Water Research Pergamon Press 1973. Vol. 7, pp. 1781-1790. Printed in Great Britain

T E M P E R A T U R E A N D T H E T O X I C I T Y O F C H R O M A T E

A N D A R S E N A T E T O T H E R O T I F E R , P H I L O D I N A

R O S E O L A

EDWARD D. SCHAEFER and WESLEY O. PIPES

Northwestern University, Evanston, Illinois 60201, U.S.A.

(Received 28 January 1973)

1. I N T R O D U C T I O N

DURING the past quarter century, there have been a number of attempts to use TErn values to predict "safe" concentrations at which the potentially toxic material would have no effect upon the aquatic organisms which are to be protected. The "safe" concentration is usually taken as an application factor (such as 0.1 or 0.03) times the 96-h TLm and is often converted into a water quality standard or an effluent limitation. Recently, questions as to whether the application factor should be changed for dif- ferent receiving water temperatures have arisen at public hearings concerned with water quality and effluent regulations.

There is general acceptance of the concept that at higher temperatures the TL m values will usually be lower. MACEK, HUTCmNSON and COPE (1969) found that an increase in temperature produced a decreased survival time of blue gill sunfish and rainbow trout in the presence of toxic concentrations of several different pesticides but there are few published data on the specific effect of temperature upon TL m values for toxic materials.

In this study the interaction of three different variables; time, temperature and TLm was investigated. The objective of the study was to determine the form of the time, temperature and toxicity relationships in order to provide a better basis for predicting "safe" concentrations from short term bioassay data and to illustrate the effect of temperature upon both short term toxicity and "safe" concentrations. The hypotheses formulated were:

(1) For short term exposure aquatic organisms can withstand higher concentrations of toxic materials at lower temperatures than at higher temperatures;

(2) The longer the exposure time the less the effect of temperature on the concentra- tion at which half of the test organisms are killed; and

(3) Short term bioassay data can be used to predict the concentration of the toxic material which will not change the median life span of the test organism.

2. MATERIALS AND METHODS

2.1 Toxic materials

Chromate and arsenate were used because they are common waste components and because their concentrations persist long enough for static bioassay procedures. The toxic solutions were sodium chromate (Na2CrO4) and sodium arsenate (NaHAsO4" 7 H20). Stock solutions were made up to give chromate ion concentrations of 5, 10, 20 and 40 mg 1-1 as Cr and arsenate ion concentrations of 4, 8, I0, 32 and 64 mg 1-1

1781

1782 EDWARD D. SCHAEFER and WESLEY O. PIPES

as As. From these stock solutions an aliquot was taken for each experiment. The same nutrients were present in all the test solutions. The concentrations of Cr +6 or As + 5 in each culture was determined by atomic absorption spectrophotometry, as described by SLAVIN (1968), at the beginning and at the end of each bioassay.

2.2 Test organism

The test organism used was the rotifer, Philodina roseola. It is a common aquatic species in the U.S.A., it has been shown to be excellent fish food (GoNZALES, 1945), and it has been used previously in studies of the environmental effects of toxic materials (ALLEE and ROSENTHAL, 1949). It was cultured in the laboratory on a lettuce infusion medium which was prepared by boiling 1.5 g of dried lettuce in a liter of distilled water, filtering off the solution, saturating with calcium carbonate, and autoclaving. The original P. roseola culture was obtained by placing a teaspoon of sand from Lake Michigan in a lettuce infusion culture and allowing it to incubate at 20°C for 6 weeks. After the P. roseola from the original culture had been washed free of protozoa and other invertebrates it was maintained in stock cultures by transfers to fresh media every 2 weeks.

2.3 Preliminary studies

Before the toxicity studies could be initiated it was necessary to determine some of the growth characteristics of P. roseola under non-toxic conditions. The growth characteristics which were measured were maximum survival temperature, thermal death times, rates of increase at different temperatures and median life span at different temperatures.

The maximum temperature and the thermal death times for P. roseola were de- termined by placing 10 ml aliquots of the culture medium in water baths at 55, 50, 45, 40 and 38°C. When the culture medium had reached the temperature of the baths, 10 rotifers from cultures held at 20°C were placed in each culture and were observed until they were dead. Cessation of motion was taken as the sign of death. The results of these experiments are presented in TABLE 1. P. roseola reproduced at 38°C but all specimens died in less than 2 days at 40°C without reproducing. Therefore, the tern-

TABLE 1. SURVIVAL TIMES OF Philodina roseola AT ELEVATED TEMPERATURES

Temperature Time required (°C) for 100K kill

55 2 min 50 9 min 45 2 h 40 2 days 38 > 3 days

perature range for the toxicity bioassays was selected at 5-35°(2 in order to be well within the normal tolerance range for the organism.

The experiments to determine the rates of increase and the median life span at various temperatures were conducted simultaneously. Forty petri dishes each with

The Toxicity of Chromate and Arsenate 1783

40 ml of the lettuce infusion were used for each test temperature (5, 15, 20, 25, 30 and 35°C). One egg of P. roseola was placed in each dish and observed daily until hatching. Thereafter the cultures were observed every other day and the number of rotifers in each culture was recorded.

The population size at each temperature was plotted versus time on semi-log graph paper for each culture. The points plotted were approximately linear, indicating that the population growth fitted the equation

In Nt = rt, (1) No

where No is the initial population, Nt is the population at time t, and r is the rate of increase. The slope of the plotted line was taken as the rate of increase, r. Several estimates of r from the several cultures at each temperature were made and the mean rate of increase and the standard deviation of the rates of increase were calculated. The mean doubling time, d, for each temperature was calculated from

In 2 r

The older rotifers were larger and darker than their offspring and could be identi- fied separately, The day of mortality of the rotifer produced from the original egg in each culture was recorded. After at least 80 per cent mortality of the original rotifers in the cultures at each temperature, the median life span was calculated. The method of LITCHFIELD and WILCOXON (1949) was used to calculate the 95 per cent confidence limit for the median life span at each temperature.

2.4 Short term bioassays

One hundred and twenty rotifers were used at each concentration for each bioassay test. The test organisms were selected at random from the stock cultures and placed, 40 per culture, in three cultures. The cultures were placed in incubators held at the desired temperature. The maximum temperature variation in any of the incubators during the testing was d:0.5°C. The cultures were observed daily and percentage of mortality was recorded. The method of LITCHFIELD and WILCOXON (1949) was used to estimate the TL, and to calculate the 95 per cent confidence interval for the WLm.

2.5 Life span bioassays

The life span bioassays were conducted in the same manner as the short term bioassays except that they were started with eggs instead of adults. For each tempera- ture and concentration tested, 40 eggs were placed in each of three cultures. After one day the hatching success was about 50 per cent in each culture. The unhatched eggs were removed and the desired concentration of either As 5 + or Cr 6 + added. Reproduc- tion occurred in these cultures but the original organisms could be visually distin- guished from their offspring. The numbers of the original organisms remaining alive were counted each day until less than 25 per cent were still alive. At the end of each life span bioassay the concentration of the toxicant in each culture was checked to make sure that it had not decreased.

1 7 8 4 E D W A R D D . SCHAEFER a n d WESLEY O , PIPES

3. RESULTS

3.1 Growth characteristics of Philodina roseola

Of the 40 eggs which were placed in separate cultures at each of five temperatures, only about 50 per cent hatched, but it appeared that temperature had no influence on hatching success. Hatching success was 31/40 at 5°C, 27/40 at 15°C, 17/40 at 20°C, 20/40 at 25°C, 18/40 at 30°C and 20/40 at 35°C. The rotifers which hatched from the eggs at 5°C all remained alive for more than 60 days but none of them reproduced during this time.

The mean rates of increase for 15, 20, 25 and 35°C are plotted on a semi-log plot in FIG. 1 with the 95 per cent confidence interval for the mean rate of increase at each temperature indicated on the graph. As expected the rate of increase for P. roseola

I ' 0

0 8

O'G

0 4

0 2

T

"o O I

- 0 0 8

0 ' 0 6

"6 0.04

g:

0"02

O-01

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1 I I I I I IO 15 20 25 30 35

T e m p e r a t u r e , *C

FiG. 1.

40

populations was found to be an exponential function of temperature. The mean doubling times which were calculated for the various temperatures were 5.1 days at 15°C, 4.2 days at 20°C, 3.1 days at 25°C and 2.0 days at 35°C. The median life span of P. roseola was determined at 15, 20, 30 and 350C. These data are presented in TABLE 2. The decrease in median life span is approximately exponential with increasing temperature.

3.2 Short term bioassay

The results of these bioassays are presented in TABLE 3. The 96-h TL~ values at 30°C and the 72-h zt~ values at 35°C are questionable because the test period is within the confidence interval for the median life span of the test organism and these

The Toxicity of Chromate and Arsenate 1785

values just indicate mortality which is within the normal range for P. roseola in non- toxic media. These data do show that Tim values are lower (greater toxicity) at higher temperatures and that the differences between the TI m values at different temperatures decrease with increasing exposure time. For example, the difference between the TI m values for Cr 6+ at 15 and 35°C at 1 day is 25 nag 1-1 but at 4 days it is 4.9 rag 1-1

TABLE 2. LIFE SPAN DATA FOR Philodina roseola AT DIFFERENT TEMPERATURES

Temperature (°c)

95 ~o Confidence limits Mean for median life span

doubling Median Base time life span Lower limit Upper limit

population (days) (days) (days) (days)

5 31 > 60.0 15 27 5.1 28.0 25.5 30.5 20 17 4.2 10.2 8.9 11.6 25 20 3.1 5.9 5.4 6.5 30 18 3.7 3.1 4.5 35 20 2.0 3.0 2.6 3.4

T~1.~ 3. ~.~ VALUES Fog CHROMATE AND ARSENATE

Time (11)

Temperature (°c)

Chromate as Cr (rag 1-1) 95 ~o contidenc¢ limits

Arsenate as As (rag l - t ) 95 Yo cordidenca limits

lower upper TL~ lower upper

24

48

72

96

5 65 50 84 150 104 215 15 43 39 52 95 71 130 20 37 30 45 84 64 110 25 28 23 33 62 47 82 30 23 19 28 56 42 75 35 18 15 22 40 29 57

5 31 27 36 78 58 105 15 22 19 26 57 42 77 20 18 15 21 44 34 57 25 14 12 17 32 26 39 30 11 9.2 13 26 19 35 35 9.1 7.4 11 21 16 29

5 16 14 19 29 23 37 15 12 lO 14 26 20 32 20 11 9.1 13 21 17 25 25 7.0 5.7 8.7 16 13 20 30 6.4 5.2 7.9 11 9.0 14 35 5.3 4.5 6.3 5 3.9 6.4

5 12 10 14 18 15 22 15 8.9 7.4 12 16 13 19 20 7.4 6.2 9.1 13 11 16 25 5.5 4.3 7.0 8.2 6.5 10 30 4.4 3.5 5.6 6.6 5.4 8.1

1786 EDWARD D. SCHAEFER and WESLEY O. PIPES

The difference between the TL m values for As 5+ at 15 and 35°C at 1 day is 55.5 mg 1-! but at 4 days it is 11.2 mg 1 - t . These data support the first two hypotheses stated in the Introduction to this paper.

3.3 Prediction of"safe" concentrations

In this study "safe" concentration was defined as the concentration which had no effect on the median life span of the organism. No effect on the median life span of the test organism does not necessarily mean no physiological effect on the organisms but it is one simple method of defining a "safe" concentration. In order to predict the "safe" concentration, it was decided to try to fit the data to a line described by the equation

a t-day T L m - - (2)

t

where t is exposure time in days and a is a constant to be determined empirically. The TI.m concentrations for Cr 6 + and As 5 + are plotted vs the reciprocal o f time in

FIGS. 2 and 3. From these graphs it can be seen that the data points lie close to a series of straight lines. The lines plotted were fitted to the data by a least-squares regression.

7 0

Legend o / 5°C • Originol biocssoy /

60 -- x Predicted life span TL m / • Measured life s p a n T L m /

5O / , / 15 °c

20°C

30 - • 25°C

I 0 - -

, 0 : 2 5 0 "5 0 ' 7 5 I 0

I / t , day -E

FIO. 2.

The values of the slope a of the prediction equation and confidence interval estimates for a were calculated according to the procedures outlined in NXrRELLA (1963). The values of a calculated for pentavalent arsenic were 96.3 mg 1-1 at 15°C, 82.8 mg I -a at 20°C, 59.8 mg 1-1 at 25°C, 52.2 mg 1-1 at 30°C and 38.1 mg 1-1 at 35°C. The values for hexavalent chromium were 42.3 mg 1-1 at 15°C, 36.1 mg 1-1 at 20°C, 27.1 mg1-1 at 25°C, 22.1 mg 1-1 at 30°C and 17.7 mg 1-1 at 35°C. In this equation, the value of the

The Toxicity of Chromate and Arsenate 1787

v_,

F

¢.

140

12C

80

6(;

40

20

Legend

• Original bioassay

× Predicted time spanrL _ • Measured time SDORTL_

~" I ~ r 0-25 0 "5 0 '75 I.o

I / t , day - I

FIG. 3.

• i=5*C

, I =150C

t=25"C

t=30*C

slope, a, in each ins tance is also the best s tat is t ical es t imate o f the 24 h TL m. In each ins tance the measured value o f the 24 h TLm was well wi th in the 95 per cent confidence in terva l fo r the ca lcula ted value o f a, ind ica t ing a good fit o f the equa t ion to the da ta . Cor re l a t ion coefficients for the fit o f the line to the da ta would also be h igh bu t would have no stat is t ical mean ing in this case because it is k n o w n tha t there is a func t iona l re la t ionsh ip between TLm and t ime.

TABLE 4. CoMPARISON OF PREDICTED AND MEASURED LIFE SPAN Tim VALUES

Toxic ion

Median Predicted Measured 95 ~0 Confidence limits Temperature life span Tr~ T ~ for measured TEn,

(°CT) (days) (mg I -I) (mg I -I) (rag I -i)

Cr • +

AsS+

15 28.0 1.5" 3.5 2.2-5.6 10.2 3.5 4.6 2.8~.7

25 5.9 4.6 4.5 3.1~.4 30 3.7 5.9 3.8 2.5-5.9 35 3.0 5.9* 3.7 2.7-5.0

15 28.0 3.4" 6.0 3.8-9.5 20 10.2 8.1 9.0 5.0-16 25 5.9 10.0 10 7.8-13 30 3.7 14.1 13 7.1-23 35 3.0 12.7 11 7.2-18

* Exceeds 95 ~o confidence limits.

1788 EDWARD D. SCHAEFER and WESLEY O. PIPES

3.4 Life span bioassays

The purpose of fitting the lines to the data plotted in FIGS. 2 and 3 was to predict the IL,, values for the median life spans at the various temperatures. The predictions were made by substituting the appropriate values of a and t for each toxicant and temperature into equation (2), and a new set of bioassays were run to measure the life span TLm values for C r 6 + and As 5 ÷ at the various temperatures. The results of the predictions and the measurements are presented in TABLE 4. For seven of the 10 predictions the predicted life span TLm was within the 95 per cent confidence interval for the measured life span TLm. The TLm values at end points of the temperature range, 15 and 35°C, showed the greatest deviations between the predicted values and the measured values. For the middle of the temperature range used, 20-30°C, there was close agreement between the predicted and measured values.

4. DISCUSSION 4.1 Growth characteristics

The results of the studies of the growth characteristics of P. roseola were in line with generally accepted theory. The thermal death point of this organism was found to be between 38 and 40°C but the very short median life span at 35°C indicates that this organism is under considerable thermal stress at this temperature even though it was able to reproduce. The lower end of the temperature range was not determined. Although no reproduction was observed in the cultures at 5°C before they were dis- carded after 60 days there is no assurance that they would not have reproduced had they been held longer. The exponential increase in the rate of increase and the exponen- tial decrease in median life span with increasing temperature were both expected.

4.2 Short term bioassay

The medium provided for the growth of P. roseola in these studies provided enough nutrients that no limitation of growth was observed at the population densities used. All the bioassays were run using growing cultures. Reproduction in the cultures used in this investigation was parthenogenetic and thus the generalization of these results to sexual organisms is open to question.

The results clearly show that for 1-, 2-, 3- and 4-day exposure times TLI values are much lower at higher temperatures for P. roseola living in a lettuce infusion medium. Since this result is in accord with results obtained by other investigators working with other organisms (WARREN, 1971) it is reasonable to generalize and state that these data support the hypothesis that in general for aquatic organisms toxic materials are more toxic at higher temperatures when the exposure time is relatively short. How- ever, the data also indicate that the effect of temperature on the TLm values of Cr 6 + and As 5 + for P. roseola becomes rather small as the exposure times are lengthened. Thus, for long term toxicity bioassay procedures the temperature selected for the test appears to be relatively less important than for short term tests.

4.3 Predictions from short term bioassays

One purpose of short term toxic bioassays using aquatic organisms is to determine "safe" or threshold concentrations of the toxic materials in question which can be

The Toxicity of Chromate and Arsenate 1789

tolerated in receiving waters. It is not the purpose of this paper to examine the philo- sophical basis for the concept of threshold concentrations but to present and evaluate one method of mechanically predicting such a concentration.

The prediction equation used in this study is a variation of the equation

TL m X t ~ constant

which is one of the five equations cited by WARREN (1971) as having been used for describing the relationship between time of exposure and concentration of toxic material. The results obtained for 25°C show a very close agreement between the predicted value and the measured value for the life span TErn. The results obtained at 20 and 25°C show the predicted values to be within the 95 per cent confidence intervals for the measured values but at 15 and 35°C three of the four predicted values were outside the 95 per cent confidence interval for the measured value.

The comparisons between the predicted and measured TEn values can be explained in terms of the expected results from an empirical extrapolation. The agreement between the predicted and measured values at 15°C is poor because the prediction was made for a 28-day median life span which is several times as long as the longest bioassay period (4 days) used to obtain the data which were used to make the predic- tion. The prediction of a TEn value for 10.2 days (20°C) was closer than the prediction for 28.0 days but not as good as the prediction for 5.9 days (25°C). The shorter the extrapolation the better the prediction as would be expected for any empirical predic- tion. There is no indication that the prediction equation has any theoretical meaning.

P. roseola is a hardy organism. ALLEE and ROSENTHAL (1949) have shown that it has a rather wide tolerance range for a variety of environmental factors. Thus, it would be expected that it would have a higher tolerance for toxic materials such as Cr 6 + and As 5+ than many other aquatic organisms. The measured "safe" concentrations of Cr e+ and As 5 + for P. roseola are expected to be higher than those for more sensitive aquatic organisms and do not represent "safe" levels for these organisms in the receiving waters. However, despite its lack of suitability for establishing criteria for receiving waters P. roseola is still a suitable organism for examining the form of time, temperature, and concentration relationships. The "safe" concentrations measured in this investigation were those which did not change the median life span of the test organism. The measured life span TErn values for the various temperatures are listed in column 5 of TABLE 4. The values listed indicate that as temperature is increased the measured life span TEn may either increase of decrease; that is, no general pattern is evident. However, for Cr 6 + the life span TErn values measured at the various tempera- tures range from 3.5-4.6 mg 1-1 but the 95 per cent confidence interval for the life span TL,~ at 20°C is 2.8-7.7 mg 1-1. The variation is the measured life span TLm values at all temperatures is less than the expected variation in the life span TErn measured at 20°C. The same observation holds for the As s + data, the range of measured life span TErn values being 6.0--13.0 mg 1-1 while the 95 per cent confidence interval for the life span "rLm measured at 20°C is 5.0-16.0 mg 1-1. The conclusion from these data is that temperature has no effect on the life span TLm concentrations.

5. CONCLUSIONS (1) In a lettuce infusion medium all specimens of Philodina roseola were killed in

less than 2 days at 40°C but some specimens lived long enough to reproduce at 38°C.

1790 EDWARD D. SCHAEFER and WESLEY O. PIPES

(2) The rate o f increase o f Philodina roseola increased exponentially with tempera- ture in the range, 15-35°C, but its median life span decreases exponentially with temperature in the same range.

(3) The data presented support the hypothesis that for short exposure times aquatic organisms can withstand higher concentrat ions o f toxic materials at lower tempera- tures than at higher temperatures.

(4) The data presented support the hypothesis that the longer the exposure time the less the effect o f temperature upon the concentrat ion o f toxic material which will kill half o f the test organisms.

(5) The data presented support the hypothesis that life span TLm values are inde- pendent o f temperature.

Acknowledgement--During the period when this investigation was undertaken Mr. SCHAEFER was supported in a traineeship from the U.S. Environmental Protection Agency.

R E F E R E N C E S ALLEE W. C. and ROSENTHAL G. M. (1949) Group survival value of Philodina roseola, a rotifer.

Ecology 30, 395-397. GONZALES J. L. 0945) Bugs you should know (for the aquarium), All-pets Magazine 16, (3) 38-39. LITCHnELD J. T. and WILCOXON F. (I949) A Simplified method for evaluating dose-effect experiments.

J. Pharmac. exp. Therap. 96, 99-113. MACEK K. J., HUTCHINSON C. and COPE O. B. (1969) The effect of temperature on the susceptibility

of bluegills and rainbow trout to selected pesticides. Bull. of environ. Contain. Toxic. 4, (3), 174-183.

NATRELLA M. G. (1966) Experimental Statistics, 485 pp. National Bureau of Statistics, U. S. Depart- ment of Commerce, Washington, D.C.

SLAVIN W. 0968) Atomic Absorption Spectroscopy, 307 pp. Interscience, New York. WARREN C. E. (1971) Biology and Water Pollution Control, 434 pp. Saunders, Philadelphia.