NOTES AND COMMENT CHANGES IN INORGANIC PHOSPHATECONCENTRATION OCCURRING DURING SEAWATER SAMPLE STORAGE' The measurement of dissolved inorganic phosphate is an integral part of many oceanographic research programs, but it is only practical to perform the analyses at sea on larger research vessels. The storage of samples for later analysis is often required. Unfortunately, if samples are stored without treatment, marked changes in the level of inorganic phosphate occur. These changes may be increases arising from the bacterial or enzymatic decomposition of organic phosphorus or decreases from utilization by growing bacteria and plankton or by adsorption on detritus or sample bottle walls, or both. The problem of sample storage for phosphate determinations is therefore of importance. Various solutions to this problem have been recommended (for example, Ibanez Gomez 1933; Harvey 1948; Heron 1962). Currently, the two most commonly used techniques utilize polyethylene storage bottles, with chloroform treatment for short-term storage (Harvey 1955) and quick-freezing for long-term storage (Collier and Marvin 1953). However, the effectiveness of even these methods has been questioned (Murphy and Riley 1956; Hassenteufel, Jagitsch, and Koczy 1963; Jones 1963 ) . In the initial stages of a survey of estuarine waters of Ecuador, it was noted that preservation by quick-freezing was providing erratic results, and it was hypothesized that significant changes were taking place during the freezing and thawing processes. Experiments were conducted to establish a method of preservation suitable for samples of waters characterized by high phosphate levels, dense bacterial and plankton population, and heavy particulate organic l This research was partially supported by the Inter-American Tropical Tuna Commission, Scripps Institution of Oceanography, La Jolla, California. detrital loads. The experiments were designed to test whether the addition of chloroform to the sample immediately after collecting and before freezing: a) would stabilize the samples between thawing and analysis, b ) would act as a safety factor should the freezing be delayed or should the samples accidentally thaw before analysis, and c) would not significantly increase the concentration of inorganic phosphate. EXPERIMENTAL TECHNIQUE The seawater used for all experiments was collected from the Ester0 Salado of the Golfo de Guayaquil, Ecuador. The water characteristics of the samples at the time of collection were: 24.4-28.2C, 27%32.9a/co S, and 4.12-6.58 pg-at. PO,-P/liter. Samples were transported (within 20 min) to the laboratory in 5-gal ( 19-liter ) polyethylene carboys and transferred to a lo-gal (38liter) Pyrex carboy where they were kept gently agitated by a magnetic stirrer. The drawing of loo-ml subsamples for the various experiments was started within 45 min of collection. Subsamples were usually placed in 4-0~ ( 112 ml) polyethylene screwtop bottles and, depending upon experimental design, were either quick-frozen in an ethyl alcohol-Dry Ice bath or maintained at room temperature. The frozen samples were stored at -5 to -lOC until thawed for analysis. Subsamples incubated at room temperatures were exposed to ambient temperatures between 18.2 and 21.3C. Chloroform was added to the subsamples with a volumetric syringe and Whatman No. 1 filter paper was used in those cases where experimental design required filtration. All analyses were conducted using a Beckman model DU spectrophotometer following the method presented by Strickland and Parsons ( 1960). Statistical tests for significance were made at the ;p = 0.05 level. 325 326 NOTES AND COMMENT Seven experiments were conducted and 845 samples were analyzed. Depending upon the expected variability, three to five samples made up a subset. The complete data, including statistical treatment and a detailed description of each experiment, are available from the author. Only the most pertinent results are discussed here. Seawater is saturated with chloroform at approximately 0.7% v/v. The addition of this quantity of chloroform effectively inhibits changes in the phosphate concentration of unfrozen seawater samples for up to 6 hr, even under the extreme conditions represented by the Gulf of Guayaquil samples (Fig. 1). This experiment was based on 125 subsamples, with the first subset receiving no treatment, the second a 0.4% v/v treatment, etc. The level of phosphate within the carboy (represented by the “zero time” values) also decreased with time similar to the subsamples receiving no treatment. A sample of seawater may be likened to a loosely organized but actively metabolizing system in which bacterial and planktonic growth, death, and regeneration and various extracellular enzymatic and absorptive processes bring about a series of simultaneous increases and decreases in the phosphate content of the seawater samples. The complex cycle of changes that may develop from the interactions of system components is well illustrated by one of the experiments where both frozen and unfrozen samples showed a decrease for the first 3 hr, followed by a significant increase before entering the characteristic longer-term decrease in phosphate concentration ( Fig. 2). The analyses of samples representing the unfrozen and frozen subsets were 25 hr out of phase, indicating that the cycle of change was not an artifact introduced by the experimental technique. It further indicates that the cycle of change is characteristic of a particular system and does not change even when the system is frozen, thereby delaying the development of the cycle for more than 24 hr. FIG. 2. A comparison between the variation in phosphate concentrations of unfrozen and frozen/ thawed seawater samples receiving no chloroform treatment. FIG. 3. A comparison between the variation in phosphate concentration of unfrozen and frozen/ thawed a prefreezing __ ^ seawater samples receiving chloroform treatment, c 6CtiC : 90I FIG. 1. A comparison between the variation in uhosohate concentration of unfrozen seawater samples deceiving chloroform treatments (in % v/v). The initial concentrations varied from 4.41 to 5.28 pug-at/liter. RESULTS AND DISCUSSION NOTES TABLE 1. AND 327 COMMENT A comparison of the inorganic phosphate concentration water treated with 0.770 v/v chloroform. Values of unfroxen and frozen/thawed in pg-at. P04-P/liter Unfrozen Frozen -CHCL, Time interval Initial + 35 + 70 +205 +405 +14.5 + 27 + 48 + 72 + 96 min min min min hr hr hr hr hr +CHCL, -CHCL, SD Mean SD Mean SD Mean 5.174 5.265 5.194 5.104 5.046 5.098 5.043 3.032 3.168 2.791 0.118 0.059 0.090 0.040 0.025 0.071 0.159 0.599 0.459 0.625 5.229 5.276 5.288 5.192 5.238 5.124 5.009 4.774 5.239 5.376 0.024 0.063 0.027 0.048 0.088 0.068 0.182 0.304 0.196 0.115 5.147 5.005 5.009 4.882 4.921 4.672 3.480 2.668 2.051 2.068 0.032 0.134 0.091 0.140 0.045 0.024 0.245 0.242 0.925 0.179 5.198 5.194 5.167 5.167 5.083 5.236 5.344 5.299 5.269 5.242 Total number of samples: Subsamples per treatment As a working hypothesis, it can be assumed that the two predominant system components are the increase on phosphate concentration resulting from the enzymatic decomposition of organic material and the decrease resulting from phosphate utilization by a developing bacterial population, These experiments suggest that the enzymatic process often predominates during the initial 3-hr period (Table 1 ), and thereafter an exponentially developing bacterial population commences to utilize phosphorus in excess of that released by the enzymatic process, resulting in a net decrease in phosphate concentration. Theoretically, it would be desirable to remove the particulate organic material that acts as substrate for many of the processes, either by filtering or centrifuging the samples, a concept supported by the work of Heron ( 1962). In a practical sense the time delay introduced by such set: min SD 0.071 0.043 0.075 0.080 0.122 0.095 0.195 0.056 0.107 0.053 160 4 sample treatment could allow a larger change to take place than if the entire system were quickly deactivated by, for example, quick-freezing. Unfortunately, freezing can increase the rate at which the phosphate concentrations decrease ( Fig. 2). Such an increase in net change could result from the disruption of organic material in the sample, thereby providing increased substrata for bacterial growth or adsorptive processes or both. However, since the decrease can be minimized by the chloroform treatment ( Fig. 3) it seems reasonable to assign it to the bacterial component. When samples were treated with 1.2% v/v chloroform, a significant difference was often, but not always, noted between the filtered and unfiltered samples; this indicates that leaching can take place under certain circumstances (Fig. 1 and Table 2). Of additional interest, the data suggest that values, of inorganic phosphate concentrations in filtered 1.2yS v/v chloroform treatment. Values in pg-at./liter Time +15 Treatment Unfiltered Filtered Difference Standard error of the difference +CHCL, Mean TABLE 2. A comparison, with initial unfiltered seawater, receiving sea- +45 min +90 and interval min +180 min +24 hr -0.009 +0.012 0.021 -0.012 -0.074 0.062 +0.011 -0.074 0.085 +0.046 -0.044 0.090 +O.lOS -0.091 0.199 0.012 0.019 0.021 0.028 0.016 328 NOTES ANDCOMMENT samples treated with chloroform appreciably in excess of saturation exhibit a much higher variability ( coefficient of variation at 1.2% v/v = 0.344) than samples receiving chloroform treatment at or near saturation (coefficient of variation at 0.7% v/v = 0.283), thus emphasizing the need for care during the addition of chloroform for treatment of phosphate samples. Perhaps the most important aspect of these experiments is to emphasize that the rate of change of phosphate level with time is not constant but depends upon the biological and physical characteristics of the sample. It can be expected to vary from region to region, from season to season, and it is doubtful whether there is any one sample treatment that will fulfill the requirements of all experimental designs. CONCLUSIONS a. The low levels of chloroform occasionally recommended to stabilize samples (as low as 0.05% v/v) are often insufficient to achieve stabilization in highly productive regions. The optimum chloroform treatment is slightly less (0.7% v/v) than that required to saturate the sample. b. If the samples are quick-frozen without chloroform treatment, it is imperative that they be analyzed immediately upon thawing. The process of freezing and thawing can bring about a marked increase in the rate of change of inorganic phosphate level. c. The addition of chloroform at levels near saturation (O.&OS% v/v) does not significantly increase the level of inorganic phosphate. d. The rate of change of inorganic phosphate in quick-frozen samples treated with AN ICE RECORD OF Wind-induced waves are reflected in the University of Wisconsin Laboratory of Limnology boat slip to produce a standing wave that crests approximately 3 m from the boat slip door which acts as a reflector. Ice is built up along the side of the slip by chloroform is significantly less than that in frozen or unfrozen samples without chloroform. e. The addition of chloroform to samples before freezing tends to stabilize the samples during the time between thawing and analvsis. , MALVERNGILMARTIN Hopkins Marine Station, Stanford University, Pacific Grove, California 93950. REFERENCES COLLIER, A. W., AND K. T. MARVIN. 1953. Stabilization of the phosphate ratio of sea-water by freezing. Bull. U.S. Bur. Fisheries, 79: 71-76. 1948. The estimation of phosHARVEY, H. W. phate and total phosphorus in sea waters. J. Marine Biol. Assoc. U.K., 27: 337-359. HARVEY, J. W. 1955. The chemistry and fertility of sea waters. Cambridge, London, England. 224 p. HASSENTEUFEL, W., R. JAGITSCH, AND F. F. KOCZY. 1963. Impregnation of glass surface against traces. Limnol. sorption of phosphate Oceanog., 8: 152-156. HERON, J. 1962. Determination of phosphate in water after storage in polyethylene. Limnol. Oceanog., 7: 316-321. IBANEZ GOMEZ, 0. 1933. Note on the effect of salts in the determination of phosphates in sea-water by Deniges method. J. Conseil, Conseil Perm. Intern. Exploration Mer, 8: 326-329. JONES, P. G. W. 1963. The effect of chloroform on the soluble inorganic phosphate content of unfiltered sea-water. J. Conseil, Conseil Perm. Intern. Exploration Mer, 28: 3-7. MURPHY, J., AND J. P. RILEY. 1956. The storage of sea-water samples for the determination of dissolved inorganic phosphates. Anal. Chim. Acta, 14: 818-819. STRICKLAND, J. D. H., AND T. R. PARSONS. 1960. A manual of sea water analysis. Bull. Fisheries Res. Board Can. 125. 185 p. STANDING WAVES splashing water and is undercut by wave action. The ice, therefore, serves as a record of wave height. The photographs were taken at about 1200 CST on 4 December 1964. The maximum diameter of pancake ice shown in