Arch. Environ. Contam. Toxicol. 13,403-409 (1984)

nvironmL~ranl Ceu'memirmtion 9 1984Springer-VerlagNew York Inc.

Copper Complexation and Toxicity to Freshwater Zooplankton U. Borgmann and K. M. Ralph Department of Fisheries and Oceans, Canada Centre for Inland Waters, Burlington, Ontario, Canada L7R 4A6

Abstract. The effect of copper on the growth rate of cyclopoid copepods and survival of rotifers was determined in natural water with and without addition of the complexing agent Tris. Free copper concentrations were estimated, both by cupric ion electrode and from the bioassay data, making use of the known complexing ability of Tris and the increase in total copper tolerated after Tris addition. Growth rates of copepods were directly related to free copper concentrations indicating that the copper-Tris complex was not toxic to these animals. Rotifer survival was similar at equivalent free copper concentrations in water with and without 1 mmole/L Tris, but addition of 3 mmole/L Tris resuited in slightly lower free copper at equivalently toxic total copper concentrations. Free copper concentrations calculated from bioassay data compared well with electrode measurements in all cases except when calculated using the 3 mmole/L Tris data for rotifers, when free copper concentrations were slightly overestimated.

Recent studies relating metal toxicity to speciation have demonstrated that, in most instances, toxicity is primarily a function of the free metal ion concentration. Metal complexes with organic ligands are generally non-toxic (Sunda and Guillard 1976; Anderson and Morel 1978; Jackson and Morgan 1978; Sunda and Gillespie 1979; Canterford and Canterford 1980). However, most of this work has been conducted on phytoplankton or bacteria. With a few exceptions, such as the effect of carbonate and phosphate on copper lethality to Daphnia (Andrew et al. 1977) and the effect of nitrilotriacetic acid (NTA) and salinity on cadmium lethality to grass shrimp (Sunda et al. 1978), little work has been done with invertebrates. Furthermore, these tox-

icity studies measured only lethal effects. However, copper bioaccumulation also appears to be related to free cupric ion concentration in oysters (Zamuda and Sunda 1982). While some studies have been conducted on metal speciation and toxicity to fish, they have been concerned with varying pH, hardness and alkalinity, which allows for only a small variation in the degree of complexation at any one pH (Howarth and Sprague 1978; Chakoumakos et al. 1979). One reason for the scarcity of data on free metal ion effects on aquatic organisms, especially in natural waters, is the difficulty of measuring free metal ion concentrations. Consequently, Sunda and Gillespie (1979) estimated free copper concentrations in natural sea water after copper addition by comparison of copper toxicity to bacteria in the natural sea water with toxicity in water of known complexing ability. Similarly, Borgmann (1981) estimated free copper concentrations toxic to copepods in natural water using a bioassay, but in this case by comparing toxicity before and after addition of a reference complexing agent. This latter approach has subsequently been shown to provide reliable free copper estimates in defined artificial media using a D a p h n i a bioassay (Borgmann and Ralph 1983). H o w e v e r , the accuracy of the bioassay method when applied to invertebrates in natural waters has not been extensively tested. The first objective of this study was to determine ff metal toxicity to zooplankton is relatively constant on a free metal basis, especially at sublethal concentrations. A second objective was to verify the accuracy of the bioassay method of determining free metal concentrations toxic to invertebrates in natural waters. To do this two bioassays were used, a sublethal test measuring the growth of cyclopoid copepods, and a short lethal test for rotifers. Both organisms are a major natural component of the

404

u . Borgmann and K. M. Ralph

zooplankton of local fresh waters. Copper toxicity was measured before and after addition of Tris, and bioassay determinations of free copper were compared with cupric ion electrode measurements.

bioassay were performed by the method of Borgmann (1981). The total copper concentration resulting in a 20% reduction in copepod growth (G20) or 50% mortality of rotifers (EC50) is

Materials and Methods

where [Cu] is the concentration of free cupic ions, [Cu-Tris] is the concentration of all copper-Tris complexes, and [Cu-X] is the total concentration of all other copper complexes. If [Cu] at equivalently toxic total copper concentrations is equal before and after addition of Tris, then, by equilibrium, [Cu-X] before Tris addition must be equal to [Cu-X] after Tris addition. Therefore the increase in total copper concentration required to reduce growth by 20% (AG20) or cause 50% mortality (AEC50) must be equal to [Cu-Tris]. Therefore,

Copepod (primarily Acanthocyclops vernalis and Diacyclops thomasi) growth rates were determined by incubating nauplii collected from the Burlington Canal (Canada) in canal water for one week at 20~ copepods fed on the natural food organisms in the canal water. Final copepod size was calculated from the nylon monofilament screen sizes on which copepods were retained. The procedure is described in detail in Borgmann (1980) and Borgmann et al. (1979). Rotifer bioassays were performed by incubating two L of Burlington Canal water, filtered through an 80 I~m nylon monofilament screen, in pyrex glass jars at 200C. Most Keratella cochlearis passed through the 80 wm screen, but larger carnivorous rotifers and copepods, which prey on Keratella, were retained on the screen and discarded. After 24 hr incubation, the rotifers were filtered onto a 44 Ixm screen, washed into a 15 ml graduated centrifuge tube, fixed with Lugol's solution, and permitted to settle. The volume in the centrifuge tubes was adjusted to about 5 ml by removal of the upper, rotifer free water. The sample was thoroughly mixed and a 1.85 ml subsample (the volume of the counting chamber) was removed for counting. The counting chamber consisted of a 3.5 mm thick glass plate with a 27 mm diameter hole, glued on top of another glass plate with lines etched about 1.5 mm apart. All live and dead Keratella in the chamber were counted, and the percent survival was calculated. Keratella which were alive at the time of fixing were easily distinguished from dead animals, since they contract after addition of the Lugol's solution, leaving no portion of the head extending past the anterior edge of the lorica. Keratella killed by low concentrations of copper are extended, and remain so when fixed. Copper (CuCI 2 9 2H20) was added after the addition of Tris (Tris-hydroxymethyl-aminomethane). Tris was used as the reference complexing agent because it is commonly used in bioassays, is not toxic to most organisms, and has a good cupric ion buffering ability. The pH was adjusted to 8.3 which is the approximate mean pH of Burlington Canal water in the summer. Tris did not have a significant effect on copepods or rotifers in the absence of added copper. Rotifer survival ( ___95% confidence limits) was 97.7 + 1.9, 97.6 -+ 1.3, and 94.4 -+ 1.7% in the absence of added copper with additions of 0, 1, and 3 mmoles/ L Tris respectively. At the same Tris concentrations, instantaneous copepod growth rates were 0.38 --- 0.06, 0.44 __. 0.03, a n d 0.40 -+ 0.06 per day, respectively, without added copper. Total copper concentrations were not routinely measured, because initial experiments indicated that total copper measured by graphite furnace atomic absorption spectrophotometry at the end of the experiment was identical to added copper, within the experimental error (SD/mean = 14%). Background copper concentrations were near or below 0.05 i~mole/L (Borgmann et al. t980 and unpublished data). Free copper concentrations were determined by bioassay and with an electrode (Borgmann and Ralph 1983). Electrode measurements were performed at the end of the incubation period, after removal of the animals, using an Orion | 94-29 cupric ion electrode with a Radiometer PHM 84 pH meter and Radiometer K401 reference electrode. Free copper determinations by

[Cu T] = [Cu] + [Cu-X] + [Cu-Tris]

[Cu] = A G 2 0 \

[Cu-Tris] / - 1 [Cu] }

(1)

(2)

for copepods. A similar equation, with AEC50 replacing AG20, applies to the rotifers. Equation 2 was used to calculate free copper concentrations from the bioassay data. The ratio [CuTris]/[Cu] was estimated from electrode data in artificial medium as described below. Prior to conducting the bioassay experiments, the ratio of [CuTris]/[Cu] was measured experimentally using the cupric ion electrode in artificial medium (1 mmole/L CaC12, 2 mmole/L NaHCO 3, and 5 mmole/L NaC1). This medium (hardness and alkalinity 100 mg/L as CaCO 3) has a hardness, alkalinity, and ionic strength similar to that of Burlington Canal water (hardness 180 mg/L, alkalinity 103 me/L), but contains no organic comptexing agents to interfere with electrode measurements. In this medium, the ratio of total to free copper is

[CUT] [Cu]

[Cu-Tris] + [Cu-I] + 1 [Cu] [Cu]

(3)

where [Cu-I] is the concentration of inorganic complexes of copper (eg., hydroxide and c a r b o n a t e complexes). By subtracting [CuT]/[Cu] values obtained in the presence of Tris from those obtained in the absence of Tris, [Cu-Tris]/[Cu] was calculated. At pH 8.3, this ratio was approximately equal to [Cu-Tris]/[Cu] = K([Tris T] - 2[Cu-Tris]) 2

(4)

where [Tris T] is the total Tris concentration and K has a value of 109.99 at 1 mmole/L total riffs and 109.83at 3 mmole/L Tris. Equation 4 would be expected to hold if the only copper Tris complexes formed were of the type Cu(Tris) 2. Since K was not exactly equivalent at the two different total Tris concentrations, it is likely that some other complexes are also formed. Nevertheless, equation 4 fit the observed electrode data in artificial medium well, and was therefore used to estimate [Cu-Tris]/[Cu] for predicting free copper by the bioassay technique employing equation 2. To estimate [Cu-Tris]/[Cu] in the bioassays, [Cu-Tris] on the right hand side of equation 4 was assumed to equal AG20 or AEC50. All free copper values are concentrations, not activities. Since ionic strength was not altered (I -~ 0.01), these would be directly proportional to, but slightly greater than free cupric ion activities.

Copper Toxicity to Zooplankton

405 1.1- --

1.0-

0.9-

" ~O~ 0.8-

0.7-

Fig. 1. Growth rate (g) relative to control growth (g') of copepods in Burlington Canal water as a function of total added copper for several added Tris concentrations. Each line is the average of 10 replicate experiments. Vertical bars indicate 95% confidence limits. O - - n o added riffs; A - - I mmole/L Tris, 11--3 mmole/L Tris

0.6-

0.5-

0.4

-6'.5

-61o

-si0

-515

-415

-4.'0

tog Cu T

T

I I

1.0-

0.9-

T

"~.~ 0.8-

Fig. 2. Growth rate relative to control growth of copepods as a function of free copper ion concentration. Vertical bars indicate 95% confidence limits for growth rates. Horizontal bars indicate 95% confidence limits for cupric ion electrode measurements. Confidence intervals for data obtained in the presence of Tris are omitted for clarity, but are similar in magnituide as those obtained without Tris. Q----no added Tris; A - - 1 mmole/L Tris; I - - - 3 mmole/L Tris

0.7-

0.6

II 0.5-

• 0.4

-16.o

-9'.s

-~'.o

-8'~

-~.o

log Cu §

Results

Copper Toxicity and Complexation The effect of copper on copepod growth rates at 0, I and 3 mmole/L Tris is shown in Figure 1. The presence of Tris displaces the toxicity curve consid-

erably. The total copper concentrations causing a 20% reduction in growth (G20) are 0.66 txmmole/L without Tris addition and 4.3 and 29 ixmmole/L in canal water with 1 and 3 mmole/L Tris, respectively. Figure 2 contains the same growth rate data, but plotted against the free copper concentrations as determined with the cupric ion electrode. On a free

406

U. Borgmann and K. M. Ralph

~.0-

0.8-

> 0.673

Fig. 3. Fraction of rotifers surviving for 24 hours, corrected for control survival, in Burlington Canal water as a function of total added copper for several added Tris concentrations. Each line is the average of 8 (no Tris), 7 (1 mmole Tris), or 6 (3 mmole/L Tris) replicate experiments. Vertical bars indicate 95% confidence limits. Q - - n o added Tris; A - - 1 mmole/L Tris; 11--3 mmole/L Tris

0.4-

0.2-

-6.'o

-515

-4.5

-510

-41.0

-315

log CUT

copper basis, the three toxicity curves overlap, and log G20 is approximately equal to - 9 . 3 . The curve in Figure 2 is given by

m = 2.99 x 108 x [Cu]

1

g/g' =

1 +4.48 • 108 • [Cu]

copper (Figure 3). The curve in Figure 4 was obtained from the combined data without and with 1 mmole/L Tris and is given by

(5)

where g is growth rate, g' is control growth rate and [Cu] is free copper ion concentration. This curve was derived by calculating a toxicant affected growth rate (g") from 1/g" = 1/g - 1/g' and assuming a linear relationship between log g"/g' and log free copper. The denominator in the above equation equals 1 + g'/g". Equations analogous to equation 5 have previously been found satisfactory for relating growth rates of copepods to total metal concentrations (Borgmann et al. 1980). Equation 5 is also analogous to the equation derived by Sunda and Gillespie (1979) relating glucose uptake by bacteria to free copper, although they obtained a better fit using [Cu] 2 in the denominator instead of [Cu]. The effect of copper on rotifer survival at the different Tris concentrations is shown in Figure 3. Rotifer survival in 0, 1 and 3 mmole/L Tris on a free copper basis is shown in Figure 4. The toxicity curves for 0 and 1 mmole/L Tris are identical, within experimental error. However, in canal water with 3 mmole/L Tris, survival is affected at lower free copper concentrations than in canal water with no or 1 mmole/L Tris. Nevertheless, the variation in free copper concentration resulting in a given mortality is still much less than the variation in total

(6)

where [Cu] is the free copper concentration and m is the mortality rate. m was calculated from (In NT/ N)/t where N is the number of live rotifers, N T is live plus dead rotifers and t is time (1 day). Equation 6 is of a different form than equation 5 because the growth rate decreases, whereas the mortality rate increases, with increasing copper concentrations. Apparently, the mortality rate is directly proportional to the free copper concentration. The same conclusion was obtained by Andrew et al. (1977) in studies relating Daphnia mortality rate to free copper.

Bioassay Determinations of Free Copper The displacement of the toxicity curve by Tris was used to calculate free copper concentrations for comparison with the direct electrode measurements. The G20 on a total copper basis, increase in the G20, and free copper concentrations toxic to copepods as calculated using equations 1 to 4 are shown in Table 1. Also included are the free copper concentrations obtained from the regression of free copper against total copper concentrations using the cupric ion electrode data. The bioassay data agrees well with the electrode data. Furthermore,

Copper Toxicity to Zooplankton

407

1.0-

0.8-

._>

Fig. 4. Fraction of rotifers surviving for 24 hours, corrected for control survival, as a function of free copper ion concentration. Vertical bars indicate 95% confidence limits for survival. Horizontal bars indicate 95% confidence limits for cupric ion electrode measurements. Confidence intervals for data obtained in the presence of 1 mmole/L Tris are omitted for clarity. O---no added Tris; A - - 1 mmole/L rids; 11--3 mmole/L Tris

0.6-

0.4-

0.2i

0

-lo.o

-9.'5

-9.'o

i-,<_ i

-8.b

-8; log C u §247

-z~

Table 1. Copper concentrations reducing copepod growth by 20% (G20) or rotifer survival to 50% (EC50) in Burlington Canal water with or without added Tris, free copper concentrations calculated from the bioassay data, and free copper measured with the electrode - log free Cu

Tris

G20 or EC50

AG20 or AEC50

added

(~moles/L)

(p~moles/L)

Calculated from bioassays

Measured with electrode

0.66 (0.27) a 4.27 (1.81) 28.8 (11.1)

-3.61 28.1

-9.42 9.32

9.43 9.37 9.35

1.59 (0.21) 25.0 (5.4) 78.2 (6.1)

-23.4 76.6

-8.58 8.85

8.62 8.68 9.22

Copepod experiments None 1 mmole/L 3 mmole/L

Rotifer Experiments None 1 mmole/L 3 mmole/L

a Standard deviations for 6 to 9 replicate experiments done on different days

the similarity in the bioassay data using 1 or 3 mmole/L Tris implies that the copper Tris complex is not toxic (since more of this complex is present at the higher Tris concentration). The EC50, AEC50, and free copper concentrations toxic to rotifers are shown in Table 1. The bioassay determination of free copper at the EC50 for 1 mmole/L Tris is in close agreement with the electrode determination for 0 and 1 mmole/L Tris. In the presence of 3 mmole/L Tris, however, both the bioassay and electrode data indicate a lower free copper concentration. Table 1 indicates (as does Figure 4) that when the bioassay is used in the absence of electrode data, it is preferable to verify results using several concentrations of the reference complexing agent.

The free copper G20 and EC50 data in Table 1 with similar calculations at 40% growth reduction for copepods and 25 and 75% mortality of rotifers, are shown along with electrode measurements in Figure 5. The bioassay data for copepods and rotilers and the electrode data agree fairly well, with the exception of the rotifer data obtained using 3 mmole/L Tris, which is somewhat low. Discussion The results demonstrate that after addition of Tris, copper toxicity to planktonic invertebrates is much less variable on a free copper than on a total copper basis. This is particularly striking when comparing Figures 1 and 2. These observations are in agree-

408

U. Borgmann and K. M. Ralph -8.0-

-8.5-

/" o

-9.0++ 0 0') 0

-9.5o

-10.0log Cu T

Fig. 5. Free copper ion concentrations as a function of total added copper. The line is the regression for electrode data. O - electrode data obtained for the copepod experiments; O---electrode data obtained for the rotifer experiments; A--free copper at 20 and 40% growth inhibition of copepods calculated from bioassay data using 1 mmole/L Tris; D----free copper calculated from copepod bioassay data using 3 mmole/L Tris; A - - f r e e copper at 25, 50 and 75% mortality of rotifers calculated from bioassay data using 1 mmole/L Tris; II--free copper calculated from rotifer bioassay data using 3 mmole/L Tris

ment with the majority of studies on copper speciation and toxicity after addition of organic complexing agents; most of these have been done on phytoplankton or bacteria. Little work of this type has been done with invertebrates, although Andrew et al. (1977) showed that copper lethality to Daphnia was related to free copper in the presence of inorganic complexing agents and Sunda et al. (1978) obtained similar results with cadmium induced mortality in grass shrimp after addition of NTA or chloride ions (increased salinity). These results, and the rotifer experiments, measured lethal effects. The copepod data demonstrates that the same phenomenon can be observed with sublethal toxicity to invertebrates. Although copper toxicity is related primarily to free copper ion concentrations, there are examples where toxicity is also related to the concentration of other complexes. The data in Figure 4 indicate that either the copper-Tris complex is also toxic or that Tris modifies copper toxicity in some other way. Guy and Kean (1980) indicated a reduction in free copper at toxic copper concentrations after Tris

addition when using algae. In addition, it was found that copper toxicity to Daphnia, but not to guppies (Poecilia reticulata), is strongly related to the concentration of copper amino acid complexes, as well as the free copper ion concentration, in defined media (Borgmann and Ralph 1983). Therefore, exceptions to the common observations that toxicity is related only to free copper ions do occur. In the studies with invertebrates, it is unlikely that these exceptions are due to competitive interference with other metals (as may be the case with algal studies) because addition of high concentrations of complexing agents (even strong complexing agents such as EDTA) by themselves (which reduced natural free metal concentrations) did not alter growth or mortality rates. It cannot be completely discounted that some form of metal competition, or some other interaction, is responsible for the results shown in Figure 4. Figure 5 demonstrates that free copper concentrations can be determined by a bioassay approach in which copper toxicity is compared before and after addition of a reference complexing agent (Borgmann 1981). However, more than one concentration of the complexing agent should be used, to ensure that the calculated free copper concentration is independent of the concentration of the reference ligand. This procedure determines free copper concentrations but does not indicate whether copper toxicity in the test water is related only to the free copper ion. Sunda and Gillespie (1979) also estimated free copper ion concentrations in natural water using a bioassay, but their method involved comparing copper toxicity in natural water with a standard curve developed in a different medium (U.V.-treated sea water with NTA). This approach could give erroneous free copper estimates if natural waters contain substances which produce complexes with copper which affect the relationship between free copper and toxicity. More recently, Sunda and Ferguson (1983) employed a bioassay conceptually similar to ours for estimating free copper concentrations toxic to bacteria in sea water. They assumed that the concentration of the copper-NTA complex was equal to the total concentration of copper minus the concentration of copper resulting in the same level of inhibition of amino-acid uptake in the absence of NTA. Since the bioassay procedure does not require production of a standard curve in defined medium, it is particularly useful for growth studies on zooplankton; for example, the copepods fed on natural food particles in the water. It was not necessary to culture a suitable food for growing these animals as required when developing a standard curve in defined me-

Copper Toxicity to Zooplankton

dium. In addition, the effect of the food organisms on copper speciation (through adsorption) does not interfere with the determination of the relationship b e t w e e n free and total copper in natural water, since these organisms are a natural component of this water. The data in Table 1 indicate that similar estimates of free metal ion concentrations are obtained from either the bioassay data or from direct electrode measurements. In this case, the bioassay determinations of free copper serve only to support the electrode measurements (and vice versa). However, metal ion electrodes are not sufficiently sensitive or appropriate for use under all conditions. For example, the cupric ion electrode does not provide reliable estimates of free copper when used in sea water (Westall et al. 1979; Zirino and Seligman (1981). Free copper in toxicity studies in sea water has, therefore, been estimated primarily from theoretical calculations (Sunda and Guillard 1976; Anderson and Morel 1978; Jackson and Morgan 1978). Under such conditions, a bioassay procedure can be especially valuable (Sunda and Ferguson 1983). While not designed as a tool for the chemist, the bioassay is very useful to the biologist who wishes to estimate free copper concentrations toxic to animals, because it only requires that bioassays be repeated, using the same species of animals, in the same water after addition of a reference complexing agent such as Tris.

References Anderson DM, Morel FMM (1978) Copper sensitivity of Gonyaulax tamarensis. Limnol Oceanogr 23:283-295 Andrew RW, Biesinger KE, Glass GE (1977) Effects of inorganic complexing on the toxicity of copper to Daphnia magna. Water Res 11:309-315 Borgmann U (1980) Interactive effects of metals on biomass production kinetics of freshwater copepods. Can J Fish Aquat Sci 37:1295-1302 (1981) Determination of free metal ion concentrations using bioassays. Can J Fish Aquat Sci 38:999-1002 Borgmann U, Cove R, and Loveridge C (1980) Effect of metals on the biomass production kinetics of freshwater copepods. Can J Fish Aquat Sci 37:567-575 Borgmann U, Loveridge C, Cove R (1979) A rapid method for

409 the estimation of, and some factors affecting, copepod production rates in the Burlington Canal. J Fish Res Board Can 36:1256-1264 Borgmann U, Ralph KM (1983) Complexation and toxicity of copper and the free metal bioassay technique. Water Res 17:1697-1703 Canterford GS, Canterford DR (1980) Toxicity of heavy metals to the marine diatom Ditylum brightwellii (West) Grunow: Correlation between toxicity and metal speciation. J Mar Biol Assoc U.K. 60, 227-242 Chakoumakos C, Russo RC, Thurston RV (1979) Toxicity of copper to cutthroat trout (Salmo clarki) under different conditions of alkalinity, pH, and hardness. Environ Sci Technol 13:213-219 Guy RD, Kean AR (1980) Algae as a chemical speciation monitor 1 A comparison of algal growth and computer calculated speciation. Water Res 14:891-899 Howarth RS, Sprague JB (1978) Copper lethality to rainbow trout in waters of various hardness and pH. Water Res 12:455-462 Jackson GA, Morgan JJ (1978) Trace metal-chelator interactions and phytoplankton growth in seawater media: Theoretical analysis and comparison with reported observations. Limnol Oceanogr 23:268-282 Suuda WG, Gillespie PA (1979) The response of a marine bacterium to cupric ion and its use to estimate cupric ion activity in seawater. J Mar Res 37:761-777 Sunda WG, Guillard RRL (1976) The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J Mar Res 34:511-529 Sunda WG, Engel DW, Thuotte RM (1978) Effect of chemical speciation on toxicity of cadmium to grass shrimp, Palaemonetes pugio: Importance of free cadmium ion. Environ Sci Technol 12:409-413 Sunda WG, Ferguson RL (1983) Sensitivity of natural bacterial communities to additions of copper and to cupric ion activity: A bioassay of copper complexation in seawater. In: Wong CS, Boyle E, Bruland KW, Burton JD, Goldberg ED (eds) Trace metals in sea water. Plenum, New York, pp 871 891 Westall JC, Morel FMM, Hume DN (1979) Chloride interference in cupric ion selective electrode measurements. Anal Chem 51:1792-1798 Zamuda CD, Sunda WG (1982) Bioavailability of dissolved copper to the American oyster Crassostrea virginica. I. Importance of chemical speciation. Mar Biol 66:77-82 Zirino A, Seligman PF (1981) A note on the polarographic behavior of the Cu(II) ion selective electrode in seawater. Mar Chem 10:249-255 -

Received for publication August 29, 1983 and in revised form December 9, 1983.