Effects of nutrient trace metal speciation on algal growth in the presence of the chelator...

ELSEVIER Aquatic Toxicology 36 (1996) 253-275

Effects of nutrient trace metal speciation on algal growth in the presence of the chelator [S,S]-EDDS

Diederik Schowaneka’*, Drew McAvoyb, Don Versteegb, ArnbjCrn Hanstveitc

“Procter & Gamble, European Technical Centre, Temselaan 100. B-1853 Strombeek-Bever, Belgium

“Procter & Gamble, Ivorydale Technical Centre, 5299 Spring Grove Avenue, Cincinnati, OH 45217, USA

‘TN0 Nutrition und Food Research Institute, Department of Environmental Toxicology,

P. 0. Box 6011, NL JA Delft. Netherlands

Received 18 August 1995; accepted 2 April 1996

Abstract

This study tests the hypothesis that the apparent toxicity of strong chelators in standard algal

growth inhibition tests (e.g. method OECD 201, EC C.3., IS0 8692) is related to essential

trace metal bioavailability. This hypothesis was investigated for the chelator [S,S]-ethylene diamine disuccinate ([S,S]-EDDS) and the green alga Chlorella vulgaris. Metal speciation

calculations were used to help design the algal growth experiments and interpret the data. Results suggest that interaction of the chelator with trace metals alters the free metal con-

centration and affects algal population growth, as opposed to a direct interaction between the alga and the chelator (toxicity sensu stricto). Even low levels of [S,S]-EDDS (i.e. 3 mg 1-l or less) reduce the free pcU and pzn (pbletal = -log[Metal]) in standard OECD medium below 16 and 11, respectively, which are the minimum levels required to support algal growth. Nutrient deficiency was overcome by supplementing the medium with appropriate amounts of the

trace metals Cu, Zn and Co, but not by increasing the hardness of the medium. A short-term photosynthesis inhibition experiment with the alga Selenastrum capricornutum in metal-free medium showed only a minimal effect of [S,S]-EDDS on the ‘“C-CO2 fixation rate. About 10% inhibition was observed at 100 mg [S,S]-EDDS lP’, i.e. the ECso for COz fixation is greater than 100 mg 1-l. Results from this study illustrate that the standard algal

growth inhibition test is not well suited to the assessment of algal toxicity (sensu stricto) of strong chelators. The no-effect level and ECsO value are probably overestimated by at least one order of magnitude for [S,S]-EDDS. The study also illustrates the importance of speciation calculations when assessing algal inhibition by chelators.

Keywords: Algal growth inhibition tests; Chelators; [S,S]-EDDS; Trace metals; Speciation; Chlorella vulgaris; Selenastrum capricornutum

*Corresponding author. Tel. : 32 2 456 29 00; fax: 32 2 456 28 45

0166-445X/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved.

PIISOI 66-445X(96)00807-7

254 D. Schowanek et aLlAquatic Toxicology 36 (1996) 253-275

1. Introduction

A correct estimation of a chemical’s effect on algae is important when assessing its environmental risk, and also in the context of the current European Union (EU) legislation on Classification and Labelling of Dangerous Substances (Directive 67/ 548/EEC, 7th Amendment). Under the latter legislation, new chemicals are classified for their inherent environmental properties on the basis of standard ecotoxicity (algae, fish, invertebrates), bioaccumulation and biodegradation tests. For the algal test, the EU guidelines (Official Journal of the European Communities, 1992) re- commend a general growth medium (OECD medium) which supports the growth of the most commonly tested algal species. This medium contains a precise level of essential trace metals held in solution by a chelator (EDTA). The introduction of an additional chelating agent in the growth medium, which may or may not have higher stability constants than EDTA, can affect the algal response (i.e. growth rates, total biomass at the end of the test, cell division pattern, etc.) by changing trace metal availability. It should be noted that algal growth inhibition assays were not designed specifically for testing either heavy metals or chelators (Peterson and Nyholm, 1993).

Chelators show remarkably low EC+,0 values in the algal growth inhibition test (i.e. a high apparent toxicity) compared with the acute EC5a or LC5,, values for invertebrates and fish (Table 1). This effect seems to be much less pronounced in other categories of chemicals, with the exception of herbicides (see e.g. Verschueren (1983) or Schiiberl and Huber (1988)). Chelators, however, do not contain func- tional groups associated with herbicidal activity. It cannot be excluded that algae are inherently more sensitive to chelators than other organisms, for instance due to different cellular processes or organisation. However, another hypothesis supported by an increasing body of evidence suggests that a large part (if not all) of the inhibition can be explained by simple nutrient limitation (Gode, 1983 ; Gledhill and Feijtel, 1992; Peterson and Nyholm, 1993). Trace metal levels tend to be more important in algal growth tests than in other short-term tests on higher organisms. The main reason is the rapid multiplication rate of the algae, leading to a very significant biomass increase during the test, and a high dependence on the (limiting) amount of essential trace metals. In contrast, fish and Duphnia do not grow significantly during an acute test and in a chronic experiment these organisms can obtain sufficient trace elements through their food. Metals are also an essential part of the active centre of many components of the photosynthetic apparatus, e.g. Cu in the redox-carrier plastocyanine, Mg in chlorophyll, Fe in ferredoxine, Mn in photosystem II. Zn is a co-factor for respiration, amino acid, protein and nucleic acid metabolism, and in cell division (Ajay and Rathore, 1991; Ting et al., 1991).

Studies have shown that the free (i.e. uncomplexed) metal concentration, and not the total metal concentration, governs growth and toxicity for algae (Morris and Russel, 1973; Allen et al., 1980; Allen, 1993; Xue and Sigg, 1993; Tubbing et al., 1994). The free metal concentration is approximately equal to the sum of the ionic, hydrogen and hydroxide forms. Under conditions of limitation, the Monod equation predicts how the growth rate (u) varies with the free nutrient trace metal

D. Schowanek et aLlAquatic Toxicology 36 (1996) 253-275 255

concentration. The biomass level achieved during the exponential phase is also

directly dependent on the growth rate via the following exponential relationship

Total Biomass =

= Average Cell Volume X Total Cell Numbers = ACV X No X &-“l”) (1)

where tlag is the duration of the lag phase, and No the number of cells in the inoculum. In terms of studying free metal effects, the exponential phase (which

usually lasts up to 96 h in an algal batch test) is the most informative. While the

total biomass level at the end of a prolonged test (i.e. beyond the exponential growth period) may reveal additional information, it is expected to reflect the total

rather than the free nutrient trace metal level. This is because free and chelated

metals are in dynamic equilibrium and a significant uptake of free metals will lead

to the chelated metals becoming biologically available. Also, at the end of the

exponential phase other factors such as light and CO2 may become limiting. While algal toxicity data are available for most chelators, surprisingly few studies

seem to have systematically investigated the above-mentioned effects in the batch culture system prescribed by the testing guidelines. In this work, the chelator [S,S]-

ethylene diamine disuccinate ([S,S]-EDDS) was used as a model compound to investigate chelant effects on the growth and metabolism of algae. [S,S]-EDDS is comparable in its chelating properties to EDTA, DTPA or the polyphosphonic

acids EDTMP and DETPMP (Table 2) but has the advantage of being readily biodegradable (D. Schowanek, unpublished data, 1995). Speciation calculations

were used alongside the algal test to help design the experiments and interpret

the phenomena observed. Chemical equilibrium calculations make use of the affinity

constants of a chelator. In this context, three classes of chelating agents were

operationally defined. The first group contains weak chelators with a moderate affinity for metals (affinity constant, log K, typically less than 5). In practice, they are used mainly to bind Ca2+ and Mg2+ ions, i.e. to reduce water hardness in applications such as detergents and industrial process water (e.g. zeolites). In general, weak chelators do not show pronounced effects on algal growth and their ECso is typically above 10 mg 1-l (Table 1). Furthermore, the ECSo can vary considerably

with the hardness of the medium (Gode, 1983; Hamilton et al., 1994). The hardness

effect can be explained by the fact that their affinity constants do not vary much with the metal and these chelators can easily be loaded with Ca2i- and Mg2+ ions,

thereby releasing chelated trace metals.

The second group contains the chelators of intermediate affinity (i.e. affinity

constants typically between 6 and 12, e.g. NTA). The third group is that of the strong chelators (e.g. [S,S]-EDDS and EDTA) with affinity constants usually above 12. They have a marked affinity for trace metals such as Cu, Hg, Pb, Zn, Co, rather than for Ca and Mg. Speciation considerations predict significant and hardness independent effects on the level of free trace metals at chelator concentrations of about 100 ug l-l, which may lead to pronounced effects on algal growth.

With these concepts in mind, the objective of this paper was to investigate the

256 D. Schowanek et al.lAquatic Toxicology 36 (1996) 253-275

effect of trace metal speciation on algal growth, with the strong chelator [S,S]- EDDS as the model chemical.

2. Materials and methods

2.1. Abbreviations

The following abbreviations are used in this paper:

[S,S]-EDDS

EDTA

DTPA

NTA

HEDP

EDTMP

DETPMP

E, Go

Eb Cso

[S,S]-Ethylenediaminedisuccinic acid or trisodium salt (L-aspartic acid-NJ’-1,2-ethane-

diylbis)

Ethylenediaminetetraacetate

Diethylenetriaminepentaacetate

Nitrilotriacetate

Hydroxyethanediphosphonate

Ethylenediaminetetramethylphosphonate

Diethylenetriaminepentamethylphosphonate

50% inhibition value calculated on algal growth rate data by a parametric model (Kooij-

man et al., 1983)

50% inhibition value calculated from the area under the growth curve (i.e. biomass) as

described in OECD (1984)

2.2. Reagents and media

Tests were executed on the acid form of [S,S]-EDDS. The [S,S]-EDDS test sample was 94% pure. Contaminants included about 3% aspartic acid and 2.4% Na. The

isomeric purity of the EDDS was over 95% [S,S]-form. The concentrations in the tests were nominal, always expressed as protonated [S,S]-EDDS. All chemicals used to make the media were of the highest purity commercially available.

The standard OECD algal growth medium (OECD, 1984) was prepared with ultrapure Mini-Q water (Waters Associates, Inc., Bedford, MA) and composed of (mg 1-l): NH4C1 15, MgCls*6HsO 12, CaCls.2H20 18, MgS04.7HsO 15, KHsPOd

1.6, CsHsOrFe.3HsO 0.080 (Fe-citrate), NazEDTA?H20 0.10, HsB04 0.185,

MnClss4HsO 0.415, ZnCls 0.003, CoCls.6HsO 0.0015, CuCls*2HsO 0.00001, NazMo04.2H20 0.007, NaHCOs 150 (not 50 mg 1-l as specified in the OECD

Guideline, to improve the buffer capacity). The stock solution containing trace elements was prepared without the elements Co, Cu and/or Zn. Concentrated stock solutions of each of the latter elements and EDTA were added separately, allowing the media concentration of these metals to be varied.

Appropriate volumes of each of these trace elements and EDTA were then added to 100 ml portions of micropore (0.45 urn) filtered medium. Fe-citrate was added to

the medium because it has been observed that in the absence of properly complexed Fe, algal growth tends to be erratic (A. Hanstveit, personal communication, 1994). The media were allowed to equilibrate for a period of 3-24 h before inoculation.

River water was obtained from the River Meuse at the village of Hank. The

D. Schowanek et al.lAquatic Toxicology 36 (1996j 253-275 251

Table 1

Toxicity data for chelating agents in standard aquatic toxicity tests with fish, invertebrates and algae.

Reported ECsa and NOEC values (mg 1-r) are at 96 h for algae and fish, and at 48 h for Daphnia

Chelator Algae”

NOEC ECso

Daphnia magna Fishh

NOEC ECss NOEC ECss

Ref.

EDTA < 10

<l 50

l-10 50 1000

640

8.3

626 2040

> 100 > 100

2808 1000 1800

1535 2600

1033 2040

Schiiberl and

Huber, 1988

Schiiberl and

Huber, 1988

Zeolites HEDSET,

1994

Payne, 1973

Schoberl and

Huber, 1988

DTPA

Zeohte

Citrate

NTA

Polycarboxylates Acrylate-maleate

co-polymer

(70 kDa)

32

(EClO)

> 908 > 1000 Schumann, 1990

Polyacrylate

(4.5 kDa)

180

(EC10

> 1000 > 200 Freeman and

Bender. 1993

Phosphonates HEDP

ATMP

1.3 3 400

1.4 19.6 125

0.09 0.42 250

1.9 5.2 125

521 210

240

164

180

Gledhill and

Feijtel, 1992

Gledhill and

Feij tel, 1992

Gledhill and

Feijtel, 1992

Gledhill and

Feijtel, 1992

EDTMP

DETMP

510

242

EDDS

Racemate 0.135 0.29 Procter & Gamble,

unpublished data,

1990

Procter & Gamble,

unpublished data,

1993

[S,S]-form < 1’ < 1c 320 1000 1000 1000

“Selenastrum sp. or Scenedesmus sp. “Brachydanio rerio. ‘Estimate based on data in this paper

water was aerated, stored in the dark, and filtered before use (0.2 pm pore dia- meter). Organic and inorganic micropollutant contents were determined and found to be acceptably low for the tests. Of the three metals studied, only Cu occurred at a


140 : a A _._.__..__.,....... &

4 120

,...

,....- L

,:’ t

* 1 103 j/,’ !

x

.v 5 180 ,.I’

Q

:a 1~ :’ d :

140 ,,.f” n ?

xl ,.I’ . /.,’

0 _.._...I ...

0 20 ‘$I 60 80 ml 120 140 I60

Fig. 1. Effect of increasing water hardness on the growth of Chlorella vulgaris in the presence and ab-

sence of 1 mg 1-l [S,S]-EDDS. Standard OECD medium was augmented with CaCls*2HsO to achieve

hardness levels of 24, 50, 125, 250 and 375 mg 1-i (expressed as CaCOs). Hardness series without

[S,S]-EDDS: W, 24 mg CaCOs 1-l ; q , 50 mg CaCOs 1-l; l , 125 mg CaCOs 1-i; 0, 250 mg CaCO:{

I-‘; A, 375 mg CaCOs 1-l; dotted line, average curve. Hardness series with I mg 1-i [S,S]-EDDS: A, 24 mg CaCOs 1-l ; l , 50 mg CaCOs 1-l ; 0, 125 mg CaCOs I-’ ; X , 250 mg CaCOs 1-i ; 8, 375 mg

CaCOs 1-i; solid line, average curve.

concentration above the detection limit (3 pg 1-l). The hardness of the river water

was 165 mg CaC03 1-l.

Table 2

Stability constants (log K) for commercially important chelators, and for [S,S]-EDDS (the data for

NTA, EDTMP, DETPMP, EDTA are after Gledhill and Feijtel, 1992)

Metal Chelator

Citratea Poly- NTA [S,S]-EDDS’ EDTMP DETPMP EDTA DTPAC

carbox.”

Al(II1) ~ > 10 _ 16.1 ~

Ca(I1) 3.17 4.47 6.4 4.23 9.33 7.11 10.7 10.6 Cd(I1) 3.98 10.1 10.80 13.9 13.4 16.5 19.0

Cu(I1) 5.20 _ 12.7 18.36 19.0 19.5 18.8 20.5

Co(I1) ~ _ 14.06 _

Fe(III) 9.5 _ 15.9 22.00 25.1 27.5

Hg(II) ~ 12.7 17.50 _ 21.8 27.0

Mg(II) 3.40 ~ 5.4 5.82 8.63 6.40 8.7 9.3 Ni(II) 5.11 _ 11.3 16.79 _ 18.6 20.0

Pb(II) 3.00 ~ 11.8 12.70 _ 18.0 18.9 Zn(I1) 4.85 10.5 13.49 17.1 16.5 16.5 18.0

H 5.67 _ 10.33 9.82 11.0 10.23 10.5

“Martell and Smith (1977).

bPolycarboxylate: acrylic-maleic acid co-polymer, 70 kDa (BASF, 1994).

“Martell and Smith (1974).


120 ,

1 .e-6

0 ZnEDDS

HEDDS

1 .e-5

s,s-EDDS (M)

l.e-4

= CoEDDS = CuEDDS

m Ca+MgEDDS m MnEDDS

Fig. 2. Speciation of [S,S]-EDDS in standard OECD growth medium at chelant levels in the range of

1O-5 to 1O-6 M.

Table 3

Results of the growth inhibition test with 1 mg I-’ [S,S]-EDDS in standard OECD medium and in

OECD lM, 2M and 3M media, which are enriched in total Co, Cu and Zn concentrations. The dura-

tion of the experiment was 66 h

OECD

Medium

Concentration [S,S]-EDDS Total cell Growth rate

(mg 1-r) volume (h-t) co cu Zn (lo6 pm3 ml-r)

(10m7 M) (lo-* M) (lo-” M)

Standard 0.063 0.006 0.022 0 216

0.063 0.006 0.022 1 29

1M 2.1 1.7 1.0 0 243

2.1 1.7 1 .o 1 45 2M 6.2 5.1 2.9 0 159

6.2 5.1 2.9 1 223

3M 18.5 15.3 8.8 0 27

18.5 15.3 8.8 1 155

0.073 (0.022-0.125)

0.034 (0.021LO.047)

0.071 (0.0540.088)

0.044 (0.022-0.066)

0.064 (0.059-0.069)

0.066 (0.058-0.075)

0.037 (0.028%0.047)

0.058 (0.037-0.080)

Values in parentheses are 95% confidence limits.

260 D. Schowonek et aLlAquatic Toxicology 36 (1996) 253-275

2.3. Test organisms

The test organism used in the growth inhibition experiments was the freshwater

green alga, Chlorella vulgaris (21 l/l 1 b, Collection of Algal Cultures, Institute of Plant Physiology, University of Gottingen, Germany), belonging to the class Chlor-

ophycea, order Chlorococcales. It was selected because it is a well studied test organism, representative of algae in many locations, and is recommended in the OECD (1984) guideline. The alga was subcultured as specified in the guideline.

In the short-term photosynthesis experiment the test species was the fresh water

green alga Selenastrum capricornutum (Chlorophycea, Chlorococcales). The source was the culture collection of algae at the University of Texas at Austin (UTEX

1648). The alga was cultured in Bold’s basic medium (Carolina Biological, 1978) with subsequent liquid cultures initiated from the previous culture approximately every 6 days. Cells were harvested by centrifugation from a liquid culture for use in

the photosynthesis test.

2.4. Speciation model applied to algal media

The strength of a metal-chelator complex is defined by its affinity constant (K), also known as a stability constant. For a mononuclear monoligand complex it is defined as

Table 4

Measured growth parameters in the metal toxicity test with Chlorella vulgaris. Total Co, Cu and Zn

levels were varied in OECD medium as shown in treatments 2-10. In treatments 11 and 12 the indi-

cated metals were spiked into River Meuse water instead of OECD medium. The duration of the ex-

periment was 93 h

Medium Concentration [S,S]-EDDS Total cell Growth rate

(mg 1-l) volume (h-r) co cu Zn (lo6 pm3 ml-‘)

(10m7 M) (10-s M) (lo-’ M)

1” 0.063 0.0058 0.022 0 3854 0.057 (0.052~0.062)

2 6.3 0.0058 0.022 0 7269 0.090 (0.073-0.106)

3 0.063 0.0058 2.9 0 6031 0.061 (0.047-0.075)

4 0.063 5.1 0.022 0 8912 0.084 (0.0640.103)

5 0.063 5.1 2.9 0 4292 0.077 (0.062-0.092)

6 6.3 5.1 0.022 0 10184 0.108 (0.093-O. 123)

I 6.3 0.0058 2.9 0 5474 0.072 (0.055-0.089)

8 18.5 0.0058 0.022 0 4284 0.071 (0.051-0.091)

9 0.063 0.0058 8.8 0 13 0.006 (-0.008-0.020)

10 0.063 15.0 0.022 0 9504 0.076 (0.048-O. 104)

11 0.063 0.0058 0.022 0 18593 0.109 (0.09330.125)

12 0.063 0.0058 0.022 1 6338 0.071 (0.06550.076)

“Standard OECD medium, used as the control.

Values in parentheses are 95% confidence limits.

D. Schowanek et al.lAquatic Toxicology 36 (1996) 253-275 261

GIL = [MW([M] X IL]) (3)

Based on the above mass action equation and mass balance considerations, the concentrations of chemical species can be determined. In this study, the chemical equilibrium calculations were conducted using the speciation program MINEQL+

(Schecher and McAvoy, 1992; Schecher and McAvoy, 1994). MINEQL+ is an interactive data management system for chemical equilibrium modelling, which

uses the original MINEQL program (Westall et al., 1976) as an underlying numer-

ical engine. Thermodynamic data used in this study were identical to that in MIN-

TEQAl (Brown and Allisson, 1987), with the exception of [S,S]-EDDS which had

to be added to the thermodynamic database (Table 2). The thermodynamic data for

[S,S]-EDDS were obtained from Martell and Smith (1974). The stepwise stability constants in Table 2 were converted to overall stability constants at zero ionic strength prior to being included in the database. Calculations of the equilibrium

distributions and concentrations of the trace nutrient metals Co, Cu and Zn were performed by varying the concentrations of Ca, Cu, Co, Zn and [S,S]-EDDS in the algal medium.

2.5. Photostability test

A stability test was conducted over a 7 day period to understand the (photo)- stability of [S,S]-EDDS in OECD medium under illumination. Duplicate flasks

containing 100 ml of standard OECD medium and 30 mg 1-l of [S,S]-EDDS were incubated at a temperature of 23? 2°C and illumination of about 80 pE m-2 s-1 with and without an algal inoculum (Chlorella vulgaris). The test material

was included at a high level because the detection limit for [S,S]-EDDS in high performance liquid chromatography (HPLC) is about 10 mg 1-l. Samples were

taken at 0, 1, 3, 5 and 7 days. No algal growth occurred owing to the elevated [S,S]-EDDS concentration, reducing nutrient trace metals to deficient levels. Sam-

ples were preserved with 1% formaldehyde (final concentration) and stored in a refrigerator at 4°C prior to analysis by HPLC.

The concentration of [S,S]-EDDS in the photostability tests was determined using HPLC (Procter & Gamble, unpublished data, 1991) based on chelation with copper.

The method has the following specifications: chromatographic column Chiralpak WH (Diacel/Baker), with mobile phase 0.6 mM CuS04 at pH 3, isocratic flow of 1.5 ml min i. The injection volume is 100 yl and the detector is UV-VIS with wave-

length fixed between 232 and 254 nm.

262 D. Schowanek et aLlAquatic Toxicology 36 (1996) 253-27s

pco” pd

Fig. 3. Effect of [S,S]-EDDS concentration on the free metal levels of Co, Cu and Zn in OECD 2M medium (top series), and its effect on the growth of Chlorella vulgaris, expressed as total cell numbers (middle series), or as total cell volumes (lower series). Growth results are expressed relative to the control, i.e. standard OECD medium.

2.6. Procedures for the algal toxicity tests

2.6.1. General procedure The growth inhibition tests were carried out as described in the OECD Guide-

line No. 201 (OECD, 1984), using the test conditions described in IS0 8692 (1989) (involving changes in light intensity and temperature), unless described otherwise. Three to four days before the start of a test, a pre-culture of algae was prepared from the stock culture. The tests were carried out in autoclaved 200 ml conical flasks covered with silicone sponge caps. One millilitre of algal suspension, containing about lo6 cells ml-‘, was mixed with 100 ml of the media containing the appropriate nutrient and chelator levels. Within each experiment all treatments and controls were run in triplicate. In addition, a single series containing the test solutions without algae was also run to serve as a background for electronic particle counting.

D. Schowanek et al.lAquatic Toxicology 36 (1996) 253-275

!? 0.25

0.05

0.00

263

0 5 10 15 20 25

Time (minutes)

Fig. 4. Effect of [S,S]-EDDS on CO2 fixation (nmol pg m1 chlorophyll a) by Selenastrum capricornutum

at four concentrations of [S,S]-EDDS. The asterisk denotes a significant difference from the control

(P < 0.05).

The cultures were incubated under continuous standard illumination (about 80

pE mM2 s-l) on a shaker table at 23? 2°C. The number of cells (particles) were determined by means of an electronic particle counter (Coulter counter TAII) at the start of the incubation and at appropriate times throughout the test. The

coefficient of variation for the total cell volumes of the replicate control culture

flasks was usually less than 15%, which is normal for this type of assay. The appearance of the organisms in the various cultures was checked visually

with a microscope at the beginning and end of the test. Significant bacterial con-

tamination was never observed. The pH of the cultures was also determined at the start and end of the test. The initial pH value of the media was approximately 8.0. During a test the pH of the medium normally increases proportion-

ally with algal growth. After 3 days of incubation, the pH increase in the control cultures was less than one unit, which is in accordance with the validity criteria

of the guideline (OECD, 1984). In the extended tests described below, the highest pH value at the end of the experiment was 9.5.

The growth parameters, i.e. growth rate, cell counts, total cell volume (i.e. cell counts X average cell volume = total biomass or yield), were determined at the end of the tests and their 95% confidence interval calculated from the cell counts and/ or the total cell volumes by the method developed by Kooijman et al. (1983). Chlorella vulgaris showed a typical growth pattern. During the first 24 h of the

264 LX Seho~~ek et ai.lAqualic Toxicology 36 (1996) 253-275

test, there was an increase in cell size, without an increase in the cell density of the culture (synchronic growth). Thereafter, an exponential growth pattern was observed for 48-72 h, when the culture became CO%-limited. Owing to this tendency for synchronous growth at the start of the test, parameters estimated based on total cell volume were considered more representative than parameters based on cell counts only. The growth curves constructed with total cell volumes showed a much less pronounced lag phase and were generally smoother. The duration of a number of experiments was extended to 7 days to record a com- plete growth curve. However, growth rates were always calculated from the linear part of the logarithmically transformed growth curves (usually up to 96 h), and are independent of the test duration. All algal tests were carried out in compli- ance with the OECD principles of Good Laboratory Practice (GLP).

2.6.2. Water hardness test The standard OECD algal medium has a very low water hardness (20-25 mg

1-r as CaCOs) compared with the average hardness of 100-150 mg 1-r in Eu- ropean surface waters. Hardness-dependent chelation effects can be expected when weak chelators are tested in OECD medium. Owing to the low levels of Ca2+ and Mg ‘+ in standard OECD medium, essential trace metals are made un- available even by weak chelators. With increasing hardness, however, weak chelators readily release trace elements. For stronger chelators with a large difference (over 5 log units) in affinity constants between hardness cations (Ca’+ or Mg*+) and trace metals (Zn2+, CL?-+, Co2+), speciation calculations predict that an increase in water hardness will not affect the algal response in a realistic hardness range. The Ca2+ or Mgzf . tons will not be able to effectively replace the seques- tered heavy metals. To verify this hypothesis, a growth inhibition experiment with ~hlore~la v~~~ar~s and [S,S]-EDDS was carried out in which the standard OECD medium was augmented with CaC12*2H20 to achieve hardness levels of 24, 50, 125, 250 and 375 mg 1-l (expressed as CaCOs). This series was tested in the presence and absence of 1 mg 1-l (3.4 PM) [S,S]-EDDS. The test was extended to 163.5 h.

2.6.3. ~eta~-e~~ic~~ent tests In the first test a medium was created, based on chemical equilibrium calcula-

tions, in which the levels of free Cu, Co and Zn in the presence of 1 mg 1-l (3.4 pM) [S,S]-EDDS are the same as in standard OECD medium without the chelator. This modified medium was designated OECD 2M. Two other media were tested which contained three times less (OECD 1M) and three times more (OECD 3M) Cu, Co and Zn than OECD 2M (Table 3). This was done to cover possible inaccuracies in the determination of the [S,S]-EDDS stability constants. All media were tested in the presence and absence of [S,S]-EDDS, with standard OECD medium as the control. Note that the 1 mg 1-l level of [S,S]-EDDS was an arbitrary choice and that the same experiment could have been done with other medium~helator ratios. The test duration was that of a standard growth test, i.e. 66 h (about 3 days).

D. Schowanek et al.lAquatic Toxicology 36 (1996) 253.-275 265

A second experiment was performed using metal enriched medium. The objec-

tive was to further demonstrate the validity of the nutrient depletion hypothesis.

A range of [S,S]-EDDS concentrations from 0 to 10 mg 1-l was tested in the OECD 2M medium. Standard OECD medium without [S,S]-EDDS was used as reference. The test was extended to 143 h (about 6 days).

2.6.4. Toxicity tests with varying Co, Cu and Zn levels

To facilitate the interpretation of the speciation calculations, the relationship

between the concentrations of the trace metals Co, Cu and Zn in the OECD test

medium and the growth of the alga Chlorella vulguris was explored. A number of variations in trace metal concentrations were tried alone or in combination. In addition, a growth test was done in natural river water from the Meuse with and

without 1 mg 1-l [S,S]-EDDS. Duration of the test was 93 h.

2.7. Short-term photosynthesis inhibition test

A short-term photosynthesis inhibition experiment was run according to the

method described by Versteeg (1990) in an attempt to determine the toxicity sensu

strict0 on algae. The duration of the experiment was 50 min to avoid algal cell multiplication. The experiment was done in an organic mineral-free buffer, so that

[S,S]-EDDS was essentially in its free and potentially most active form (it is

generally assumed that the free form of a chelator is the most toxic as it can potentially scavenge metals from the active centre of biocatalyst molecules). 5’. cupricornutum was selected as the test species because a large set of inhibition

data was available. Algae were centrifuged out of liquid culture immediately prior to test initiation.

The algal pellet was washed and resuspended in a 460 mM HEPES/KOH buffer (pH 7.5) to achieve a cell density of about 1 X lo6 cells ml-’ in each test replicate. Cell density was determined using a Coulter Multisizer Particle counter. [S,S]-

EDDS, algae and buffer were added to the test chamber and allowed to equilibrate

to the light conditions (520 pE rnp2 s-l) for 30 min. [S,S]-EDDS concentrations were 0, 0.1, 1.0, 10 and 100 mg 1-l.

To prepare the stock solution, 0.058 g of [S,S]-EDDS was diluted with deionised

water in a 100 ml volumetric flask. One millilitre of this stock was further diluted with 9 ml of water for use as the dosing stock for the lowest concentration to be tested (0.1 mg 1-l). A radiolabelled/unlabelled sodium bicarbonate solution was made for use as the carbon source by mixing two l-ml ampules of 1 .O uCi ml-’ sodium bicarbonate with 1 ml of 2.2 mM NaHCOs in water. After the equilibration

period, 0.1 ml of the radiolabelled NaHCOs solution was added to each replicate. Each replicate was sampled (0.1 ml) at 2, 4, 8, and 20 min.

The samples were fixed with 0.1 ml 2 N HCl solution to volatilise the remaining, non-fixed carbon. After drying at 90°C scintillation cocktail was added and counts of acid-stable carbon determined by liquid scintilla@on counting (Beckmann LS6500). A second compound, Cu, with a known toxic effect was used as a positive

control. Nominal concentrations were used to estimate the level of [S,S]-EDDS


causing toxicity. Statistical analysis was conducted by ANOVA followed by Dun- nett’s multiple comparison test.

3. Results

3.1. Photostability test on [S,S]-EDDS

It was considered essential to have analytical confirmation of the stability of [S,S]-EDDS during the algal inhibition tests. Not only is the material biodegradable, but photodegradation of metal complexes of chelators has been reported (e.g. Matthijs et al., 1989). Results from the photostability test showed than on average 90 rf: 7% of the [S,S]-EDDS was recovered in the test solutions containing algae, and 91 + 3% in the algae-free medium after 7 days with no trends detected over time (individual data not shown). Degradation, if any, is therefore not likely to have exceeded 10% during the algal tests.

3.2. Effect of water hardness on algal growth in the absence and presence of [S,S]-EDDS

The effect of water hardness on the growth of Chlorel~a va~gari.~ in the presence and absence of 1 mg 1-l fS,S]-EDDS was investigated (Fig. 1). Algal growth was independent of medium hardness, both in the presence and absence of the chelator. The addition of 1 mg [S,S]-EDDS 1-l reduced algal growth in the OECD medium, as expected based on the data for the racemate form (Table 1). Average growth rates (i.e. for all levels of water hardness) as determined for the 24-96 h exponential period were 0.065 h-l in the absence of the test material and 0.031 h-l in its presence (53% reduction). Total cell volume (i.e. biomass) was inhibited by 65%. However, this effect was thought to be due to nutrient deficiency and not [S,S]- EDDS toxicity sensu stricto.

3.3. ~ro~lth experiments in metal-enriched UECD algal medium

Speciation calculations were carried out to determine the trace metal distribution in standard OECD medium with increasing [S,S]-EDDS levels. Below about 3 mg 1-l (equal to or less than 10e5 M), [S,S]-EDDS is mainly associated with Zn, Co and Cu, and Zn-[S,S]-EDDS is the dominating complex (Fig. 2). With the addition of increasing amounts of [S,S]-EDDS the concentration of free metals decreases fast, and nutrient trace metal concentrations may become insufficient to support algal population growth (see also Fig. 3, top series). The growth of ChlorelZa vu& garis was tested in metal-enriched OECD lM, 2M, and 3M media, and compared with the standard OECD medium. In the OECD 2M medium the levels of free Cu, Co and Zn in the presence of 1 mg 1-l (3.4 FM) [S,S]-EDDS were the same as in standard OECD medium without the chelator. In the OECD 1M and 3M media the


Cu, Zn and Co levels were a factor of 3 below and above, respectively, those in the

2M medium. In the absence of the chelator, impaired growth due to metal toxicity was ob-

served in the OECD 2M and 3M media (Table 3). Total cell volume at the end of

the test was reduced by 26% compared with the control. The effect was most pronounced (about 87% inhibition of total cell volume) in the OECD 3M medium.

Growth rate was less sensitive to this metal toxicity effect than total cell volume, but a marked decrease in growth rate (about 50%) was also seen in the 3M medium.

In the presence of 1 mg 1-l [S,S]-EDDS algal growth, as measured by total cell volume and growth rate, was reduced by 86% and 53%, respectively, in standard

OECD medium. Hence, the estimated Ei,Css and &C’s0 in this medium are 1 mg 1-l or less. Relative to the algal population growth at 1 mg [S,S]-EDDS 1-l in OECD

medium, growth in OECD lM, 2M and 3M was greater in the presence of [S,S]- EDDS (Table 3). The best growth, almost identical for both growth parameters to

the OECD medium control without chelator, was obtained with the OECD 2M medium. In medium OECD lM, the level of trace elements was apparently still

insufficient to support good growth, while in the OECD 3M medium the levels were supraoptimal.

To better understand the interactions between chelator, trace metals and algae, a second experiment with the metal-enriched OECD 2M medium was executed by

varying the [S,S]-EDDS concentration from 0 to 10 mg 1-l. Fig. 3 (top series)

illustrates how the levels of free Cu, Co and Zn levels vary with the [S,S]-EDDS

concentration in OECD 2M medium. Free concentrations of all metals change by

more than six orders of magnitude as the chelator concentration ranges from 0 to 3 mg 1-l (O-10 PM). In the case of Cu, the range exceeds eight orders of magnitude. The free metal concentrations in turn drive the response of the alga. As the [S,S]- EDDS concentration increases from 0 to 10 mg 1-l (O-35 PM), algal cell numbers

are initially low relative to the OECD medium control. A maximum is reached at approximately 1 mg [S,S]-EDDS l-l, which corresponds to a free pcO

(PM&d = -log[Metal]) of 9-11 M, a free pcU of 11-13 and a free pzn of 7.559. A

further increase in [S,S]-EDDS concentration leads to a decrease in algal cell numbers. At the extremes of the free metal concentrations, algal growth is impaired or

completely inhibited. Most consistent is the low growth at the left side of the curves. This corresponds to the high end of the [S,S]-EDDS range of concentrations (3-10 mg 1-l) and the low levels of free metals (PC” = 16-17; pcO = 13-14; pzrl =: 1 l-12.5)

and is the zone of nutrient depletion. Growth is also impaired at low levels of the

chelator (O-O.5 mg l-l), caused by toxic concentrations of free metals in this metal- enriched medium. These effects are evident at 72, 96 and 143 h (Fig. 3, middle series).

A slightly different behaviour is observed if the data are expressed as total cell volumes (Fig. 3, lower series). Total cell volume is apparently less sensitive to metal

toxicity. When comparing the deficiency effects on algal cell volume at 72 and 143 h, a shift toward less pronounced effects can be observed over time.


3.4. Toxicity test with varying Co, Cu and Zn levels

The effect of increased metal levels on the growth of the alga Chlorella vulgaris

was explored. A number of variations in trace metal concentrations were tested to illustrate which metal or combination of metals were causing the toxic effects. In addition, a growth test was done in river water, with and without 1 mg 1-l [S,S]- EDDS, to better understand laboratory versus field effects (Table 4). The highest algal growth rate and biomass level achieved (0.108 h-l, 10184 X lo6 urn3 ml-i) was obtained in medium 6. Total Co and Cu concentrations were 6.3 x 10e7 and 5.1 X 10e8 M, respectively, or about 100 and 1000 times higher than in the standard OECD medium. The corresponding free pcO and pcll levels were 7.04 and 7.35. At these levels, the growth rate was identical to that obtained in River Meuse water enriched with OECD nutrients. It was observed that under those optimised conditions, Chlorella vulgaris showed little or no tendency for synchronous growth. This also illustrates the influence that trace metal levels have on algal growth rate and growth pattern. In fact, in all treatments, with the exception of medium 9, growth rates were above that of the control.

For Co, no significant toxicity was observed, even at the highest tested total concentration of 18.5 x 1O-7 M in medium 8 (free pcO = 7.04). Similarly, Cu was found to support algal population growth up to a total concentration of 15 X 1OP M in medium 10 (free pcu = 7.47). In medium 3, the presence of 2.9 X 1OP M total Zn (free pZn = 5.73) supported normal growth. However, a clear inhibition of algal growth was noted if the total Zn level was raised to 8.8 X 1OP M (medium 9). This corresponded to a free pzn of 5.21.

In River Meuse water spiked with OECD nutrients, [S,S]-EDDS was also found to reduce algal population growth. However, the Meuse water medium in the presence of 1 mg 1-l [S,S]-EDDS supported an algal growth rate (0.071 h-l) which was significantly higher than in standard OECD medium without chelator (0.057 h-l, Table 4).

3.5. Short-term photosynthesis inhibition test

The effect of [S,S]-EDDS on photosynthesis was tested in a short-term experiment with the alga S. capricornutum. Fixation of CO:! into acid-stable counts was linear from test initiation to approximately 8 min (Fig. 4). After 8 min the supply of 14C02 became limiting. [S,S]-EDDS at 100 mg 1-l caused a minimal reduction in the CO2 fixation rate of approximately 10% after 4 min, and 14% after 8 min incubation with 14COs. The observed reduction was statistically significant from the control at 8 min (P = 0.022) but not at 4 min (P = 0.30). All other exposure concentrations produced no reduction, or a stimulation of photosynthesis. No EC& for photosynthesis could be estimated, but it is clearly greater than 100 mg 1-l. Testing at higher concentrations was considered irrelevant for the environmental situation. The positive control CL? produced a pronounced effect; about 50% inhibition of the photosynthesis rate at 0.1 mg 1-i.


4. Discussion

To produce results interpretable as toxicity in a standard effects test, only the toxicant concentration should vary. Increased effects can then be related to increased toxicant concentration. However, in an algal test with a chelator as the toxicant, the free nutrient trace metal concentrations can change with chelator

concentration in a non-linear way over several orders of magnitude (Fig. 3). Algal growth rate and biomass levels during the exponential phase depend directly on the

availability of the essential trace metals. With strong chelators, the bioavailable fraction of trace metals can be easily reduced to a level below which algal growth

will be impaired. This can potentially be confused with toxic effects. It is therefore of great practical importance to understand trace metal speciation along with algal

population changes to predict the toxic level.

4.1. Photostability

During the development of the chelator EDDS, the racemic material (a mixture of 25% [R,R-1, 50% [R,S-1, and 25% [S,S]-form) was tested first and was found to

have an algal EbC50 of 0.29 mg 1-l with Chlorella vulgaris. This contrasted with the favourable acute toxicity data for fish and invertebrates, and triggered further

research on the [S,S]-form, which is the fully biodegradable enantiomer. A photostability pre-test was executed with [S,S]-EDDS because some chelators such as

phosphonates and EDTA are known to form metal complexes which are photo- labile. In particular, the Fe3+ and Cu2+ complexes of these chelators were found to

be sensitive to UV and sunlight (Matthijs et al., 1989; Frank and Rau, 1990;

Gledhill and Feijtel, 1992). In this study, [S,S]-EDDS was found to be stable under

the test conditions. Thus, algal effects testing could be conducted in the high light conditions of the laboratory environment.

4.2. Hardness eflects

The most important conclusion from the hardness experiment is that the growth

pattern of Chlorella vulgaris was not significantly affected by the water hardness in the presence or absence of [S,S]-EDDS. This is what would be predicted for a

strong chelator by speciation calculations and the nutrient depletion concept.

4.3. Metal enrichment tests

The experiments in metal-enriched media were aimed at demonstrating the validity of the nutrient depletion hypothesis in a quantitative way. By varying the level of free trace metals via the chelator, the typical pattern for a limiting nutrient, i.e. a bell-shaped curve, was produced (Fig. 3). The poor growth at the left and right sides of such curves (e.g. Fig. 3, middle and lower series) can be explained by nutrient (trace metal) deficiency and toxicity, respectively. The experiment with OECD 2M

medium showed that growth increased with increasing chelator level, up to 1 mg


1-l. This is exactly the [S,S]-EDDS level at which the concentration of trace metals is the same as in standard OECD medium without chelator, as calculated by chemical equilibrium. It should be noted also that as the level of [S,S]-EDDS increases, the free, i.e. potentially the most toxic form of the chelator, becomes relatively more important. Such a dose-response relationship is the reverse of what would be observed in a normal toxicity test and clearly illustrates that [S,S]-EDDS is not inherently toxic to algae in the O-l mg 1-l concentration range. The decreased growth beyond 1 mg 1-l is ascribed to suboptimal free nutrient levels (i.e. lower than in standard OECD medium).

4.4. Nutrient requirements and metal toxicity

To validate these conclusions, the nutrient requirements and metal sensitivity of Chlorella vulgaris were reviewed. A very good correlation between our observations and the literature was found. Deficiency can probably be ascribed to suboptimal Cu levels. Sunda (1975) reported that Chlorella vulgaris becomes Cu-limited at a free pcU of 16. Knauer et al. (1994) found that optimal growth of Chlorella fusca occurs at a free pcU of 10-13, with toxicity above pcU = 10.

The inhibition effects in OECD 2M medium in the absence of [S,S]-EDDS (cf. Fig. 3 and Table 3) are primarily related to Zn toxicity. According to our metal toxicity test, Co would not be involved, at least up to a pcO of 7.04. However, Zn has been demonstrated in the metal toxicity test (Table 4) to produce a sharp decrease in algal growth between a free pzn of 5.73 and a pzn of 5.21. In the experiments with metal-enriched OECD medium (Fig. 3, middle series), a sharp decrease in algal cell numbers is indeed observed in that pzn zone. Another obser- vation is that at higher free trace metal concentrations the cells are still able to increase their size, but cell division does not follow (Fig. 3, differences between middle and lower series). This decoupling between cell volume and cell division in these treatments also hints at Zn toxicity. Ting et al. (1991) reported that Chlo-

rella vulgaris forms enlarged cells as a result of a disturbance of cell division at a pZn

below 5. This is at a slightly higher concentration than observed in our tests, but it relates to total Zn levels. The free levels were not reported by Ting et al. (1991).

Rachlin and Gross0 (1993) found that the EC 50 for Chlorella vulgaris corresponded with a free pcU of 5.1. In our metal toxicity test, a free pcU of 7.47 was non-toxic. In Fig. 3 (middle series), it can be observed that the growth of Chlorella

vulgaris is reduced as of a pcll of 10-l 1. This suggests that Cu is not directly involved in the toxicity. However, because of the high affinity constant of [S,S]- EDDS for Cu relative to Zn, small changes in Cu levels may indirectly affect free Zn levels in a significant way.

4.5. Nutrient deficiency

Several other authors have observed chelator effects comparable to those described in this paper and also concluded that nutrient depletion is the most plausible cause. Some examples are discussed here. Zeolites are insoluble sodium alumino-


silicates effective in sequestering Ca2+ ions. Algal growth is initially inhibited in the concentration range 0.1-10 mg 1-i (LOEC values; Zeohtes HEDSET, 1994, and references cited therein). However, when zeolites were pre-saturated with Ca2’ and Mg2+ ions, or when the medium was enriched in nutrients, the inhibition level was markedly raised (Code, 1983). A combination of macronutrients containing Ca2+,

Mg2+, and K” ions was more effective in avoiding algal growth inhibition by zeolites than a solution of trace elements. This is exactly what would be expected for a weak chelator in light of the nutrient depletion hypothesis.

Studies by Hamilton et al. (1994) on polycarboxylates have demonstrated that algal effect levels may be increased by at least lo-fold when Ca2+. and/or Mg2+ are added to the test medium.

Some growth inhibition of ~~le~ustr~rn c~p~ico~n~t~rn in Lake Erie water was observed with the weak chelator citrate at concentrations between I and 10 mg 1-l. Inhibition was not found when primary or secondary effluent was added (Payne, 1973). The author postulates that the effect is due to the chelation of trace metals, as citrate is known as a compound with a very low inherent toxicity.

The inhibitory effect of NTA, a chelator with intermediate affinity constants, towards algae is not very pronounced and varies considerably with the species. Yet the algal EC5s values are significantly below those for other aquatic test organisms. Cyclotella nana was found to be one of the most sensitive organisms with a LOEC value between 1 and 5 mg 1-l (Anderson et al., 1985). A clear effect of water hardness was also observed for NTA; the EC50 for Navicula semulinum was 185 mg 1-l in soft water, and 477 mg 1-l in hard water. Also for the class of suc~inate-tartrate builders, which are ‘intermediate type’ chelators, similar hardness effects were observed (Pittinger et al., 1992).

Despite its widespread use, few data have been published on the effect of the strong chelator EDTA on algae. The algal EC30 (l-10 mg 1-i) is significantly below that for fish and Daphnia (over 500 mg 1-l). In the OECD medium the EDTA concentration is about 0.1 mg I-’ (0.3 FM). This is sufficient to keep essential trace metal ions in solution, but is below the effect threshold. In one experiment, Soria Dengg and Horstmann (1993) demonstrated that uptake of ““Fe is markedly inhibited by 14.6 mg 1-l EDTA (50 PM) in a medium very low in trace elements. This can possibly be explained by the reduction of the free Fe concentration by EDTA. Gledhill and Feijtel (1992) reported that, up to 10 mg l-l, the effects on algal growth of phosphonate chelators in general is algistatic and not algicidal. This could also be an indication that these strong chelators at moderate concentrations act through nutrient depletion, but not as toxicants. Overall, effects that can be explained by nutrient depletion have been reported for all types of common com- mercial chelators.

We compared the results of the growth inhibition tests with a short-term photosynthesis assay. This test assesses the interaction of the chelator with the photosynthesis process, a relevant endpoint integrating various cellular processes and an

212 D. Schowanek et dlrlquatic Toxicology 36 (1996) 253-275

attractive alternate endpoint to growth (Peterson and Nyholm, 1993). Because the time interval is short, algae will survive on stored nutrients, eliminating any con- founding or secondary effect of the chelator on nutrient availability. Versteeg (1990) has tested a suite of chemicals by means of the short-term photosynthesis inhibition test and compared the results with those from a standard growth test. The short- term test was found to be somewhat less sensitive, but overall correlated well with longer growth tests (Versteeg, 1990; Peterson and Nyholm, 1993). Our tests showed no effect at 10 mg I-‘, and only a minimal effect at a level of 100 mg 11” [S,S]- EDDS. This indicates that even the free form of [S,S]-EDDS has a low inherent toxicity to algae.

4.7. Environmental relevance

Although by itself nutrient depletion is a reproducible effect in the laboratory, it is not necessarily relevant for the environmental situation. In the case of [S,S]- EDDS, the predicted environmental concentration in the aquatic compartment is in the order of 1 pg 1-l (D. Schowanek, unpublished, 1995). At this chelator concentration the levels of free Cu, Zn and Co are not reduced significantly. Secondly, algae appear to be able to overcome conditions of nutrient deficiency via biochemical acclimation mechanisms. Algae, Like other classes of microorganisms, can influence metal bioavailability by synthesising siderophore-type chelators in order to satisfy their requirements for trace elements (McKnight and Morel, 1979). At the other extreme, natural or man-made chelators can serve to mitigate metal toxicity towards microorganisms by reducing free metal levels (Morris and Russel, 1973; Peterson and Nyholm, 1993; Xue and Sigg, 1993; Tubbing et al., 1994). This was also illustrated in our experiments (test in OECD 3M medium).

Some authors (e.g. Gode, 1983; Hamilton et al., 1994) have pointed out that over-chelation of nutrients may be a laboratory artifact only, because many chelators used in industrial or domestic applications are dosed as the acid or sodium form, but will be released to the natural environment loaded with Ca2+, Mg’+, or other ions. For those chelators that have not been pre-exchanged with metal ions, the hardness of the receiving waters (120-- 150 mg 1-l on average) may cause a mitigation of toxicity relative to standard tests. This hardness effect is, however, not expected to be important for [S,S]-EDDS, but could play a role for many weaker chelators. Finally, Steber and Wierich (1987) and Horstmann and Groh- mann (1988) observed in outdoor flow-through systems a stimulation of algal growth by phosphonates, whereas the same concentrations produced an inhibitory effect in the laboratory.

5. Conclusions

This work supports the existing body of evidence that ‘over-chelation’ of essential nutrient trace metals can create an imbalance in the media used for algal growth inhibition tests. This leads to deficiency of essential trace elements, and therefore


shows an apparent toxic effect on algal growth. The toxicity sensu strict0 of a

chelator towards algal growth and metabolism is difficult to separate from effects on nutrient bioavailability, unless one understands the trace metal speciation in the test medium. For [S,S]-EDDS, standard algal growth tests probably overestimate the ‘true’ EC50 by at least one order of magnitude, as shown in the short-term

photosynthesis test. The depletion effect can be overcome by supplementing the

medium with appropriate amounts of growth-limiting nutrients. These are a func-

tion of: (1) the type of chelator and its specific affinity pattern, (2) chelator concentration, and (3) the intrinsic nutrient requirements of the algae. This work

illustrates that speciation modelling is a useful, if not essential element to evaluate the effects of chelators in algal tests. The use of a short-term photosynthesis test provides an additional approach to understanding the interaction of the chelator

with the algae. Nutrient availability effects of chelators seen in the laboratory may be difficult to

extrapolate to the real world because anthropogenic chelator concentrations will

typically fall in the low ppb range, biochemical adaptation mechanisms in the algae may be triggered, and other factors such as water hardness, higher essential trace

metal levels in natural water versus OECD test medium, or photodegradation may

alter the effects. Results from this study illustrate the inappropriateness of the algal growth inhibition test for assessing the toxicity sensu strict0 of chelators.

Acknowledgements

The algal growth inhibition tests were skilfully carried out at TN0 by Harry Oldersma.

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