0.8). DeJormities

Newly hatched alevins were examined for gross physical deformities. In all cases, the incidence of deformities was low, ranging from 0-3.1 ~o (Table 3). The most common type of deformity observed was malformation of the spine, but there was no correlation between incidence of deformity and methanol concentration. An inspection of Table 3 also shows that the incidence of deformity was not related to the two exposure times tested. The overall proportions of deformed alevins in each series were almost identical (1-1-1.2 ~o). TABLE 3 INCIDENCE OF DEFORMITIES AMONG CHUM ALEVINS SUBJECTED TO VARIOUS METHANOL TREATMENTS DURING FERTILISATION. DEFORMITIES ARE LISTED BY NUMBER: 1, SHORT LOWER JAW: 2, TWO BODIES ATTACHED TO SAME YOLK SAC: 3, CROOKED SPINE; 4, ABNORMAL YOLK SAC (DROPSY)

Exposure time

Methanol concentration

32-sec series

Controls 0"001 0.01 0.1 I 10

30-min series

Totals Controls 0-001 0.01 0-1 1 10 Totals

Deformity 2 3

1 -1 . . . . -. . .

--. -.

.

.

--+-1 ---. . . . . --

5 3 1 5 .

--

4

Total

2

7 4 1 5 0

--

.

0

6 5 I 1 l --

2 --

17 6 8 1 1 1 0

17

No. examined 513 267 264 251 249 69 1613 482 254 246 192 243 1

1418

o~, 1.4 1.5 0.4 2"0 0.0 0"0 1" 1 1.2 3-1 0.4 0-5 0.4 0.0 1-2

DISCUSSION

The observations of the ova used as controls in these tests are representative of standard hatchery results for chum salmon. Withler & Morley (1970) report a similar hatching time (mean 541-1, range 528.7-557.5C ° days), size at hatching (mean 25-2, range 24.0-25.6 mm) and occurrence of physical deformities (1.2 ~o) for chum salmon eggs incubated at 11 °C. The fertilisation rates of the control ova also fall within the normal range for hatchery practice (85-100 ~o)-

EFFECTS OF METHANOL ON FERTILISATION OF SALMON OVA

91

The observations of gametes exposed to methanol demonstrate that, under experimental conditions, exposure to methanol for up to 30 rain at concentrations up to and including I ~o does not adversely affect the fertilisation process or survival to hatching of chum salmon, although it is known that continuous exposure to 1 ~0 methanol over the entire incubation period produces 100~o mortality among grayling eggs (McMahon & Cartier, 1974). A 10~o concentration of methanol was lethal in most cases, even for short exposures. An average mortality of 73 ~o occurred after an exposure time of only 32 sec; 99.6 ~o died after an exposure time of 30 min. The nature of the lethal effect in the 10 ~ treatments is not known, but it was observed that this concentration was potent enough to kill some eggs before the end of the 30-min exposure period (the eggs were already turning opaque). McMahon & Cartier (1974) report that juvenile Arctic char and grayling suffer 100 ~, mortality at this concentration (24-h test). For the several parameters examined to detect sublethal effects (hatching time, alevin size at hatching and physical deformities), no significant differences were found between treatment concentrations of 1 ~o or less and control observations. Whilst these results suggest that a short exposure to spilled methanol diluted to 1 ~o or less in a receiving stream would not affect the viability of deposited ova and sperm, it should be borne in mind that the methanol used in these tests was reagent grade. Methanol is the major constituent of antifreeze used in pipeline testing, but combined effects due to contaminants which vary in kind and proportion in industrial grade antifreeze are not known. ACKNOWLEDGEMENT

This study was funded by Canadian Arctic Gas Study Limited through Northern Engineering Services. REFERENCES ELSON, M. S. (1975). Enumeration of spawning chum salmon (Oncorhynchus keta) in the Fishing Branch River, 1971-74. In Northern Yukon Fisheries Studies 1971-74, ed. by L. Steigenberger, M. Elson and R. DeLury, Vol. I, Chap. 11. Environmental-Social Programme, Northern Pipelines, Environ. Canada. MCMAHON, B. & CARTIER, L. (1974). Methanol toxicity in northern fishes--Preliminary report. Canadian Arctic Gas Study Ltd, Calgary, Alberta, Biological Report Series, 15(5), 1-35. MCPHAIL, J. D. & LINDSEY,C. C. (1970). Freshwater fishes of northwestern Canada and Alaska, Fish. Res. Bd Can. Bull., 173, 1-381. MORLEY,R. B. & WITHLER, F. C. (1969). Observations on sockeye and pink salmon ova and milt relative to spawn collection. Fish. Res. Bd Can. Tech. Rep., 111, 1 11. ROSENTHAL, H. & ALDERD1CE, D. (1976). Sublethal effects of environmental stressors, natural and pollutional, on marine fish eggs and larvae. J. Fish. Res. Bd Can., 33, 2047-65. ROSENTHAL, H. & SPERLING,K. (1974). Effects of cadmium on development and survival of herring eggs. In The early liJe history offish, ed. by J. Blaxter, 383-96. New York, Springer-Verlag. WITHLER, F. C. 8/, MORLEY,R. B. (1970). Sex-related parental influences on early development of Pacific salmon. J. Fish. Res. Bd Can., 27, 2197-214. YOSHIHARA, H. (1973). Monitoring and evaluation of Arctic waters with emphasis on North Slope drainages. Alaska Dep. Fish Game A. Rep., 14, 1 83.