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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:
From 2015-06-15 to 2015-06-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD Guideline No. 201 without any deviation. However, the study is considered reliable with restriction based on the non-reproducible results (analytical and biological data) between the range-finder and the main study, and the unexplained increase of recovery values in the main study from 0 to 48 hours.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on June 03-05, 2013 / signed on November 05, 2013
Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via GC-MS at the start (0 hours), after 24 and 48 hours, and at the end of the exposure (72 hours).
Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 24, 48 and 72 hours, separate replicates were prepared with algae at the beginning of the exposure and incubated under test conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Stock solution: 4.00 mg/L were freshly prepared with dilution water. This concentration was taken based on the highest measured concentrations in the Daphnia studies, very close to 4.00 mg/L. Due to the higher complexity of the Alga medium compared to the Daphnia medium regarding salts and trace elements it's not assume that much higher solubility in the Alga medium can be achieved.
- Dispersion treatment: The stock solution was stirred with approximately 1100 rpm for 24 ± 1 hours at room temperature. Thereafter the stock solution was treated with ultrasound for 5 minutes.
- Controls: Six replicates (without test item) were exposed under the same test conditions as the test loadings.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE/m2/s for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Dilution water medium has a nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
22-23 °C (22.5 °C)
pH:
7.93-8.20 (t=0 h); 8.38-8.96 (t=72 h)
Dissolved oxygen:
None
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 0.25, 0.50, 1, 2 and 4 mg/L
Geometric mean measured concentrations: 0.209, 0.389, 0.767, 1.53 and 2.67 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 119 mL, with aluminium tops with PFTE seals
- Test volume: 119 mL
- Initial cells density: Nominal: approximately 5000-50000 cells/mL; Current: 5526 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Application: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Incubation: The flasks were positioned randomly and repositioned daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the OECD guideline 201

OTHER TEST CONDITIONS
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Photoperiod: 24 hours/day light
- Light intensity and quality: 67.9 to 75.6 (71.8) µE/m2/s
- Light homogeneity: The light homogeneity over the incubation area was within -5.43 to 5.29 % of the average light intensity (required: within ± 15 % from the mean light intensity).

EFFECT PARAMETERS MEASURED:
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the concentration level of 4.00 mg/L in the range finding test.
- Microscopic evaluation: Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.
Physical Chemical Parameters:
- The pH-value at the beginning and end of the test were measured from pooled replicates per concentration and control after measurement of the cell density, respectively. The room temperature was measured continuously. Light intensity was measured prior to test start.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP preliminary range finding test, three nominal test concentrations of 0.04, 0.4 and 4 mg/L were tested. These concentrations correspond to 0.031, 0.35 and 3.79 mg/L based on geometric mean measured concentrations. Inhibition of growth rate was 0, -2 and 16% at test concentrations of 0.031, 0.35 and 3.79 mg/L after 72 h. Respective inhibitions of yield were 2, -7 and 52 %.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of growth rate
Remarks on result:
other: 95% CL: 1.02 – 2.28 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.67 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of growth rate
Details on results:
Biological data:
Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control. All effect values are given based on geometric mean measured test item concentrations.

Measured Exposure Concentrations during the Definitive Test:
The concentrations of the test item were analytically verified by GC-MS at the start (without algae), after 24 and 48 h and the end of the exposure (with algae) in all test concentrations and the control. The measured concentrations of the test item were in the range of 57 to 106% of the nominal concentrations during the test. The geometric mean measured concentrations were 2.67, 1.53, 0.767, 0.389 and 0.209 mg/L for the nominal concentrations of 4.0, 2.0, 1.0, 0.5 and 0.25 mg/L, respectively
Results with reference substance (positive control):
Results with reference substance (potassium dichromate) valid
- The ErC50 value based on Growth Rate Inhibition: 0.613 mg/L (with Headspace) and 0.774 mg/L (without Headspace)
- The EyC50 value based on Yield Inhibition: 0.281 mg/L (with Headspace) and 0.502 mg/L (without Headspace)
Reported statistics and error estimates:
EC-values and statistical analyses: EC10, EC20 and EC50 values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses: The NOEC/LOEC were determined by calculation of statistically significant differences of growth rate and yield inhibition. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05.

Table 6.1.5/1: Cell Densities

 

Geometric mean measured test item concentrations (nominal test item concentrations) mg/L

[mg/L]

Replicate No.

Cell Density [cells/mL]

0 hours

24 hours

48 hours

72 hours

2.67 (4.0)

1

5526

6482

23486

42904

2

5526

15664

44585

48527

3

5526

15959

47799

109889

Mean

5526

12702

38623

67107

1.53 (2.0)

1

5526

13316

9759

273930

2

5526

20619

38877

393625

3

5526

17841

12594

455492

Mean

5526

17259

20410

374349

0.767 (1.0)

1

5526

7966

29463

749586

2

5526

20660

77544

754666

3

5526

15564

21131

405993

Mean

5526

14730

42713

636748

0.389 (0.5)

1

5526

6946

71924

726718

2

5526

18713

69074

972104

3

5526

13686

140675

686439

Mean

5526

13115

93891

795087

0.209 (0.25)

1

5526

8621

133823

697378

2

5526

14043

61757

674623

3

5526

16358

31849

726530

Mean

5526

13007

75810

699510

Control

1

5526

26126

76105

589775

2

5526

20845

60773

583754

3

5526

19130

89210

635511

4

5526

21099

88953

508942

5

5526

26744

86814

608717

6

5526

24517

89966

513909

Mean

5526

23077

81970

573435

 

Table 6.1.5/2: Evaluation after 72 h             

 

Geometric mean measured test item concentrations (nominal test item concentrations) [mg/L]

Replicate No.

Growth Rate [d-1]

Inhibition of Growth Rate [%]

Yield [cells/mL]

Inhibition of Yield [%]

2.67 (4.0)

1

 

0.683

56

 

37378

93

2

 

0.724

53

 

43001

92

3

 

0.997

36

 

104363

82

Mean

(+)

0.801

48

(+)

61581

89

1.53 (2.0)

1

 

1.30

16

 

268404

53

2

 

1.42

8

 

388099

32

3

 

1.47

5

 

449966

21

Mean

(-)

1.40

10

(-)

368823

35

0.767 (1.0)

1

 

1.64

-6

 

744060

-31

2

 

1.64

-6

 

749140

-32

3

 

1.43

7

 

400467

29

Mean

(-)

1.57

-1

(-)

631222

-11

0.389 (0.5)

1

 

1.63

-5

 

721192

-27

2

 

1.72

-11

 

966578

-70

3

 

1.61

-4

 

680913

-20

Mean

(-)

1.65

-7

(+)*

789561

-39

0.209 (0.25)

1

 

1.61

-4

 

691852

-22

2

 

1.60

-4

 

669097

-18

3

 

1.63

-5

 

721004

-27

Mean

(-)

1.61

-4

(-)

693984

-22

Control

1

 

1.56

 

 

584249

 

2

 

1.55

 

 

578228

 

3

 

1.58

 

 

629985

 

4

 

1.51

 

 

503416

 

5

 

1.57

 

 

603191

 

6

 

1.51

 

 

508383

 

Mean

 

1.55

 

 

567909

 

negative inhibitions = increase of growth

*) statistical significance caused by stimulation of growth

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Table 6.1.5/3: Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 h)

 

Replicate No.

Specific Growth Rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.55

1.07

2.05

1.56

0.489

31.4

24.7

2

1.33

1.07

2.26

1.55

0.627

40.4

3

1.24

1.54

1.96

1.58

0.363

22.9

4

1.34

1.44

1.74

1.51

0.211

14.0

5

1.58

1.18

1.95

1.57

0.385

24.6

6

1.49

1.30

1.74

1.51

0.222

14.7

NA

Mean

1.55

NA

SD ±

0.03

CV [%]

1.96

SD = Standard deviation          CV = Coefficient of variation

Table 6.1.5/4: Environmental Conditions

Geometric mean measured test item concentrations (nominal test item concentrations)

pH-value

[mg/L]

Start; 0 hours

End; 72 hours

2.67 (4.0)

7.93

8.38

1.53 (2.0)

8.09

8.79

0.767 (1.0)

8.10

8.81

0.389 (0.5)

8.17

8.92

0.209 (0.25)

8.24

8.96

Control

8.20

8.76

Room temperature [°C]:

min.: 22.0

max.: 23.0

mean value: 22.5

 

Light intensity
[µE∙m-2∙s-1]

n = 24

mean value: 71.8

range of the measured values: 67.9 to 75.6 equalling -5.43 to 5.29 %

CV [%]: 3.36

CV = Coefficient of variation                             

n = number of measuring points

The light homogeneity over the incubation area was within -5.43 to 5.29 % of the average light intensity (required: within ± 15 % from the mean light intensity).

 

Validity Criteria

The study meets the validity criteria of the guideline:

Validity Criterion

Required

This study

Increase of the cell growth in the control cultures

Exponentially16-fold corresponding to a specific growth rate of 0.92 day-1

104-fold
(specific growth rate 1.55 day-1)

Mean coefficients of variation for section-by-section specific growth rates (days           0-1, 1-2 and 2-3) in the control cultures

35 %

24.7 %

Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures

7 %

1.96 %

Validity criteria fulfilled:
yes
Conclusions:
In this study, test substance was found to inhibit the growth rate of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values (geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) after 72 hours was > 2.67 mg/L. The EC10-values for inhibition of growth rate (ErC10) after 72 hours was 1.54 mg/L.
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at nominal concentrations of 0.25, 0.50, 1, 2 and 4 mg/L for 72 h. The study was conducted under static conditions with an initial cell density of 5526 cells/mL. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. 

All test concentrations and the control were analytically verified by GC-MS at the start of the exposure and after 24, 48 and 72 hours. The measured concentrations of test item at the start of the exposure were in the range of 58 - 78 % of the nominal test item concentrations. At the end of the exposure, the measured concentrations were in the range of 72 - 88 % of the nominal test item concentrations. However, when compared to the initial measured concentrations, the mean measured concentrations were 107 to 128%. Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control. All effect values were based on the geometric mean measured concentrations of the test item (0.209, 0.389, 0.767, 1.53 and 2.67 mg/L).

 

All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates).

 

In this study, test substance was found to inhibit the growth rate of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values (geometric mean measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) after 72 hours was > 2.67 mg/L. The EC10-values for inhibition of growth rate (ErC10) after 72 hours was 1.54 mg/L.

Description of key information

OECD Guideline 201, EU Method C.3, GLP, key study, validity 2:

72h-ErC50 (P. subcapitata) > 2.67 mg/L (the highest tested concentrations), based on geometric mean measured concentrations.

72h-ErC10 (P. subcapitata) = 1.54 mg/L (95% CL: 1.02 - 2.28 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
1.54 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, one experimental study is available.

This valid study (NOACK, 2015) was assessed as the key study and was performed on the registered substance according to OECD Guideline 201 and EC Method C.3 in compliance with GLP, on Pseudokirchneriella subcapitata. The unicellular freshwater green algae (with an initial cell density of 5526 cells/mL) were exposed to five concentrations of the test substance: 0.25, 0.50, 1.00, 2.00 and 4.00 mg/L plus a control, under static conditions over a period of 72 hours. The maximum tested concentration of 4.00 mg/L was taken based on the highest measured concentrations in the Daphnia studies, very close to 4.00 mg/L. Due to the higher complexity of the algae medium compared to the Daphnia medium regarding salts and trace elements, it's not assume that much higher solubility in the algae medium can be achieved. Three replicates were tested for each test substance concentration and six repicates for the control. The envionmental conditions were within the acceptable limits. All test substance concentrations and the control were analytically verified by GC-MS at the start of the exposure and after 24, 48 and 72 hours. The measured concentrations of the test substance at the start of the exposure were in the range of 58 to 78% of the nominal test substance concentrations. At the end of the exposure (72 hours), the measured concentrations of the test substance were in the range of 72 to 88% of the nominal test substance concentrations. As the measured concentrations were lower than 80% of the nominal values during the test, the geometric mean measured concentrations were determined to be 0.209, 0.389, 0.767, 1.53 and 2.67 mg/L for the nominal concentrations of 0.25, 0.50, 1.00, 2.00 and 4.00 mg/L, respectively. These geometric mean measured concentrations were in the range of 107 to 128% of the initial measured concentrations. All effects values were based on the geometric mean measured concentrations of the test substance. According to the biological data, the inhibition of grow rate after 72 hours of exposure were -4%, -7%, -1%, 10% and 48% for the geometric mean measured concentrations of 0.209, 0.389, 0.767, 1.53 and 2.67 mg/L, respectively. Therefore, based on these results, the 72h-ErC50 value was determined to be greater than the highest tested concentration, 2.67 mg/L, and the 72h-ErC10 value was determined to be 1.54 mg/L (95% CL: 1.02 - 2.28 mg/L).