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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-06-2016 to 06-08-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met..
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WAF (water accommodated fractions). Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h, t=24 h, t=48 and t=72 h; further maintenance of actual concentrations was demonstrated by running a test vessel at 3.2 mg/L (WAF) substance concentration but without algae and samples for analysis were taken at the start, after 24 hours, 48 hours of exposure and at the end of the test period.
- Sampling method: 4 mL volume samples were extracted; the replicates were not pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days pre-culture.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 7.1 ± 0.3
- Any deformed or abnormal cells observed: None reported.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
In accordance with the OECD TG 201 guideline.
Hardness:
Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 23 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
pH:
0 hours: pH 7.3 ± 0.1 ; 72 hours: pH 7.7 - 8.0 (definitive test concentrations) and pH 8.0 (controls). pH did not vary more than 1.5 units.
Nominal and measured concentrations:
Range-finder test: 0 (Control), 0.2, 1.0, 5.0, 25.0 and 100.0 mg/L prepared as WAF
Final test: 0 (Control), 0.32, 1.0, 3.2, 10 and 32 mg/L prepared as WAF
See table 3 for WAF and measured concentrations at initial exposure and during the course of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 120 mL, containing 120 mL test solution ; closed airtight (capped)
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/mL.
- Control end cells density: Mean (of replicates after 72 hours) 164.1 x10^4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 67 to 79 µE/m2/s.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 680 nm with immersion probe (pathlength = 10 mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WAF (water accommodated fractions) in the range-finding test. In the definitive test 5 concentrations were used at 0.32, 1.0, 3.2, 10, and 32 mg/L prepared as WAF.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.517 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 7.719 - 7.923 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.59 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 2.453 - 2.730 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.208 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.998 - 2.410 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.969 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.797 - 1.120 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.471 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells at the concentration closest to the EC50 when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.187, 0.471 and 2.035 mg/L GMM growth inhibition was negative between 24-48h and/or for 0.187 and 0.471 mg/L GMM at 0-72h. This was low dose stimulation and was limited to < 6% and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from initial values. Analyses of samples taken on a daily basis showed that the measured concentrations gradually decreased by approximately 50% over the 72-hour test period. The decrease for the highest test concentration was slightly less with 35%. The decrease observed in the intermediate test group incubated without algae showed a similar decrease indicative of the fact that adsorption to algae did not cause the decrease. No further observations were made to account for the differences.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L. The historical ranges for growth rate inhibition range between 0.82 and 2.6 mg/L. Hence, the 72h-ERC50 for the present test in closed systems is within the historical range. The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. Hence, the 72h-EYC50 for the present test closed systems was within range.
- Other: The sensitivity of the test system was in agreement with the historical data.
Reported statistics and error estimates:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

Growth rates were in the range of the controls at average measured concentrations of 0.187 and 0.471 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 2.035 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at test concentrations of 2.035 mg/L and higher. Note that the 9.0% inhibition as found for 2.035 mg/L can be considered as not biologically relevant based on the fact that the effect is below 10%.

Inhibition of yield increased with increasing average measured concentration of test item from 0.471 mg/L upwards resulting in 97% inhibition at 10.450 mg/L. Statistically significant inhibition of yield was found at test concentrations of 2.035 mg/L and higher.

Table 1. Mean cell densities (x10^4 cells/mL) during the range-finding test

Time (h)

Test item, WAF concentration (mg/L) 

 

Control

0.2*

1.0

5.0

25.0

100.0

0

1.0

1.0

1.0

1.0

1.0

1.0

72

109.8

114.0

97.2

59.4

5.4

1.4

 

 

 

 

 

 

 

* 5-fold dilution of the 1.0 mg/L WAF

 

Table 2. Percentage inhibition of growth rate and inhibition of yield during the range-finding test

 

Growth rate (0 – 72h)

Yield (0 – 72h)

Test item, WAF concentration (mg/L) 

mean

Std. Dev.

n

% inhibition

mean

Std. Dev.

n

% inhibition

Control

1.566

0.0044

3

-

108.8

1.45

3

-

0.2*

1.579

0.0185

3

-0.8

113.0

6.26

3

-3.9

1.0

1.526

0.0063

3

2.6

96.2

1.84

3

11.6

5.0

1.361

0.0208

3

13.1

58.4

3.69

3

46.3

25.0

0.490

0.2565

3

68.7

4.4

4.45

3

96.0

100.0

0.105

0.1251

3

93.3

0.4

0.56

3

99.6

 

 

 

 

 

 

 

 

 

* 5-fold dilution of the 1.0 mg/L WAF

 

Table 3. Measured concentrations versus nominal concentrations: final test

Test item, WAF concentration (mg/L) 

Measured concentrations (mg/L) at time point

Geometric Mean Measured average (mg/L)

t=0h

t=24h

% initial

t=48h

% initial

t=72h

% initial

0 (Control)

n.d.

n.d.

n.a.

n.d.

n.a.

n.d.

n.a.

0

0.32

0.252

0.215

85

0.156

62

0.135

54

0.187

1.0

0.615

0.531

86

0.299

49

0.333

54

0.471

3.2

2.84

2.29

81

1.99

70

0.138

49

2.035

10

5.15

3.56

69

3.51

68

2.49

48

3.412

32

12.8

11.3

88

8.91

70

8.26

65

10.450

 

 

 

 

 

 

 

 

 

3.2 #WA

2.98

2.61

88

1.53

51

1.47

49

 

 

 

 

 

 

 

 

 

 

n.d. = not detected

n.a. = not applicable

#WA = without algae

 

Table 4. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test item, WAF concentration (mg/L) 

Geometric Mean Measured average (mg/L)

Growth rate (0 – 72h)

Yield (0 – 72h)

mean

Std. Dev.

n

% inhibition

mean

Std. Dev.

n

% inhibition

0 (Control)

0

1.699

0.0312

6

-

163.1

15.80

6

-

0.32

0.187

1.713

0.0199

3

-0.8

169.5

10.32

3

-3.9

1.0

0.471

1.692

0.0108

3

0.4

159.4

5.22

3

2.3

3.2

2.035

1.546

0.0047

3

9.0*/#

102.3

1.45

3

37.3*

10

3.412

1.358

0.0506

3

20.1*

58.2

8.82

3

64.3*

32

10.450

0.619

0.0703

3

63.6*

5.5

1.28

3

96.6*

 

 

 

 

 

 

 

 

 

 

*= effect was statistically significant

# = effect not biologically relevant (<10%)

 

Table 5. Mean cell densities (x10^4 cells/mL) during the final test

 

Replicate

Geometric Mean Measured average (mg/L)

0 (Control)

0.187

0.471

2.035

3.412

10.450

t=0h

1

1.000

1.000

1.000

1.000

1.000

1.000

2

1.000

1.000

1.000

1.000

1.000

1.000

3

1.000

1.000

1.000

1.000

1.000

1.000

4

1.000

 

 

 

 

 

5

1.000

 

 

 

 

 

6

1.000

 

 

 

 

 

n

6

3

3

3

3

3

Mean

1.0

1.0

1.0

1.0

1.0

1.0

Std. Dev.

0.0

0.0

0.0

0.0

0.0

0.0

 

CV

0.0

0.0

0.0

0.0

0.0

0.0

t=24h

1

6.248

6.645

8.048

10.267

4.241

3.169

2

7.752

6.378

6.967

7.208

5.750

2.948

3

6.373

6.378

6.665

4.723

7.279

2.938

4

5.448

 

 

 

 

 

5

6.373

 

 

 

 

 

6

6.781

 

 

 

 

 

n

6

3

3

3

3

3

Mean

6.5

6.5

7.2

7.4

5.8

3.0

Std. Dev.

0.8

0.2

0.7

2.8

1.5

0.1

CV

11.6

2.4

10.1

37.5

26.4

4.3

t=48h

1

36.957

38.165

36.243

23.934

17.400

3.315

2

44.292

41.233

37.169

24.603

16.675

3.521

3

36.082

36.978

34.000

26.127

15.433

3.189

4

35.871

 

 

 

 

 

5

35.710

 

 

 

 

 

6

35.695

 

 

 

 

 

n

6

3

3

3

3

3

Mean

37.4

38.8

35.8

24.9

16.5

3.3

Std. Dev.

3.4

2.2

1.6

1.1

1.0

0.2

CV

9.1

5.7

4.6

4.5

6.0

5.0

t=72h

1

147.970

167.075

165.762

104.962

67.577

7.068

2

191.492

182.141

160.139

102.854

50.006

7.389

3

155.989

162.382

155.330

102.185

60.051

5.025

4

152.447

 

 

 

 

 

5

167.669

 

 

 

 

 

6

168.992

 

 

 

 

 

n

6

3

3

3

3

3

Mean

164.1

170.5

160.4

103.3

59.2

6.5

Std. Dev.

15.8

10.3

5.2

1.4

8.8

1.3

CV

9.6

6.1

3.3

1.4

14.9

19.7

 

Table 2. Specific growth rate (per day) during the final test

 

Replicate

Geometric Mean Measured average (mg/L)

0 (Control)

0.187

0.471

2.035

3.412

10.450

t=0h - t=24h

1

1.832

1.894

2.085

2.329

1.445

1.153

2

2.048

1.853

1.941

1.975

1.749

1.081

3

1.852

1.853

1.897

1.552

1.985

1.078

4

1.695

 

 

 

 

 

5

1.852

 

 

 

 

 

6

1.914

 

 

 

 

 

n

6

3

3

3

3

3

Mean

1.866

1.867

1.974

1.952

1.726

1.104

Std. Dev.

0.115

0.0237

0.0986

0.3888

0.2708

0.0428

% Inh.

-

0.0

-5.8

-4.6

7.5

40.8

t=0h - t=48h

1

1.805

1.821

1.795

1.588

1.428

0.599

2

1.895

1.86

1.808

1.601

1.407

0.629

3

1.793

1.805

1.763

1.631

1.368

0.58

4

1.79

 

 

 

 

 

5

1.788

 

 

 

 

 

6

1.788

 

 

 

 

 

n

6

3

3

3

3

3

Mean

1.81

1.829

1.789

1.607

1.401

0.603

Std. Dev.

0.0425

0.028

0.023

0.0224

0.0304

0.025

% Inh.

-

-2.1

8.6

28.1

38.7

94.2

t=0h - t=72h

1

1.666

1.706

1.704

1.551

1.404

0.652

2

1.752

1.735

1.692

1.544

1.304

0.667

3

1.683

1.697

1.682

1.542

1.365

0.538

4

1.676

 

 

 

 

 

5

1.707

 

 

 

 

 

6

1.71

 

 

 

 

 

n

6

3

3

3

3

3

Mean

1.699

1.713

1.692

1.546

1.358

0.619

Std. Dev.

0.0312

0.0199

0.0108

0.0047

0.0506

0.0703

% Inh.

-

-0.2

-1.5

3.6

13.9

55.9

Validity criteria fulfilled:
yes
Conclusions:
The test item 72h-ErC50 for growth rate reduction was 7.517 (C.I. 7.517 – 7.923) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 2.208 (C.I. 1.998 – 2.410) mg/L and the NOEC was 0.471 mg/L.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0 (control) 0.2, 1.0, 5.0, 25.0 and 100.0 mg/L concentrations prepared as WAF, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WAF (water accommodated fractions). The resulting suspensions were left to settle for an hour, were after the WAFs were collected from the middle of the settled suspensions. All WAFs were clear and colourless and used for testing. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at 0 hours (test start), 24, 48 and at 72 hours (test end) of the exposure. The samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure. Analyses of samples taken on a daily basis showed that the measured concentrations gradually decreased by approximately 50% over the 72-hour test period. The decrease for the highest test concentration was slightly less with 35%. The decrease observed in the middle test group incubated without algae showed a similar decrease indicative of the fact that adsorption to algae did not cause the decrease. The range tested based on geometric average measured concentrations was 0.187, 0.471, 2.035, 3.412 and 10.450 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 7.517 (C.I. 7.517 – 7.923) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 2.208 (C.I. 1.998 – 2.410) mg/L and the NOEC was 0.471 mg/L.

Description of key information

EC50 (aquatic algae; growth rate) = 7.517 (C.I. 7.149 – 7.923) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2016

EC10 (aquatic algae; growth rate) = 2.208 (C.I. 1.998 – 2.410) mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, 2016

NOEC (aquatic algae; growth rate) = 0.471 mg/L based on GMM concentrations, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2016

Key value for chemical safety assessment

EC50 for freshwater algae:
7.517 mg/L
EC10 or NOEC for freshwater algae:
0.471 mg/L

Additional information

Key study : OECD TG 201, 2016 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0 (control) 0.2, 1.0, 5.0, 25.0 and 100.0 mg/L concentrations prepared as WAF, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L prepared as WAF (water accommodated fractions). The resulting suspensions were left to settle for an hour, were after the WAFs were collected from the middle of the settled suspensions. All WAFs were clear and colourless and used for testing. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via GC-MS at 0 hours (test start), 24, 48 and at 72 hours (test end) of the exposure. The samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure. Analyses of samples taken on a daily basis showed that the measured concentrations gradually decreased by approximately 50% over the 72-hour test period. The decrease for the highest test concentration was slightly less with 35%. The decrease observed in the middle test group incubated without algae showed a similar decrease indicative of the fact that adsorption to algae did not cause the decrease. The range tested based on geometric average measured concentrations was 0.187, 0.471, 2.035, 3.412 and 10.450 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 7.517 (C.I. 7.517 – 7.923) mg/L based on geometric mean measured concentrations. The corresponding ErC10 was 2.208 (C.I. 1.998 – 2.410) mg/L and the NOEC was 0.471 mg/L.