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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:
14 - 19 July 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled. This study is considered reliable with restrictions as loss of test substance was observed and the analytical monitoring was performed only at t0 and t72h.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on June 03-05, 2013 / signed on November 05, 2013
Specific details on test material used for the study:
- Stability under test conditions: Stable at pH 2 - 8.5 until day 5 under test conditions similar to OECD 111
Analytical monitoring:
yes
Details on sampling:
Determination of the test item: All. concentration levels and the control were analytically verified via GC-MS at the start (0 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 72 hours samples were taken out of the pooled test replicates with algae.

Quality criteria for the analytical monitoring: Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations. The effect levels should given based on the geometric mean measured concentrations, if the initially measured concentrations differ more than 20% from the nominal concentrations or a decrease of more than 20% from the initially measured concentration is observed. Otherwise, the nominal concentrations should be used.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Stock solution: A stock solution of 50.0 mg/L was freshly prepared with dilution water.
Dispersion treatment: The stock solution was treated with ultrasound for 15 minutes at room temperature. Thereafter the stock solution was stirred with approximately 1100 rpm for 24 hours at room temperature.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
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
- 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)
- Preculture: A four day old preculture, prepared in dilution water, was used as inoculum.
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:
7.60-8.06 (t=0 h); 8.00-9.02 (t=72 h)
Dissolved oxygen:
None
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 3.13, 6.25, 12.5, 25 and 50 mg/L
Geometric mean measured concentrations: 2.00, 4.38, 9.29, 19.6 and 41.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Test volume: 100 mL
- Initial cells density: Nominal: approximately 5000-50000 cells/mL; Current: 7085 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: 60.5 to 69.5 (65.8) μE/m2/s
- Light homogeneity: The light homogeneity over the incubation area was within -8.11 to 5.56 % 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 in the preliminary range finding test at the concentration level of 100 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.
Physical Chemical Parameters:
- The pH-value at the beginning of the exposure was measured in one additional replicate of each concentration level and the control. At the end of the exposure, it was measured from pooled replicates per concentration level and control, respectively. The room temperature was measured and recorded continuously. Light intensity was measured prior to the start of the test.

TEST CONCENTRATIONS
- Range finding study: In a non-GLP preliminary range finding test, nominal test concentrations of 0.01, 0.1, 1, 10 and 100 mg/L were tested. Inhibition of growth rate was 2, 4, 4, 26 and 100% 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Respective inhibitions of yield were 12, 19, 15, 74 and 100%.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 8.17-9.18 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
9.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 9.62-11.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 11.1-13.7 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Biological data: Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values are given based on the geometric mean measured test item concentrations.
Measured Exposure Concentrations during the Definitive Test: The recovery rate of the test item at the start of the exposure was in the range of 88 to 104 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 39 to 77 % of the nominal values. All effect values are given based on the geometric mean measured concentration of the test item.
Results with reference substance (positive control):
Results with reference substance (potassium dichromate) valid
- The ErC50 value based on Growth Rate Inhibition: 0.661 mg/L (95% CI: 0.638-0.685 mg/L)
- The EyC50 value based on Yield Inhibition: 0.341 mg/L (95% CI: 0.286-0.377 mg/L)
Reported statistics and error estimates:
EC-values and statistical analyses: EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of the EC10-, EC20- and EC50-values was carried out using standard procedures.
NOEC and LOEC values: The NOEC /I LOEC was determined by calculation of statistical significance of growth rate and yield. As 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. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. 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: Evaluation after 72 hours 

Geometric mean measured test item concentration [mg/L]

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

No.

[d-1]

[%]

[cells/mL]

[%]

41.0

1

 

n.a.

100

 

n.a.

100

2

 

n.a.

100

 

n.a.

100

3

 

n.a.

100

 

n.a.

100

Mean

 

n.a.

100

 

n.a.

100

19.6

1

 

0.008

100

 

173

100

2

 

-0.014

100

 

-289

100

3

 

0.075

96

 

1796

100

Mean

(+)

0.023

99

(+)

560

100

9.29

1

 

1.70

13

 

1164211

54

2

 

1.67

15

 

1049572

59

3

 

1.72

12

 

1217597

52

Mean

(+)

1.70

14

(+)

1143793

55

4.38

1

 

1.83

6

 

1731732

32

2

 

1.81

8

 

1597803

37

3

 

1.82

7

 

1678811

34

Mean

(+)

1.82

7

(+)

1669449

34

2.00

1

 

1.88

4

 

2001050

21

2

 

1.96

0

 

2515717

1

3

 

1.92

2

 

2224951

12

Mean

(-)

1.92

2

(-)

2247239

12

Control

1

 

1.98

-

 

2654360

 

-

 

 

2

 

1.97

 

2610934

3

 

1.99

 

2753761

4

 

1.94

 

2386768

5

 

1.93

 

2302838

6

 

1.96

 

2540351

Mean

 

1.96

 

2541502

n.a. = not applicable, cell density < Limit of detection (2280 cells/mL)

Validity Criteria

Validity Criterium

Required

This study

Increase of the cell growth in the control cultures

Exponentially > 16-fold corresponding to a specific growth rate of 0.92 day-1

3600-fold

(specific growth rate 1.96 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 %

15.5 %

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

< 7 %

1.14 %

Validity criteria fulfilled:
yes
Conclusions:
In this study test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 12.0 (11.1 - 13.7) mg/L and 7.45 (6.05 - 8.85) mg/L, respectively. The EC10-values for inhibition of growth rate after 72 hours was 8.7 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 3.13, 6.25, 12.5, 25 and 50 mg/L corresponding to the geometric mean measured concentrations of the test item: 2.00, 4.38, 9.29, 19.6 and 41.0 mg/L for 72 h. The study was conducted under static conditions with an initial cell density of 7085 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.

 

The concentrations of the test item were analytically verified by GC-MS at the start and the end of the exposure of all test concentrations and the control. The recovery rate of the test item at the start of the exposure was in the range of 88 to 104 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 39 to 77 % of the nominal values. All effect values are given based on the geometric mean measured concentration of the test item.

 

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values were based on the geometric mean measured test item concentrations.

 

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 item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 12.0 (11.1 - 13.7) mg/L and 7.45 (6.05 - 8.85) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 2.00 mg/L, respectively. However, the preferred long term endpoint value is the EC10 for growth rate which was determined as 8.72 mg/L

Description of key information

OECD Guideline 201, GLP, key study, validity 2:

EC50 (72h-growth rate)= 12.0 (11.1 - 13.7) mg/L (geometric mean measured test item concentrations)

EC10 (72h-growth rate)= 8.72 (8.17 - 9.78) mg/L (geometric mean measured test item concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:
12 mg/L
EC10 or NOEC for freshwater algae:
8.72 mg/L

Additional information

One key study is available (NOACK, 2014) to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to OECD Guideline 201 with GLP statement.

The algae were exposed to the test substance at nominal concentrations of 3.13, 6.25, 12.5, 25 and 50 mg/L corresponding to the geometric mean measured concentrations of 2.00, 4.38, 9.29, 19.6 and 41.0 mg/L for 72 h. The study was conducted under static conditions with an initial cell density of 7085 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.

The concentrations of the test item were analytically verified by GC-MS at the start and the end of the exposure of all test concentrations and the control. The recovery rate of the test item at the start of the exposure was in the range of 88 to 104 % of the nominal values at all tested concentration levels. The recovery rate at the end of the exposure was in the range of 39 to 77 % of the nominal values. All effect values are given based on the geometric mean measured concentration of the test item.

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).

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values were based on the geometric mean measured test item concentrations.

In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 12.0 (11.1 - 13.7) mg/L and 7.45 (6.05 - 8.85) mg/L, respectively. The EC10-values for inhibition of growth rate was 8.72 mg/L (8.17 -9.78 mg/L).