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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-11 to 2016-01-15
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
- Test Item: Licocare RBW 106 FL TP
- Physical state: Slightly yellow flakes
- Purity test date: 2015-04-29
- Lot/batch No.: DEF2105728
- Expiration date of the lot/batch: 2020-01-16
- Storage condition of test material: Room temperature, protected from light in the tightly closed original container.
- Purity (certified): 99.63% (w/w)
- Water solubility: < 1 g/L
- Molecular mass: not specified

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 mg test item/Lwas prepared once with dilution water one day prior to the start of the exposure. This dispersion was stirred for 24 hours with 1100 rpm at room temperature. After stirring, the saturated solution was taken from the homogenous liquid phase. At the end of the stirring time undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered. The solution was checked for undissolved test item with a laser beam for a Tyndall effect.

Test concentrations (nominal)
The saturated solution and further four dilution levels out of the saturated solution was tested in a geometrical series with a dilution factor of 1.78: 10.0 - 17.8 - 31.6 - 56.2 - 100 % of the saturated solution. The dilution levels were based on the results of a preliminary range finding test (non GLP, see section 8.1).

Control
Six replicates (without test item) were exposed under the same conditions as the test item replicates.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism
Pseudokirchneriella subcapitata HINDÁK, SAG 61.81

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE•m-2•s-1 for 24 hours per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 21.0 max.: 23.0, mean value: 22.0
pH:
Saturated Solution pH-value
[%] Start; 0 hours End; 72 hours
100 7.37 7.99
56.2 7.47 8.08
31.6 7.58 8.16
17.8 7.66 8.21
10.0 7.85 8.29
Control 8.03 8.63
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
nominal: 10.0 - 17. 8 - 31.6 - 56.2 - 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 6153 cells/mL
- Control end cells density: Mean 1444109 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 66.7



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:

Biological Data

The results of the range finding test are presented below:

Results of the Range Finding Test (0 - 72 hours)
Saturated Solution [%] Growth Rate Inhibition [%] Yield Inhibition [%]
100 15 55
10.0 -6 -34
1.00 0 0


Analytical Data

The analytical data of the range finding test are presented below:

TOC Content of the Test Media at the Start and the End of the Exposure (Range Finding Test)

Saturated Solution
[%] TOC mg/L
Test start (without algae), 0 hours Test end (without algae), 72 hours
100 2.08 < 2.00
10 < 2.00 < 2.00
1.00 < 2.00 < 2.00
Control < 2.00 < 2.00
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.6 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
56.2 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
56 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 50.8 - 64.1
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
79.7 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 69.1 - 89.5
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.6 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
56.2 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
33.5 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 25.1 - 41.9
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
41 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 32.5 - 47.9
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
61.9 other: saturated solution %
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 53.9 - 73.7
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015-10-13 to 2015-10-16. The reference item toxicity is in the valid range following test facility SOPS. For results, see Table 10.

Table 10: EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.564 0.797 ± 0.428
95 % confidence interval 0.546 - 0.582
Yield inhibition
EyC50 0.322 0.415 ± 0.275
95 % confidence interval 0.239 - 0.342
SD = Standard deviation





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 and LOEC were determined by calculation of statistically significant differences of growth rates and yield. 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 -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Cell Densities

Saturated Solution

Replicate

Cell Density [cells/mL]

[%]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6153

11798

43277

343806

2

6153

10612

38981

365852

3

6153

10663

33113

312195

Mean

6153

11024

38457

340618

 56.2

1

6153

17932

108694

741043

2

6153

20008

161761

989226

3

6153

17327

115919

776072

Mean

6153

18422

128791

835447

 31.6

1

6153

30808

239114

1501241

2

6153

28381

222073

1280153

3

6153

26459

201764

1164936

Mean

6153

28549

220984

1315443

 17.8

1

6153

29193

266196

1410046

2

6153

34662

297011

1487505

3

6153

36321

331569

1668013

Mean

6153

33392

298259

1521855

 10.0

1

6153

33468

266987

1415189

2

6153

34954

283382

1594913

3

6153

31599

286286

1351277

Mean

6153

33340

278885

1453793

Control

1

6153

35816

284617

1550163

2

6153

35443

310270

1727880

3

6153

37344

295919

1464550

4

6153

29987

199425

1146403

5

6153

32671

248284

1363539

6

6153

34415

263750

1412116

Mean

6153

34279

267044

1444109

 

 


Evaluation after 72 hours

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

 

Saturated Solution

Replicate

Growth Rate

Inhibition of Growth Rate

Yield

Inhibition of Yield

[%]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.34

26

 

337653

77

2

 

1.36

25

 

359699

75

3

 

1.31

28

 

306042

79

Mean

(+)

1.34

26

(+)

334465

77

 56.2

1

 

1.60

12

 

734890

49

2

 

1.69

7

 

983073

32

3

 

1.61

11

 

769919

46

Mean

(+)

1.63

10

(+)

829294

42

 31.6

1

 

1.83

-1

 

1495088

-4

2

 

1.78

2

 

1274000

11

3

 

1.75

4

 

1158783

19

Mean

(-)

1.79

2

(-)

1309290

9

 17.8

1

 

1.81

0

 

1403893

2

2

 

1.83

-1

 

1481352

-3

3

 

1.87

-3

 

1661860

-16

Mean

(-)

1.84

-1

(-)

1515702

-5

 10.0

1

 

1.81

0

 

1409036

2

2

 

1.85

-2

 

1588760

-10

3

 

1.80

1

 

1345124

6

Mean

(-)

1.82

0

(-)

1447640

-1

Control

1

 

1.84

 

 

1544010

 

2

 

1.88

 

 

1721727

 

3

 

1.82

 

 

1458397

 

4

 

1.74

 

 

1140250

 

5

 

1.80

 

 

1357386

 

6

 

1.81

 

 

1405963

 

Mean

 

1.82

 

 

1437956

 

 


Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

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

1.76

2.07

1.69

1.84

0.202

10.9

11.3

2

1.75

2.17

1.72

1.88

0.252

13.4

3

1.80

2.07

1.60

1.82

0.236

12.9

4

1.58

1.89

1.75

1.74

0.156

 8.93

5

1.67

2.03

1.70

1.80

0.198

11.0

6

1.72

2.04

1.68

1.81

0.196

10.8

 

 

 

Mean

1.82

 

 

 

 

 

SD ±

0.05

 

 

 

 

CV [%]

2.52

 

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, LICOCARE RBW 106 FL TP was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal, percent saturated solutions of the test item): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 and 61.9 (53.9 – 73.7) mg/L. The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 31.6 mg/L. All effect values given are based on the nominal, percent saturated solutions of the test item LICOCARE RBW 106 FL TP.
Executive summary:

 

The toxicity of LICOCARE RBW 106 FL TP to the unicellular freshwater green alga seudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 from 2016 -01 -11 to 2016 -01 -15 with the definitive exposure phase from 2016-01-12 to 2016-01-15, at the test facility. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6153 cells/mL. Based on a preliminary range finding test a saturated solution with a nominal loading of 100 mg test item/L was prepared in dilution water. The dispersion was stirred for 24 hours with approximately 1100 rpm at room temperature. At the end of the stirring period, undissolved test item was removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated with the test item dispersion prior to the filtration in order to avoid adsorption during the filtration. The first 25 mL of filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration the filter was always kept covered with the test item dispersion. The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with adilution factor of 1.78:10.0 - 17.8 - 31.6 - 56.2 - 100 % of the saturated solution.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.The test media were clear throughout the test period.

 

The concentrations of LICOCARE RBW 106 FL TP were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (old media, 72 hours). In the saturated solution, 3 % of the nominally applied organic carbon was found.

All effect values given are based on the nominal, percent saturated solutions of the test itemLICOCARERBW 106 FL TP.

 


 

 NOEC, LOEC, EC-values and 95 % Confidence Intervals ofLICOCARE RBW 106 FL TP(0 - 72 hours)

                  based on percent of the saturated solution [%]         

 

Growth Rate Inhibition
saturated solution [%]

NOEC

31.6

LOEC

56.2

ErC10

56.0 (50.8 – 64.1)

ErC20

79.7 (69.1 – 89.5)

ErC50

> 100

 

Yield Inhibition

saturated solution [%]

NOEC

31.6

LOEC

56.2

EyC10

33.5 (25.1 – 41.9)

EyC20

41.0 (32.5 – 47.9)

EyC50

61.9 (53.9 – 73.7)