DDT in trout and its possible effect on reproductive potential

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1 New Zealand Journal of Marine and Freshwater Research ISSN: (Print) (Online) Journal homepage: DDT in trout and its possible effect on reproductive potential C. L. Hopkins, S. R. B. Solly & A. R. Ritchie To cite this article: C. L. Hopkins, S. R. B. Solly & A. R. Ritchie (1969) DDT in trout and its possible effect on reproductive potential, New Zealand Journal of Marine and Freshwater Research, 3:2, , DOI:.80/ To link to this article: Published online: 30 Mar 20. Submit your article to this journal Article views: 440 Citing articles: 24 View citing articles Full Terms & Conditions of access and use can be found at

2 220 [JUNE DDT IN TROUT AND ITS POSSIBLE EFFECT ON REPRODUCTIVE POTENTIAL C. L. HOPKINS Fisheries Research Division, Marine Department, Wellington S. R. B. SOLLY and A. R. RITCHIE Wallaceville Animal Research Centre, Department of Agriculture, Wellington (Received for publication 25 October 1968) SUMMARY Eggs of rainbow trout (Salmo gairdneri Richardson) from five different lakes in North Island, New Zealand, were reared to discover whether they showed significant differences in survival which could be linked with DDT levels in the tissue. The muscle and gonads of pre-breeding season fish and the parent females were assayed for DDT, as were the whole fry at the end of rearing. The highest DDT levels were found in fish from, and eggs from these fish showed the least viability. Mortality was relatively low among eggs from fish out of the other lakes. Thus, DDT may possibly contribute to the high mortality found in eggs from fish. INTRODUCTION It has been known for many years that DDT used as a pesticide on land can kill aquatic vertebrates and invertebrates (e.g., Eide, Deonier, and Burrell 1945; Everhart and Hassler 1948; Hoffman and Surber 1948, 1949). In New Zealand, direct poisoning of fish by insecticides except by accident or misuse has been rarely recorded (unpublished, Marine Department file). DDT has been suspected to cause deaths in hatchery trout, but this has never been proved because the levels of DDT in those fish assayed were all too low. Not much is known of the general levels of DDT likely to be found in fish in New Zealand fresh waters. Most fish so far assayed for DDT content have come from populations suspected to be contaminated. The Departments of Agriculture and of Scientific and Industrial Research have analysed a few samples of fish taken from supposedly DDT-free waters or waters in which there is no suspicion of adverse effects. Such analyses have shown tissue levels of DDT ranging from nil to 0.7 ppm per whole fish, well below toxic levels reported by overseas authors (Graham and Scott 1959; Cope 1961; Warner and Fenderson 1962; Bridges, Kallman, and Andrews 1963). However, death among adult fish is not the only criterion of pesticide contamination. Other N.Z. Jl mar. Freshwat. Res. 3: 220-9

3 1969] HOPKINS, SOLLY, & RITCHIE DDT IN TROUT 221 harmful effects may arise such as reduced growth (Anderson and Everhart 1966) or reduced fecundity and death of fry from contaminated eggs (Burdick, Harris, Dean, Walker, Skea, and Colby 1964). Despite the lack of evidence for lethal quantities of DDT in fish, fishery interests have feared that the widespread use of DDT in New Zealand may have adverse effects on such factors as fecundity. These fears have been focused by the work of Burdick et al., who found that sublethal doses of DDT in adult trout could cause the eventual death of fry from such contaminated parents. Because of this work, in 1966 we tried to discover whether there was evidence of any reproductive losses among trout populations in New Zealand which might be caused by DDT residues. The study was based on the hatchery rearing of eggs from rainbow trout (Salmo gairdneri Richardson) inhabiting lakes in the central region of the North Island. Services for the programme were provided by four government departments Marine, Agriculture, Internal Affairs, and Scientific and Industrial Research. METHODS AND MATERIAL The trout used were all taken from lakes important to the inland fishery. Three of the lakes,,, and, receive drainage from considerable areas of agricultural land which has been topdressed with DDT from time to time in the past. The other two, and, are surrounded by native forest and are virtually free of agricultural contamination. The areas and maximum depths of the lakes are shown in Table 1. TABLE 1 Surface area and maximum depth of lakes Area (hectares) Depth (m) 1,3 5,439 6,941 64, In April 1966, fish were taken from each lake to check levels of DDT in the tissues, particularly in the developing gonads, before the breeding season. In October, females netted from each lake were stripped of 500 eggs each. At each lake, five batches of 1,000 eggs were taken, each batch containing eggs from two females. The eggs were fertilised with sperm collected from males of the same lake. The parent females were frozen for eventual DDT assay. Table 2 shows the mean weights and range of weights of the trout analysed. Sig. 4

4 222 N.Z. JOURNAL OF MARINE & FRESHWATER RESEARCH [JUNE TABLE 2 Mean weights of trout; ranges shown in parentheses Pre-breeding season trout (April 1966) Body weights Gonad weights 11 1 Immature Male Female 0.47( ) 0.78 ( ) 4 ( ) Immature Male Female 0.33 ( ) 1.03 ( ) 3 ( ) 2.05 ( ) ( ) 0 Immature Male Female 0.81( ) 0.83 ( ) 0(4-6) 2( ) Immature Male Female 0.96 ( ) 1.93( ) 1.66( ) 7(1-0.0) 0.145( ) Immature Male Female 0.54( ) 0.59 ( ) ( ) 9 Parent female trout (October 1966) Body weight (after 500 eggs removed) 1.40( ) 1.55( ) 1.31 ( ) 1.72( ) 1.53( ) The eggs and young were reared to the stage of final yolk-sac resorption at the Department of Internal Affairs trout hatchery at Ngongotaha, near. Rearing was by standard hatchery methods, except that the normal dosing of eggs with fungicide and "kill-off" treatment by mechanical shock to separate less viable ova were omitted. Deaths of eggs and fry in each batch were carefully noted. The timing of the end of rearing for each batch of fry could not be exact; surviving fry were frozen when they became listless from apparent starvation, as decided by the hatchery manager. A disease known as blue sac caused the death of some fry, which were discarded (figures omitted from Table 5). ANALYTICAL METHODS Each fish was weighed, a sample of muscle removed from the dorsal fin region, and, in mature fish, the whole gonads removed and weighed.

5 1969] HOPKINS, SOLLY, & RITCHIE DDT IN TROUT 223 Fry were weighed in batches of 400. Tissue samples and whole fry were stored in airtight jars at 18 c until they were analysed. Where appropriate, the adult fish were analysed in pairs, identifiable with the original egg batches. Each pair was selected to be of the same sex, from the same lake, and of approximately the same body weight. Assay of whole fry was made on the pooled tissue of complete batches. A 20 g composite sample comprising g of tissue from each fish of a pair, or 20 g of fry, was ground with 60 g of anhydrous sodium sulphate in a mortar, and the mixture was transferred to a Soxhlet thimble and extracted with acetone in a Soxhlet apparatus for 12 hours. Solvents were removed from the extract in a rotary vacuum evaporator at a temperature not exceeding 40 c. The residue in the evaporator flask was transferred to a beaker with ml of hexane, the flask was washed with two 20 ml portions of hexane, and the washings were added to the beaker. The extract was dried over anhydrous sodium sulphate and the hexane solution decanted through Whatman No. 1 filter paper into a 0 ml measuring cylinder. The sodium sulphate remaining in the beaker was washed several times with hexane and filtered washings were added to the solution in the measuring cylinder to make the volume to 80 ml. Twenty ml of hexane extract (equivalent to 5 g of tissue) was purified by passage through a 12.5 g Davidow column (Davidow 1950). The column was eluted with 0 ml of hexane and the hexane solution was collected and evaporated in a beaker under a stream of air on a water bath to a volume of about ml; the final volume was adjusted to give a suitable concentration of insecticides for injection into the gas chromatograph. The gas chromatograph used was a Pye "Panchromatograph" fitted with an electron capture detector. A 1.5 m X 4-mm-diameter glass column packed with 3% SE-30 on 0/120 mesh "Gaschrom Z" was used to separate the organochlorine compounds in the extracts. Quantitative measurement of the insecticide residues was made by comparison of peak heights obtained from samples with peak heights from standard solutions. The method of analysis was tested at intervals throughout the programme, using the reagents alone. The following recoveries of pp'-dde, pp'-ddd, pp'-ddt (corrected for insecticide present before fortification) were obtained from fortified tissue samples analysed by the above method: Female gonad (1.0 ppm added) Male gonad (0.1 ppm added) Muscle (0.1 ppm added) pp'-dde pp'-ddd pp'-ddt % % % The limit of quantitative measurement of pp'-dde, pp'-ddd, and pp'-ddt was ppm. The presence of pp'-dde, pp'-ddd, and pp'-ddt in tissue samples from the fish was confirmed by paper chromatography with the methods of Mitchell (1956).

6 224 N.Z. JOURNAL OF MARINE & FRESHWATER RESEARCH [JUNE In the rest of this paper the term "pp'" is omitted in reference to the metabolites pp'-ddd and pp'-dde, but is retained where reference is specifically to the unmetabolised insecticide. Where appropriate the sum of results for all these compounds is expressed as "total DDT" in the tables. In the text the term "DDT" refers to total DDT. All results in this paper are expressed as parts per million (ppm) by weight on a fresh tissue basis. RESULTS The 20 trout taken from each lake before the 1966 breeding season commenced were assayed for DDT in muscle tissue and gonad. The mean levels of DDT and its metabolites are shown in Table 3, as well as the sums of the metabolites and unmetabolised pp'-ddt expressed as total DDT. The assumption is that much of the DDE and DDD present arise as breakdown products in the fish (Burdick et al. 1964). However, some DDE and DDD is likely to be taken up as such from the external environment (Jones and Moyle 1963); DDE may be present even in the DDT formulations used for topdressing (Hopkins, Brewerton, and McGrath 1966). Total DDT readings may therefore be slightly higher than the original levels of DDT in the fish. According to Holden (1962), the greatest quantities of recoverable DDT are found in muscle, which should therefore be the most suitable tissue for DDT assessment. On this basis the quantity of DDT in the fish samples is fairly low. However, DDT is concentrated to different degrees in different organs, depending mainly on the lipid content of each organ (Holden 1962). Our results indicate that considerably more TABLE 3 Mean values of DDT and metabolites (ppm) in muscle and gonads of prebreeding season trout (April 1966) Sex Number offish Muscle tissue DDE DDD pp'ddt DDE Gonad Total DDT DDD pp'ddt Muscle Gonad Immature; 8 Males 111 Females Immature 2 Males 9 Females 9 Immature: 0 Males Females o.6i<o.of< o.6i << CO o'.oi Immature: Males 113 Females Immature 13 Males 6 Females o'.oi 0. o'.o o'.oi 0. o'.o o'.oi '

7 1969] HOPKINS, SOLLY, & RITCHIE DDT IN TROUT 225 DDT is stored in the ovaries than in muscle or testes. Premdas and Anderson (1963) also noted this; Anderson and Everhart (1966) found that the gonads of the female hold more DDT than do those of the male, because of the higher lipid content in the ovaries. Higher levels of DDT in both muscle and gonads are recorded for the three lakes situated in agricultural land, of which the highest levels occur in fish from. This result was not unexpected, because pastures round had received greater quantities of DDT per acre than any land in the other lake basins. The ovaries give a somewhat higher DDT assay during the breeding season than earlier in the year. This can be correlated with the increased lipid content of the mature ova. Table 4 shows the results of DDT assay of the females taken in October and also of the resultant fry. For parent fish the differences in DDT levels between s,, and are not significant at P =, but the levels in s and fish are significantly higher than in those from the other three lakes. Among fry, only those from parents had DDT levels significantly different from levels found in the other fry. Table 5 lists the mean mortality of eggs and fry reared in the hatchery. In reference to the eggs, the term mortality also includes infertility. Mortality among both eggs and fry is highly variable. Standard deviations for egg mortality values varied between 44 and 118% of the means, and, for fry mortality values, between 31 and 120% of the means. Nevertheless, analyses of variance showed that though the differences between egg mortalities for the first four lakes tabled cannot be treated as significant, the mortality found among eggs is significantly higher than in the other lakes. Testing by t for the difference between the means of eggs on the one hand and those of the remaining lakes on the other showed a significant difference at P = (t = 5.34, v = 23). There was no significant differences in fry mortality between lakes even at P =.05. During rearing, the hatchery manager reported that the fry had hatched early and looked appreciably smaller than usual. Measurements of fry were therefore made at the end of rearing; unfortunately, before these were done the fry had already been frozen and measurements are therefore available for only four lakes. Table 6 shows the period in days of egg development before hatching, the mean lengths, and mean weights (calculated from standard errors of the means) of the fry at the end of rearing. The figures clearly indicate that the fry were relatively small. DISCUSSION The results indicate that the losses among eggs from parents are clearly greater than those in eggs from the other lakes. The levels of DDT occurring in fish are also

8 TABLE 4 Mean values of DDT and metabolites (ppm) in parent female trout taken in October 1966 and in their fry Muscle tissue DDE DDD pp'ddt DDE Ovary DDD pp'ddt DDE Fry DDD pp'ddt Total DDT Muscle Ovary Fry is o a, Z o T1 z w TABLE 5 Mortality of eggs and fry per batch and total survival. Five batches came from each lake, each batch initially containing 1,000 eggs w en Egg mortality Mean Standard deviation S 141 Fry mortality Mean Standard deviation Survival w cc w c Z a

9 1969] HOPKINS, SOLLY, & RITCHIE DDT IN TROUT 227 TABLE 6 Commencing date of hatching and range of mean lengths and weights of fry at time of yolk-sac resorption Date of hatch (days after fertilisation) Mean fry lengths (mm) Mean fry weights (g) much higher than among fish from other sources. The question of whether there is any link between these two circumstances therefore arises. Anderson and Everhart (1966) discussed the possibility that the reproductive potential of fish may be affected by high concentrations of DDT in the ovaries. The work of Burdick et al. (1964) showed how the insecticide causes mortality in fry; although their results showed much higher levels of DDT in the eggs than we found, they also showed a much higher mortality. The syndrome reported in fry by Burdick et al. was not seen in the fry at Ngongotaha. Indeed, statistically significant differences in mortality can be demonstrated for eggs alone, not for fry; the large losses in the brood were mostly at the egg stage. We have found no reference in the literature to mortality or infertility in fish eggs blamed on DDT; Burdick et al. make no mention of egg losses. However, the viability of birds' eggs can be affected by DDT (Mitchell, Blagbrough, and Van Etten 1953; Cramp, Conder, and Ash 1964), so a similar effect in fish eggs is possible. King (1962) reported that DDT caused abnormalities in the growth of newly born guppies (Lebistes reticulatus) and possibly affected the rate of growth. If the relatively stunted growth of fry is caused by the presence of DDT, there may also be effects on other growth processes, perhaps in the embryonic stages of cell cleavage and organogenesis. No effect on the fertility of the eggs has been demonstrated in these trials. The term "mortality" has been used to denote both true mortality of the embryo and infertility. The hatchery manager noted, however, that many of the eggs which did not hatch had begun development and reached the "eyed" stage. Thus, only part of the losses of eggs were due to original infertility. Possibly the early hatch of fry was related to their DDT content, because pp'-ddt (but not DDE) enhances protein biosynthesis in rat liver (Sanchez 1967) and in nymphal forms of the hemipteran Triatoma infestans (Agosin, Aravena, and Neghme 1965). The order in which the eggs hatched is the same as the decreasing order of p/ / -DDT content of the parents' ovaries (Table 4 and Table 6 compared).

10 228 N.Z. JOURNAL OF MARINE & FRESHWATER RESEARCH [JUNE In conclusion, a possible link between DDT in the egg and failure to develop normally is demonstrated. More work is needed, using larger numbers of fish from sources highly contaminated by DDT, for this to be definitely established or disproved. REFERENCES AGOSIN, M., ARAVENA, L., and NEGHME, A. 1965: Enhanced protein synthesis in Triatoma infestans treated with DDT. Expl Parasit. 16: ANDERSON, R. B. and EVERHART, W. H. 1966: Concentrations of DDT in landlocked salmon (Salmo salar) at Sebago, Maine. Trans. Am. Fish. Soc. 95: BRIDGES, W. R., KALLMAN, B. J., and ANDREWS, A. K. 1963: Persistence of DDT and its metabolites in a farm pond. Ibid. 92: BURDICK, G. E., HARRIS, E. J., DEAN, H. J., WALKER, T. M., SKEA, J., and COLBY, D. 1964: The accumulation of DDT in lake trout and the effect on reproduction. Ibid. 93: COPE, O. B. 1961: Effects of DDT spraying for spruce budworm on fish in the Yellowstone River system. Ibid. 90: CRAMP, S., CONDER, P. J., and ASH, J. S. 1964: "The Risks to Bird Life from Chlorinated Hydrocarbon Pesticides, September 1962-July 1963." The Royal Society for Protection of Birds, London. 24 pp. DAVIDOW, B. 1950: Isolation of DDT from fats. J. Ass. off. agric. Chem. 33: EIDE, P. M., DEONIER, C. C., and BURRELL, R. W. 1945: The toxicity of DDT to certain forms of aquatic life. J. econ. Ent. 38: EVERHART, W. H. and HASSLER, W. W. 1948: Aquarium studies on the toxicity of DDT to brown trout, Salmo trutta. Trans Am. Fish. Soc. 75: GRAHAM, R. J. and SCOTT, D. O. 1959: "Effects of an aerial application of DDT on fish and aquatic insects in Montana." Final Report. U.S. Forest Service, Missoula, Montana. 35 pp. HOFFMAN, C. H. and SURBER, E. W. 1948: Effects of an aerial application of wettable DDT on fish and fish-food organisms in Back Creek, West Virginia. Trans. Am. Fish. Soc. 75: : Effects of an aerial application of DDT on fish and fish-food organisms in two Pennsylvania watersheds. Progve Fish Cult. 11: HOLDEN, A. V. 1962: A study of the absorption of 14 C-labelled DDT from water by fish. Ann. appl. Biol. 50: HOPKINS, C. L., BREWERTON, H. V., and MCGRATH, H. J. W. 1966: The effect on a stream fauna of an aerial application of DDT prills to pasture land. N.Z. Jl Sci. 9:

11 1969] HOPKINS, SOLLY, & RITCHIE DDT IN TROUT 229 JONES, B. R. and MOYLE, J. B. 1963: Populations of plankton animals and residual chlorinated hydrocarbons in soils of six Minnesota ponds treated for control of mosquito larvae. Trans. Am. Fish. Soc. 92: KING, S. F. 1962: Some effects of DDT on the guppy and the brown trout. Spec, scient. Rep. U.S. Fish Wildl. Serv., Fisheries No pp. MITCHELL, L. C. 1956: Separation and identification of chlorinated organic pesticides by paper chromatography. VIII. Technical DDT, P,P'-DDT, DDA, DDD, DDE, 4,4'-Dichlorobenzophenone, and 2,4'-Dichlorobenzophenone. J. Ass. off. agric. Chem. 39: MITCHELL, R. T., BLAGBROUCH, H. P., and VAN ETTEN, R. C. 1953: The effects of DDT upon the survival and growth of nestling songbirds. J. Wildl. Mgmt 17: PREMDAS, F. H. and ANDERSON, J. M. 1963: The uptake and detoxification of C 14 -labelled DDT in Atlantic salmon. J. Fish. Res. Bd Can. 20: SANCHEZ, E. 1967: DDT-induced metabolic changes in the rat liver. Can. Jl Biochem. 45: WARNER, K. and FENDERSON, O. C. 1962: Effects of DDT spraying for forest insects on Maine trout streams. J. Wildl. Mgmt 26: