Effect of linear alkylbenzene sulfonate (LAS) and atrazine on marine microalgae

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Marine Pollution Bulletin 57 (2008) 559–568 www.elsevier.com/locate/marpolbul

Effect of linear alkylbenzene sulfonate (LAS) and atrazine on marine microalgae B. Debelius a,*, J.M. Forja a, A. Del Valls a, L.M. Lubia´n b a

Departamento de Quı´mica-Fı´sica, Facultad de Ciencias del mar y ambientales, Universidad de Ca´diz, Avda Repu´blica Saharaui, s/n, 11510, Puerto Real, Ca´diz, Spain b Instituto de Ciencias Marinas de Andalucı´a, Avda Repu´blica Saharaui, 2, 11510, Puerto Real, Ca´diz, Spain

Abstract Five marine microalgae (Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana), in the same biovolume quantity, were exposed to 72 h growth-inhibition tests with atrazine and LAS. In all cases, the inhibition effect of atrazine was higher than that of LAS up to two orders of magnitude higher in the case of T. chuii. In a second part of the study, initial cellular densities for each microalga strain and fixed organic compound concentration were varied, and results show density has a clear influence in growth inhibition tests. Finally, the sum of all data obtained in the study was expressed in terms of ‘‘toxic cellular quota” (mass of chemical substance per cell). The result was a sigmoid curve with a good fit, including the two main factors in toxicity tests, initial cellular density and contaminant concentration. This toxic cellular quota exhibits a general tendency to increase with cell volume/size. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Atrazine; Las; Marine microalgae; Toxic cellular quota; Flow cytometry; Toxicity tests

1. Introduction The effect of toxic substances on organisms, and especially on aquatic ecosystems, is a very important component for environmental and ecotoxicology sciences. The effect on phytoplankton communities is complex, they are taxonomically more variable than periphyton communities. Their function as primary producers makes them key targets for main environmental contaminants such as herbicides and detergents. Generally, toxicity bioassays on marine microalgae are undertaken in laboratory toxicity tests following the directives of protocols written mostly for freshwater microalgae (OECD, 1984; APHA, 1985; USEPA, 1985; EEC, 1987; ISO, 1987): there are few tests that can be described as ‘‘standard” for marine microalgae, although guidelines have been published and some marine

*

Corresponding author. E-mail address: [email protected] (B. Debelius).

0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.01.040

species have been recommended as test strains (Walsh, 1988, 1994). The triazine herbicides are among the most widely used, representing nearly 40% of the herbicides in use in European countries (Mason et al., 2003). Atrazine (2-chloro-4ethylamino-6-isopropylamino-s-triazine) is a herbicide used to control broadleaf weeds in agricultural crops; it inhibits photosynthesis by blocking electron transport between photosystems II and I (Hull, 1967) and as a herbicide it mostly ends up in surface waters. The prevalence of atrazine in aquatic systems and its potential toxicity in many aquatic organisms, particularly phytoplankton, make it an important chemical to study (Weiner et al., 2004). Also, since atrazine directly affects phytoplankton, many zooplankton species show a reduction in reproduction and growth when their ecosystems are exposed to this compound in single species laboratory tests (Graymore et al., 2001). Atrazine concentration values found in surface water range from 0.12 to 0.30 ppb with peaks in waters directly adjacent to treated fields of 62.5 ppb to 300 ppb

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(Weiner et al., 2007) or even up to 1000 ppb (Graymore et al., 2001). Linear alkylbenzene sulfonate (LAS) is one of the most common organic chemicals used in detergents and personal care products. Due to its widespread use, it has been detected at various concentrations in river water, seawater (2–510 ppb) and sediment (0.4–49 lg g 1) (Go´nzalez-Mazo et al., 1997; Matthijs et al., 1999). It is known that LAS produces alterations in membrane permeability, enzyme activity, and tissue structure in organisms (Blasco et al., 1997). Published information regarding the effect of LAS on marine microalgae is scarce (Hampel et al., 2001), although there are more studies on freshwater microalgae. It has been confirmed that inhibition of growth and of photosynthesis, as well as other variables closely related to photosynthesis, reflect the toxic effects of pollutants on microalgae (Franqueira et al., 2000). Flow cytometry has been applied in the study of aquatic environments since the 1980s (Yentsch and Pomponi, 1986); it allows rapid, multiparameter analysis of individual cells in moving fluid, and has sufficient sensitivity for the analysis of cell densities that are more typical of algal concentrations in natural systems, such as 102 cells mL 1 (Franklin et al., 2002). These factors make flow cytometry an excellent instrument for analysis of microalgae in toxicity tests. This study has several objectives. The first is to assess the toxicity of two organic compounds, following the guidelines of most of the standard methods, for five marine microalgae representing different taxonomic families (brought to the same biovolume quantity): Tetraselmis chuii, Rhodomonas salina, Chaetoceros sp., Isochrysis galbana (T-iso) and Nannochloropsis gaditana. The second is to consider the influence of initial cellular density on the EC50 values; different densities were used to demonstrate the importance of this parameter in toxicity tests. Finally, the expression of all data as ‘‘toxic cellular quota” combining the two factors studied in the first and second parts of this study, contaminant concentration and cellular density. 2. Materials and methods 2.1. Algal cultures and culture conditions Species corresponding to different classes (included in Table 1), T. chuii (Prasinophyceae), R. salina (Cryptophyceae), Chaetoceros sp. (Bacillariophyceae), I. galbana (Tiso) (Prymnesiophyceae) and N. gaditana (Eustigmatophyceae) were obtained from the Marine Microalgal Culture Collection of the Instituto de Ciencias Marinas de Andalucia (Lubia´n and Yu´fera, 1989). These algal strains were maintained with natural seawater from the Bay of Cadiz, previously filtered by GF/F (Whatman) filters and sterilized by autoclaving. The medium was enriched with a final concentration composed of: NO3 (123.88 lM), PO34 (4.01 lM) and, in the case of Chaetoceros sp., SiO2 (50 lM). This medium, rather than another standard medium, such as Guillard´s f/2 formulation, was chosen because

Table 1 Main characteristics of the five microalgae used in the study Volume (lm3)

Surface (lm2)

Eustigmatophyceae

7.7

18.69

42.18

Prymnesiophyceae

48.9

64.72

6.64

Bacillariophyceae Cryptophyceae

76.7 212.3

87.53 171.94

4.23 1.52

Prasinophyceae

324.8

227.93

1.00

Microalgae species

Classes

Nannochloropsis gaditana Isochrysis galbana (T-iso) Chaetoceros sp. Rhodomonas salina Tetraselmis chuii

Initial density (104 cell/ mL)

we were particularly concerned to conduct experiments in conditions as close as possible to those found naturally. 2.2. Toxicity tests All five microalgae were inoculated from cultures in a log growth phase to obtain the initial cellular densities shown in Table 1. The OECD (1984) algal growth inhibition test guideline for testing chemicals was followed, although medium composition and recommended species were modified for this particular assay. All glassware was cleaned with dilute nitric acid (10%) and rinsed several times with Milli-Q ultrapure water before experiments. was supplied by Petresa S.A. LAS C12, (M = 348.4 g Mol 1) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) was obtained from Sigma–Aldrich (M = 215.69 g Mol 1). Based on the volume of each strain, measured with a Coulter Counter, strains were brought to the same biovolume quantity, 324  104 lm3 mL 1; T. chuii was chosen as the reference. This allows the results of toxicity sensitivity obtained for the different strains to be compared. The five stock cultures and all toxicity tests were kept at 20 ± 1 °C, under continuous white light, in a culture chamber. In prior tests, a wide range of LAS and atrazine concentrations was assayed in order to find the appropriate range of toxicity for each species of microalga. For the 72 h toxicity tests, glass Pyrex tubes were used to hold 5 mL of test medium and were placed on an orbital shaker to homogenise the medium. Exponentially-growing populations of each microalga species were exposed to 10 progressively increasing concentrations of each organic compound, including a control. All toxicity tests were performed in triplicate. 2.3. Influence of cellular density In order to study the influence of cellular density on toxicity tests, concentrations of LAS and Atrazine were set at

B. Debelius et al. / Marine Pollution Bulletin 57 (2008) 559–568

the approximate EC50 values obtained in the toxicity tests for each strain. Tests with five different initial cellular concentrations were then analysed. The initial cellular concentrations for each microalgae strain established were the result of multiplying the initial cellular concentrations used for the toxicity tests, by the factors 0.1, 0.5, 1, 5, and 10, and all experiments were performed in triplicate. 2.4. Flow cytometry analysis Samples for analysis by flow cytometry were collected from the toxicity tests after 72 h of treatment. These were analysed using a FACSCalibur (Becton–Dickinson) flow cytometer, equipped with a blue argon-laser (488 nm) and a three colour photomultiplier with fluorescence emission filters (FL1 515–545, FL2 564–606 nm, FL3 > 650 nm; forward light scatter (FSC) and side scatter (SSC)). Data were computed with CellQuest software (Beckton–Dickinson). Each culture was analysed for 30– 60 s (6000–10,000 events per measurement, at a flow rate previously calibrated by an established sample weight during a constant period of time) from samples previously fixed with 3–4% formaldehyde. The reason of fixing the samples was due to the big difference in time (in some cases up to 5 h) when measuring by flow cytometry from the first to the last sample. This way all samples were fixed at the same time (at the hour 72). Counts, signals of side-angle light scatter (SSC), and autofluorescence (FL3 > 650 nm) obtained as arbitrary units were recorded. SSC signal was used as cellular size indicator, previous studies show that this signal has a better correlation with the cellular size than FSC (Sobrino et al., 2005), FL3 was used as indicator of chlorophyll fluorescence and FL2 as phycoeritrin signal. 3. Results 3.1. Toxicity of atrazine and LAS The degree of growth inhibition was affected in different ways by atrazine or LAS, depending on the microalgal strain and organic contaminant concentration in the media (as shown in Fig. 1). Data results obtained by flow cytometry, after 72 h of exposure to atrazine and LAS for each organic compound and microalga tested, were fitted to sigmoid equations (Table 2). Then EC50 values were obtained from the sigmoid equation corresponding to each contaminant and microalga (Table 3). From the EC50 values calculated for each microalga, it is found that the contaminant effect of atrazine results in lower EC50 concentrations than that of LAS. Of the five strains studied, T. chuii showed the lowest value, and is therefore the most sensitive to atrazine exposure. Ranked from the lowest to the highest rank of sensitivity to atrazine, the rest of the microalgae studied are the following: N. gaditana, R. salina and I. galbana (T-iso)  Chaetoceros sp. However, in the case of LAS, the order in increasing sensitivity is:

561

T. chuii, R. salina, N. gaditana, I. galbana (T-iso) and Chaetoceros sp. Tetraselmis is already established as one of the most resistant genera to some toxicants such as trace metals and LAS (Maeda and Sakaguchi, 1990; Moreno-Garrido et al., 2001), but this is not the case with exposure to atrazine, as is shown in this study. On the other hand, Chaetoceros sp. has already shown to be very sensitive to some toxicants (Moreno-Garrido et al., 2001). In this study, Chaetoceros sp. is found to be one of the most sensitive to the two organic compounds studied. As observed, there is a difference in species-sensitivity, with significant differences in the case of the two organic compounds studied. 3.2. Flow cytometry data (FL2 and FL3) Since all data were obtained by flow cytometry, lightscattering (SSC) and fluorescent properties (FL2 and FL3) of algal cells were also determined; these provide additional information regarding the toxic mode of action of atrazine and LAS, which differs between species. Data obtained from the SSC signal (light-scattering), corresponding to cell size for the five marine microalgae studied, showed different variations. Tetraselmis chuii and R. salina showed a slight decrease in the SSC signal, corresponding to a decrease in cell size, when exposed to atrazine (Fig. 2). This tendency, in the case of LAS, was also observed but only in the case of N. gaditana. However, the other microalgae studied, Chaetoceros sp. and I. galbana, showed hardly any change in SSC data. The result of the average signal value multiplied by the respective cell density in each case, termed ‘‘total FL3”, was represented as degree of inhibition, in function of the toxic concentration. The curves obtained for the relationship between total FL3 and atrazine or LAS concentration showed similarity to those obtained in Fig. 1. As shown in Table 3, EC50 values calculated from total FL3 inhibition are slightly higher compared to those obtained from the growth inhibition effect, and standard deviation data also show higher values. Statistical T-test analysis were followed and did not show significant differences for most of the strains studied of EC50-LAS calculated by the two different ways. When comparing EC50-atrazine,in this case, statistical significant differences were found for two of the five strains studied Chaetoceros sp. and T. chuii (P = 0.010 and P = 0.0005, respectively). Most protocols and several authors state that growth inhibition is the best effect to study in toxicity tests with microalgae (Stauber, 1995; Hampel et al., 2001). 3.3. Influence of cellular density The second objective of this study was to demonstrate that growth inhibition for all five microalgae exposed to atrazine and LAS decreases with an increase of the initial cellular density of the bioassay. This can be seen in Fig. 3 where, for the same organic compound concentration, at

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B. Debelius et al. / Marine Pollution Bulletin 57 (2008) 559–568 100

Nannochloropsis gaditana

% Growth Inhibition

% Growth Inhibition

100 80 60 40 20 0 0

200

400

600

800

% Growth Inhibition

% Growth Inhibition

80 60 40 20

Isochrysis galbana

20

400

800

1200

80 60 40 20

Isochrysis galbana

0 0

200

400

600

0

1000

2000

3000

100

% Growth Inhibition

100

% Growth Inhibition

40

100

0

80 60 40 20

Chaetoceros sp.

80 60 40 20

Chaetoceros sp.

0

0 0

200

400

600

800

0

1000

100

400

800

1200

100

% Growth Inhibition

% Growth Inhibition

60

0 0

1000

100

80 60 40 20

Rhodomonas salina

0

80 60 40 20

Rhodomonas salina

0 0

200

400

600

800

1000

0

1500

3000

4500

6000

100

% Growth Inhibition

100

% Growth Inhibition

Nannochloropsis gaditana

80

80 60 40 20 Tetraselmis chuii 0

80 60 40 20 Tetraselmis chuii 0

0

200

400

600

0

1000

Atrazine (ppb)

2000

3000

LAS (ppb)

Fig. 1. 72 h Growth inhibition test for five marine microalgae exposed to different concentrations of atrazine and LAS. The line plot corresponds to the fitted sigmoid curve used to calculate EC50 values.

lower values of initial density, higher values of inhibition percentage are obtained. For example, in the case of I. galbana and LAS contaminant, the same LAS concentration was almost 10 times more toxic at an initial cellular density of 104 cells mL 1 (90% growth inhibition) than at a cellar density of 106 cells mL 1 (10% growth inhibition).

4. Discussion 4.1. Toxicity of atrazine and LAS The EC50 values determined for atrazine in this study (20–209 ppb) are within the upper range of atrazine con-

B. Debelius et al. / Marine Pollution Bulletin 57 (2008) 559–568 Table 2 Parameters for the sigmoid equations of data results obtained by flow cytometry, after 72 h of exposure to atrazine and LAS for each organic compound and microalga tested r2

a

b

x0

Atrazine N. gaditana I. galbana Chaeotoceros sp. R. salina T. chuii

63.85 97.22 89.19 91.27 89.74

55.23 22.34 28.02 178.05 10.97

116.14 36.26 46.34 423.18 22.83

0.92 0.94 0.95 0.94 0.97

LAS N. gaditana I. galbana Chaeotoceros sp. R. salina T. chuii

82.57 87.62 81.45 93.17 67.61

85.32 3.69 52.97 288.26 375.24

634.06 869.96 252.07 1155.15 1170.88

0.97 0.98 0.99 0.98 0.92

Table 3 EC50 values obtained from growth inhibition and total FL3 inhibition for five microalgae strains Microalgae

Nannochloropsis gaditana Isochrysis galbana (T-iso) Chaetoceros sp. Rhodomonas salina Tetraselmis chuii

EC50 growth inhibition

EC50 total FL3 inhibition

Atrazine (ppb)

LAS (ppb)

Atrazine (ppb)

LAS (ppb)

209 ± 40

776 ± 50

185 ± 36

609 ± 97

30 ± 4

480 ± 85a

32 ± 20

43 ± 5a 165 ± 40 20 ± 4a

242 ± 34 1211 ± 176 1840 ± 54

107 ± 50a 284 ± 127 72 ± 25a

834 ± 220a 340 ± 130 1331 ± 429 1911 ± 530

a

Values resulted with significant t-tests differences between EC50 calculations from growth inhibition or FL3 inhibition (P < 0.01).

centrations that have been measured in water environments. Typical surface water concentrations range from 0.1 to 30 ppb with peaks in waters near to treated fields of 62.5 ppb to 1000 ppb (Weiner et al., 2007; Graymore et al., 2001). On the other hand, in the case of LAS, the range of EC50 values obtained in this work (242– 1840 ppb) do include those found in natural environments, such as range concentrations of 2–510 ppb found in the Bay of Cadiz (Go´nzalez-Mazo et al., 1997). Therefore,

atrazine, in spite of demonstrating lower EC50 values, which is traduced in a bigger toxic effect on marine microalgae, whereas the higher concentration values are found for LAS in natural water environments, LAS is thought to have a bigger influence and consideration in marine environments than atrazine. When comparing EC50 values obtained in this work with others already published we find several problems in finding the same microalgae species and same conditions or contaminant exposition time in toxicity tests. There is still controversy over the effects of atrazine and LAS as different studies have given contrasting results. Moreno-Garrido et al. (2001) obtains LAS EC50 values for different marine microalgae including Chaetoceros sp. and R. salina with results very similar to those obtained in this study 300 ppb and 900 ppb, respectively. Atrazine toxicity tests carried out with marine microalgae are more difficult to find, authors such as Weiner et al. (2004) obtained EC50 (96-h) of 91.10 ppb for I. galbana. This value is three times the one obtained in this study, this is probably due to the different exposition time periods and conditions of the toxicity tests. Other recent studies made on microalgal assemblages such as the established by Garcı´a-Villada and Reboud (2007) obtain significant growth rate inhibitions at atrazine concentrations greater than 60 ppb. Among phytoplankton, sensitivity to LAS and atrazine has been shown to vary across phyla (Guanzon et al., 1996). Algal responses to contaminants vary widely depending on species tested, contaminant concentrations, and endpoints measured. The difference in species-sensitivity to atrazine and LAS of this study covers a wide range, with significant differences in EC50 values. The high variability in sensitivity of different algal species to the same chemical substance can be explained: first of all by the morphology, cytology, physiology and phylogenetics of the organisms and finally, by the mode of action of the toxic. Atrazine and LAS have big differences in the mode of toxic action in microalgae. Atrazine inhibits photosynthesis by blocking electron transport between photosystems II and I. Besides, Gala and Geisy (1990) suggest that the mode of action of atrazine is algistatic, causing inhibition of growth without a corresponding increased mortality rate. Gustavson and Wa¨ngberg (1995) indicate that atrazine has

400

200

0

200

400

600

Atrazine (ppb)

800

N. gaditana

SSC (arbitrary units)

SSC (arbitrary units)

600

Tetraselmis chuii Rhodomonas salina

563

16

12

8

0

300

600

900

LAS (ppb)

Fig. 2. Decrease of SSC (volume/size) for three different microalgae exposed to increasing concentrations of atrazine and LAS.

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B. Debelius et al. / Marine Pollution Bulletin 57 (2008) 559–568 100

100

N. Gaditana Atrazine = 209 ppb LAS = 776 ppb

% Growth inhibition

% Growth inhibition

80

I. galbana

60

40

20

80

Atrazine = 30 ppb LAS = 480 ppb

60

40

20

0

0 0

1

2

3

4

0

5

5

10

15

5

20

25

-1

x10 cells mL

x106 cells mL-1 100

Chaetoceros sp.

% Growth inhibition

80

Atrazine = 43 ppb LAS = 242 ppb

60

40

20

0 0

1

2

3

5

4

5

-1

x10 cells mL 100

100

T. Chuii

R. Salina Atrazine = 165 ppb LAS = 1211 ppb

60

40

20

80

% Growth inhibition

% Growth inhibition

80

Atrazine = 20 ppb LAS = 1840 ppb

60 40 20 0

0 0

4

8 4

x10 cell mL

12

16

-1

0

2

4 4

6

x10 cell mL

8

10

-1

Fig. 3. Growth inhibition curves for different initial cellular densities in five marine microalgae, exposed to a constant concentration of atrazine (d) and LAS (.).

the same effect than decreased light levels. At low light levels, when the electron transport rate is limited for photosynthesis, a long-term compensation occurs through an enlarged antenna, which is seen as an increased chlorophyll concentration. On the other hand, in the case of surfactants, the lipophilic structure is responsible for the inhibition of cell growth (Rieb and Grimme, 1993); species-sensitivity to the same surfactant can vary as much as three orders of magnitude and the effects of different surfactants on the same algal species, can vary as much as four

orders of magnitude (Lewis, 1990). Little is found about LAS toxicity tests on marine microalgae, but previous authors such as Maeda and Sakaguchi (1990) and Moreno-Garrido et al. (2001) agree with results obtained in this work considering Tetraselmis as one of the most resistant genera to some toxicants such as trace metals and LAS. The sensitivity difference observed between different classes of algae is already established. In the case of atrazine, Hamilton et al. (1987) found that chlorophyta (green algae) appear to be the most sensitive. Considering that

B. Debelius et al. / Marine Pollution Bulletin 57 (2008) 559–568

4.2. Flow cytometry data Changes in SSC values traduced as cellular size variations depend on the strain, concentration and contaminant exposure, already established by Franklin et al. (2001). Several authors, using optical and/or electron microscopy (Fisher et al., 1981; Stauber and Florence, 1987; Bolan˜os et al., 1992) and flow cytometry (Abalde et al., 1995; Cid et al., 1996; Franqueira et al., 2000; Franklin et al., 2001,2002), have previously observed an increase in cell volume of several species of microalgae in response to toxic levels of metals. On the other hand, very little information about cellular size variations is found for the effect of LAS and atrazine (Franqueira et al., 2000). A recent study, in agreement with this work, followed by Weiner et al. (2007) observed significant decreases in both cellular biovolume and dry weight in four of the five species used in the study following atrazine exposure.

other words, it allows cellular density and contaminant concentration values to be combined in one single expression, and therefore fixes two parameters that clearly influence inhibition growth in one. This is a similar concept to that proposed by Droop (1968) for nutrients: that algal growth rates are related to intracellular nutrient concentrations, and uptake of nutrients is not coupled with growth under nutrient-deficient conditions. Moreover, Stratton and Giles (1990) state that toxicity values which consider the exposure of each organism to toxicity, in terms of weight per organism or total weight of organisms, are of more interest than calculated values of EC50.With the values expressed in terms of Q we can consider how much organic compound per cell is necessary to inhibit a particular % of growth (Q values and growth inhibition for the five microalgae are shown in Fig. 4). Q calculated in the second part of the bioassay, on the influence of cellular densities, showed very similar curves to those obtained in the first part of toxicity tests. From the overlap data in terms of Q from both bioassays, an exponential curve can be fitted for each strain and toxic compound, this enables the EQ50 to be calculated for each particular microalgae. This value describes the amount of chemical substance that inhibits a 50% of growth of a microalga for an established initial cellular density. As an example, Fig. 5 shows overlap data for the test specie I. galbana

100 80

% Growth Inhibition

Tetraselmis genera was previously included as a chlorophyta, results obtained in this study agree with this author, as T. chuii results to be the most sensitive to atrazine of all five strains studied. Be´rard et al. (2003) established that the ability of diatoms to tolerate atrazine may be due to the specificity of the high PSII/PSI ratio, their pigment composition (fucoxanthin predominance in diatoms), or alternative carbon fixation pathways that could compensate for the shutdown of PS II-based photosynthesis, and allow algae metabolism to continue. This was not observed in this study, Chaetoceros sp. did not show big tolerance to atrazine. Previous work undertaken by Tang et al. (1997) and Herman et al. (1986) established a relationship between algal pigment classification and sensitivity to atrazine. Following a pigment classification in this study such as these authors suggest, R. salina is the only strain with phycobilins and this strain results as the most tolerant to atrazine of all five strains studied. But other authors, such as Weiner et al. (2004) establish that results of his experiments suggested that sensitivity to atrazine cannot be predicted by algal pigment classification. Future researches should also consider the uptake of degradation products and their metabolism for a big number of algal species.

565

60 40 N. gaditana I. galbana Chaetoceros sp. R. salina T. chuii

20 0 0

10

20

30

40

50

60

Q (10-12 g atrazine cell-1)

4.3. Influence of cellular density and atrazine or LAS concentration It is clear that growth inhibition for all five microalgae exposed to atrazine and LAS decreases with an increase of the initial cellular density of the bioassay. Therefore, different EC50 values are obtained according to the initial cellular density used of a determinate microalga in the toxicity assay. Moreno-Garrido et al. (2000) introduces the term ‘‘toxic cellular quota” (Q), which refers to the mass of chemical substance that corresponds to each cell; this proves to be an easier way of viewing the relationship between chemical substance concentration and initial cellular density. In

% Growth Inhibition

100 80 60 40 N. gaditana I. galbana Chaetoceros sp. R. salina T. chuii

20 0 0

100

200 -12

Q (10

300 -1

g LAS cell )

Fig. 4. Growth inhibition in five marine microalgae for increasing values of toxic cellular quota (mass of atrazine/LAS per cell).

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100

% Growth inhibition

% Growth inhibition

100 80 60 40 20 r 2 = 0.84

0

80 60 40 20 r 2 = 0.91 0

0

2

4

6

8

10

0

Q (10-12 g atrazine cell-1)

10

20

30

40

50

Q (10-12 g LAS cell-1)

Fig. 5. All growth inhibition data for I. galbana expressed in terms of toxic cellular quota of atrazine and LAS. (d) toxicity tests fixing an initial cellular density of 6.64
104 cell/mL; (N) toxicity tests with different initial cellular densities, fixing atrazine and LAS concentration (30 ppb and 480 ppb, respectively). Dotted line corresponds to the fitted exponential curve.

authors such as Weiner et al. (2004) have established that features relating to algal cell size (e.g. dry weight, biovolume, and surface area) did not appear to be reliable predictors of species-specific sensitivity to atrazine exposure. Anyway, to be able to establish a tendency, a larger range of sizes and taxonomies than that used in this study should be monitored. 5. Conclusions The results of this study establish that atrazine has a more severe contaminant effect than LAS for the five marine microalgae studied, and that initial cellular density has a clear influence on the EC50 results for these two organic compounds. Of the five marine microalgae studied, Chaetoceros sp. was found to be the most sensitive to both atrazine and LAS; this higher sensitivity together with its ease of culture makes it a good subject for toxicity tests on ocean water. The overlap of all data obtained in the study (from toxicity tests and the influence of cellular density), expressed as ‘‘toxic cellular quota” (Q), allows the calculation of EQ50. This term describes the amount of chemical substance that inhibits 50% the growth of a microalga for a known initial cellular density; this allows a better comparison between

EQ50 (10-12 g LAS cell-1)

11 10 2 1

200 150 100 50

T. chuii

R. salina

Chaetoceros sp.

I. galbana

T. chuii

R. salina

Chaetoceros sp.

I. galbana

N. gaditana

0

0 N. gaditana

-1 EQ50 (10-12g atrazine cell )

exposed to atrazine and LAS. From the overlap of all data, for all strains studied exposed to atrazine and LAS, good fits of exponential curves resulted with r2 > 0.77. Thus, from the EQ50 (LAS/atrazine) value for a particular algae, by knowing the initial cellular density, we can calculate the atrazine or LAS concentration that inhibits growth in a 50%, or vice versa. From what has been concluded, in toxicity tests the quantity of contaminant taken up per cell has a bigger influence on toxicity than the number of cells in the medium. If we consider the mean cellular volumes for each strain, the cellular volumes of these five microalgae have a clear influence on the Q value to increase with increasing volume: more LAS or atrazine per cell is necessary for a larger volume cell. Values of EQ50 for the five microalgae in order of increasing volume are shown in Fig. 6. Authors such as Tang et al. (1998) found a significant negative correlation between cellular biovolume and atrazine sensitivity in freshwater algae with a large range of sizes, 119–10,052 lm3. In this work this tendency is not so clear in the case of atrazine, where R. salina with a smaller size than T. chuii shows a much higher EQ50 value. Rhodomonas is already reported to be tolerant to atrazine (Be´rard, 1996), therefore the importance of considering other cellular parameters that can possibly have influence in the toxicity tolerance of each cell. Other,

Fig. 6. Values for 50% growth inhibition in terms of EQ50 for the five microalgae studied, ranked by increasing cell volume/size.

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different toxicity tests. It is true that initial cellular density and contaminant concentration are not the only factors that have an influence in microalgae toxicity tests. The development of toxicity tests depend on several factors. Some of these factors, such as the composition of the test medium, temperature, illumination and toxic exposition time are easily fixed in these tests. The EQ50 allows to unify two of these factors that can suffer major variations in bioassays (initial cellular density and contaminant concentration). Therefore, the use of this value makes easier the normalization of established protocols for marine microalgae toxicity tests. EQ50 values obtained show a slight tendency to increase with volume of microalgae. However, the expression of results in terms of toxic cellular quota provides an easier way to standardise protocols, through the use of a single expression containing the values for contaminant concentration and number of cells. The use of this term offers a better comparison between different toxicity tests and therefore represents a potential scope for improvement in toxicity testing. In any case, account should always be taken of the overall effect that the physiology of the organism itself may have on the way in which the chemical substance is accumulated in the cell. The flow cytometry technique provides data on chlorophyll a fluorescence, cell size and other pigments, which makes it extremely useful for assessing toxicity to algae. These parameters provide further evidence of the toxicity effects of atrazine and LAS onto microalgae, and may also be useful as alternative endpoints in acute and chronic toxicity tests. The results of FL3 values (chlorophyll), determined as ‘‘total FL3”, did not show more sensitive than growth inhibition values. In the case of SSC values (cell size), different variations were found for the two organic compounds and the five microalgae studied. When exposed to increasing concentrations of atrazine the two larger cells, T. chuii and R. salina, showed a slight decrease in size. For LAS exposure it was the smallest cell, N. gaditana, which showed a slight decrease in size. The rest hardly showed any change. Taking all the results into consideration, it is concluded that growth inhibition is the most sensitive endpoint for calculating toxicity effects of chemical substances on marine microalgae. Acknowledgement This work was supported by the Spanish CICYT (Spanish Commission for Research and Development) of the Ministerio de Educacio´n y Ciencia under Contracts PTR1995-0971-OP-02. References Abalde, J., Cid, A., Reiriz, S., Torres, E., Herrero, C., 1995. Response of the marine microalgae Dunaliella tertiolecta (Chlorophyceae) to copper toxicity in short time experiments. Bull. Environ. Contam. Toxicol. 54, 317–324.

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