Registration Dossier

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:
14-17 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern guideline conform study performed in GLP certified laboratory

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: RW 2013-10-22
Color: Orange
Storage conditions: Room temperature
Expiring date: 22. 10. 2018

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Three replicates samples (5mL each) were taken from the test vessels and from the control at the two analytical occasions during the test (at the beginning and at the end of treatment period). Five different concentrations arranged in a geometric series with a separation factor of 2.0 and a concurrent untreated control was tested in the main experiment. The nominal concentrations of the test item were 6.25; 12.5; 25; 50 and 100 mg/L.
Samples were taken by using calibrated pipettes.

Sample preparation for the analysis:
Samples were not stored, the preparation was started immediately after the sampling. The samples were diluted with ultrapure water before HPLC analysis. The control samples were directly measured by HPLC method.

Test solutions

Vehicle:
no
Details on test solutions:
The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent just before the treatment. A stock solution was first prepared by dissolving an amount of 0.0679 g test item in 679 mL dilution water (OECD medium) using in order to obtain the nominal concentration of 100 mg/L. The test solutions of the chosen test concentrations were prepared by appropriate dilution of this stock solution (see Table 1).

Table 1: Preparation of the test solutions
Nominal concentration Stock Solution OECD Medium
[mg/L] [mL] [mL]
100 320 –
50 160 q.s. ad 320
25 80 q.s. ad 320
12.5 40 q.s. ad 320
6.25 20 q.s. ad 320
q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

Untreated Control

The dilution water (OECD medium) without addition of test item was used as control solution.

Reference Control

For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.
The date of the last study (Study Code: 392.440.4633) with the reference item Potassium dichromate was: 01 – 04 October 2014.
The 72h ErC50: 1.33 mg/L, (95 % confidence limits: 1.22 – 1.46 mg/L)
The 72h EyC50: 0.90 mg/L, (95 % confidence limits: 0.81 – 1.00 mg/L

Name and data of Reference Item

Name: Potassium dichromate (K2Cr2O7)
Batch No.: MKBK2472V
Description: Orange crystalline powder
Expiry Date: 26 February, 2014
Storage: Dry and cool place in closed container
Supplier: SIGMA-ALDRICH

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Test Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

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:
N/A
Test temperature:
Temperature in the Climate Chamber and in the Test Media during the Test
Exposure Time Temperature [oC]
In the climate chamber In the test media
Day 0 (Start) 21.8 – 22.9 22.4
Day 1 21.9 – 22.7 22.1
Day 2 22.0 – 22.5 22.3
Day 3 (End) 22.2 – 23.4 23.0
pH:
Concentration pH-values
[mg/L] Start End
Control 7.09 7.66-8.23
6.25 7.68 8.05-8.24
12.5 7.79 8.08-8.24
25 7.74 7.87-8.06
50 7.70 7.69-8.89
100 7.49 7.70-7.90
Dissolved oxygen:
N/A
Salinity:
N/A
Nominal and measured concentrations:
Test Item concentration measured on 14 March 2014:
Nominal concentration Mean of the measured concentrations Measured concentration
mg/L (mg/L) with the 95% confidence interval (n=3) in % of the nominal
6.25 5.71 (RSD: 0 %) 91
12.5 11.61 (RSD: 1 %) 93
25 22.81 (RSD: 0 %) 91
50 47.23 (RSD: 0 %) 94
100 91.53 (RSD: 1 %) 92
Control not detected -

Test Item concentration measured on 17 March 2014:
Nominal concentration Mean of the measured concentrations Measured concentration
mg/L (mg/L) with the 95% confidence interval (n=3) in % of the nominal
6.25 5.93 (RSD: 1 %) 95
12.5 12.23 (RSD: 1 %) 98
25 23.84 (RSD: 0 %) 95
50 50.77 (RSD: 1 %) 102
100 99.36 (RSD: 5 %) 99
Control not detected -
Details on test conditions:
Test Water (Algal Mineral Salts Test Medium)

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Separate stock solutions were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

Table 2: Composition of the OECD medium
Stock solution Substance Final concentration in the prepared growth medium
Stock solution 1
(macro nutrients) NH4Cl 15.0 mg/L
Batch No.: PP/2012/09407
Supplier: REANAL (lach:ner)
Expiry date: March 31, 2017
MgCl2 5.6 mg/L*
Lot No.: 120M0094V
Supplier: SIGMA-ALDRICH
Retest date: January 31, 2015
CaCl2 x 2 H2O 18.0 mg/L
Batch No.: PP/2012/07312
Supplier: REANAL (lach:ner)
Expiry date: November 30, 2014
MgSO4 x 7H2O 15.0 mg/L
Batch No.: PP/2011/05896
Supplier: REANAL (lach:ner)
Expiry date: June 30, 2015
KH2PO4 1.6 mg/L
Batch No.: AM0444073
Supplier: MERCK
Expiry date: July 31, 2017
Stock solution 2
(iron) FeCl3 x 6 H2O 64.0 ug/L
Batch No.: BCBL0680V
Supplier: SIGMA-ALDRICH
Expiry date: May 28, 2016
Na2EDTA x 2 H2O 100.0 ug/L
Batch No.: K44403118
Supplier: MERCK
Expiry date: April 30, 2018

Table 3: Composition of the OECD medium (continued)
Stock solution 3
(trace elements) H3BO3 185.0 ug/L
Batch No.: A0292565
Supplier: MERCK
Expiry date: April 30, 2016
MnCl2 x 4 H2O 415.0 ug/L
Batch No.: A0250927
Supplier: MERCK
Expiry date: December 31, 2015
ZnCl2 3.0 ug/L
Batch No.: B0957416
Supplier: MERCK
Expiry date: July 31, 2015
CoCl2 x 6 H2O 1.5 ug/L
Batch No.: B0396239
Supplier: MERCK
Expiry date: April 30, 2014
CuCl2 x 2 H2O 0.01 ug/L
Batch No.: K43082433
Supplier: MERCK
Expiry date: December 31, 2016
Na2MoO4 x 2 H2O 7.0 ug/L
Lot No.: SLBC3966V
Supplier: SIGMA-ALDRICH
Retest date: May 31, 2016
Stock solution 4
(bicarbonate) NaHCO3 50.0 mg/L
Batch No.: PP/2012/05232
Supplier: REANAL (lach:ner)
Expiry date: January 31, 2017
* This corresponds to the concentration of 12.0 mg/L of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)

Environmental Conditions

The test containers were kept during the test in a climate chamber with controlled environmental conditions.

Temperature: Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.1 – 23.0 °C measured in the flask and between 21.8 and 23.4 °C measured within the climate chamber.

pH: The pH was measured in each test group at the start and in each test vessel at the end of the test. The range of the pH was 7.09 – 8.24 during the experiment. The pH of the control cultures did not vary by more than 1.5 units during the experiment.

Light Intensity: The algal culture flasks were continuously illuminated with fluorescent lamps (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the position occupied by algal culture flasks at the start of the test. The average measured light intensity was 8928 lux. The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.

Test Units

Type and Size: Erlenmeyer flasks of 250 mL volume with 50 mL test medium
Identification: Each test unit was uniquely identified with study code, test item concentration and replicate.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
130.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 88.62 – 191.23 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
27.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 24.09 – 31.63 mg/L
Details on results:
Validity of the Test

The cell density in the control cultures increased by a factor of 71.33 within 72 hours. This corresponds to a specific growth rate of 1.42 day-1.
The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
- CV for section-by-section growth rate day 0-1: 6.40 %
- CV for section-by-section growth rate day 1-2: 5.13 %
- CV for section-by-section growth rate day 2-3: 6.18 %
The mean coefficient of variation for section-by-section specific growth rates: 5.90 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.68 %.

All validity criteria were met, therefore the study can be considered as valid.

Analytical Results

Five different concentrations arranged in a geometric series with a separation factor of 2.0 and a concurrent untreated control was tested in the main experiment. The nominal concentrations of the test item were 6.25; 12.5; 25; 50 and 100 mg/L. The analytically measured test item concentrations were 5.71; 11.61; 22.81; 47.23 and 91.53 mg/L at the start and 5.93; 12.23; 23.84; 50.77 and 99.36 mg/L at the end of the test. As the performed analytical determinations confirmed that the test item concentration remained within the range of 20 % of the nominal concentration, the biological results are reported based on the nominal concentrations of the test item.
Detailed description of the analytical part of the study (method, determination, results) is attached to the Report as Appendix II.

Biological Results

The influence of the test item YELLOW LF 6881 on the growth of Pseudokirchneriella subcapitata is shown in Tables 5, 6-14 and Figures 1-3.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software. After the exposure period of 72 hours average specific growth rates and yield were significantly different from that of the control group in the concentrations of 12.5; 25; 50 and 100 mg/L. (see Table 10 and 11).

The test concentration of 6.25 mg/L was determined as the 72-hour No Observed Effect Concentration (NOEC) related to the growth rate and yield.
The test concentration of 12.5 mg/L was determined as the 72-hour Lowest Observed Effect Concentration (LOEC) related to the growth rate and yield.
The 72-hour ErC50 was determined to be > 100 (130.18) mg/L (95 % CL: 88.62 – 191.23 mg/L).
The 72-hour EyC50 was determined to be 27.60 mg/L (95 % CL: 24.09 – 31.63 mg/L).

Microscopic Examinations

The shape of the algal cells growing was obviously not affected by the test item.
The cell density in the control has increased from nominal N= 1 x 104 cells/mL at the start of the test (0 hours) to N= 71.33 x 104 cells/mL (mean value) after 72 hours by a factor of 71.33. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
The behaviour of the test item did not show any remarkable observations.
Results with reference substance (positive control):
For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.
The date of the last study (Study Code: 392.440.4633) with the reference item Potassium dichromate was: 01 – 04 October 2013.
The 72h ErC50: 1.33 mg/L, (95 % confidence limits: 1.22 – 1.46 mg/L)
The 72h EyC50: 0.90 mg/L, (95 % confidence limits: 0.81 – 1.00 mg/L
Reported statistics and error estimates:
Mean values and standard deviations were calculated for each treatment using Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test ( = 0.05) using TOXSTAT software. The data passed normality test by Chi-Square Test and homogeneity of variance test Bartlett’s Test ( = 0.05, statistical software program TOXSTAT).
For determination of the ErCx and EyCx values with 95 % confidence limits, Probit analysis was performed using TOXSTAT software.

Any other information on results incl. tables


The Specific Growth Rates during the Test Period of 72 Hours

Concentration (mg/L)

 

Growth Rates (r)

0 – 24 h

0 – 48 h

0 – 72 h

Control

 

0.0578

0.0590

0.0600

 

 

0.0578

0.0564

0.0601

 

 

0.0578

0.0578

0.0580

 

 

0.0578

0.0564

0.0582

 

 

0.0578

0.0590

0.0601

 

 

0.0671

0.0602

0.0590

 

Mean

0.0593

0.0581

0.0592

 

SD

0.0038

0.0015

0.0010

6.25

 

0.0578

0.0602

0.0603

 

 

0.0578

0.0590

0.0603

 

 

0.0671

0.0564

0.0601

 

Mean

0.0609

0.0586

0.0603

 

SD

0.0054

0.0019

0.0001

12.5

 

0.0578

0.0578

0.0543

 

 

0.0578

0.0564

0.0549

 

 

0.0458

0.0550

0.0541

 

Mean

0.0538

0.0564

0.0544

 

SD

0.0069

0.0014

0.0004

25

 

0.0671

0.0500

0.0481

 

 

0.0578

0.0518

0.0490

 

 

0.0458

0.0500

0.0477

 

Mean

0.0569

0.0506

0.0483

 

SD

0.0107

0.0010

0.0007

50

 

0.0458

0.0405

0.0401

 

 

0.0458

0.0458

0.0429

 

 

0.0458

0.0433

0.0429

 

Mean

0.0458

0.0432

0.0420

 

SD

0.0000

0.0026

0.0016

100

 

0.0289

0.0335

0.0376

 

 

0.0458

0.0373

0.0367

 

 

0.0458

0.0433

0.0356

 

Mean

0.0401

0.0381

0.0366

 

SD

0.0098

0.0049

0.0010


Growth Rates (r) and Percentage Inhibition of r during the Test Period

Concentration
[mg/L]

Growth Rates r and % Inhibition of r

0–24 h

0–48 h

0–72 h

r

%

r

%

r

%

Control

0.0593

0.0

0.0581

0.0

0.0592

0.0

6.25

0.0609

-2.6*

0.0586

-0.7*

0.0603

-1.7*

12.5

0.0538

9.3

0.0564

3.0

0.0544

8.1

25

0.0569

4.1

0.0506

13.0

0.0483

18.5

50

0.0458

22.8

0.0432

25.7

0.0420

29.1

100

0.0401

32.3

0.0381

34.5

0.0366

38.2

*The negative value (slight stimulation) was treated in the later statistical evaluations as zero inhibition

 

 

Yield (y) and Percentage Inhibition of y during the Test Period

Concentration
[mg/L]

Yield y and % Inhibition of y

0–72 h

y

%

Control

70.3

0.0

6.25

75.7

-7.6*

12.5

49.3

29.9

25

31.3

55.5

50

19.7

72.0

100

13.0

81.5

*The negative value (slight stimulation) was treated in the later statistical evaluations as zero inhibition

 


Results of the Statistical Evaluation with the Growth Rates (Bonferroni t-Test (a=0.05))

Concentrations
(mg/L)

Calculated t-Value

Difference from Control

0–24 h

0–48 h

0–72 h

0–24 h

0–48 h

0–72 h

6.25

0.3345

0.2330

1.5060

0.0016

0.0004

0.0010

12.5

1.1977

1.0097

7.2290*

0.0056

0.0017

0.0048

25

0.5287

4.3884*

16.5162*

0.0024

0.0075

0.0110

50

2.9241

8.6991*

26.0042*

0.0135

0.0149

0.0173

100

4.1397*

11.7088*

34.0364*

0.0192

0.0201

0.0226

 

0–24 h

0–48 h

0–72 h

Minimal significant difference

0.0137

0.0051

0.0020

Percentage of control

23.0

8.7

3.3

* The mean value significantly different from the control(Bonferroni t-Test (a=0.05)).

 

Results of the statistical evaluation with the Yield (Bonferroni t-Test (a=0.05))

Concentrations
(mg/L)

Calculated t-Value (0–72 h)

Difference from Control (0–72 h)

6.25

2.3851

5.3333

12.5

9.3915*

21.0000

25

17.4413*

39.0000

50

22.6588*

50.6667

100

25.6402*

57.3333

Minimal significant difference

6.5891

Percentage of control

9.4

* The mean value significantly different from the control(Bonferroni t-Test (a=0.05)).

 


Results of Probit analysis with the Growth Rate

Concentrations
(mg/L)

Number subject

Number observed

Observed proportion

Predicted proportion

6.25

100

100

1.0000

0.9702

12.5

100

91

0.9100

0.9270

25

100

81

0.8100

0.8470

50

100

70

0.7000

0.7236

100

100

61

0.6100

0.5650

Chi2 lack of fit = 5.6588       Likelihood lack of fit = 8.5510                 Table Chi2 = 7.8147, alpha = 0.05, df = 3

 

 

Results of Probit analysis with the Yield

Concentration
[mg/L]

Number subject

Number observed

Observed proportion

Predicted proportion

6.25

100

100

1.0000

0.9157

12.5

100

70

0.7000

0.7683

25

100

44

0.4400

0.5365

50

100

28

0.2800

0.2912

100

100

18

0.1800

0.1168

Chi2 lack of fit = 19.5458    Likelihood lack of fit = 27.3405               Table Chi2 = 7.8147, alpha = 0.05, df = 3

 

 

The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate

Expressed in hours

0–24 h

24-48 h

48-72 h

0.0593

0.0570

0.0614

Expressed in days

0-1 day

1-2 day

2-3 day

1.4235

1.3674

1.4743

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with YELLOW LF 6881 the 72-h EC50 value based on growth rate was determined to be > 100 (130.18) mg/L and based on yield was determined as 27.60 mg/L.
Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be 6.25 mg/L. The overall LOEC of the test item is 12.5 mg/L.
Executive summary:

The effect ofYELLOW LF 6881test item was assessed on algal growth using the unicellular green algaPseudokirchneriella subcapitata(Selenastrum capricornutum), over an exposure period of 72 hourson the recommendations of theOECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 23rdMarch, 2006 (Annex 5 corrected: 28 July 2011).

Based on the results of a non-GLP preliminary test, the nominal test item concentrations included in the study were 6.25; 12.5; 25; 50 and 100 mg/L.The analytically measuredtest item concentrations ranged between 91 – 94 % of the nominal at the start and 95 – 102 % of the nominal at the end of the test.As the performed analytical determinations confirmed that the test item concentration remained within the range of 20 % of the nominal concentration, the biological results are reported based on the nominal concentrations of the test item.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rate and yield were significantly lower from that of the control group in the concentration range of 12.5 – 100 mg/L (Bonferroni t-Test; a=0.05).

 

The 72-hour ErC50was determined to be > 100 (130.18) mg/L (95 % CL*: 88.62 – 191.23 mg/L).

The 72-hour EyC50was determined to be 27.60 mg/L (95 % CL*:24.09 – 31.63 mg/L).

 

*CL = confidence limits

 

Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be 6.25 mg/L. The overall LOEC of the test item is 12.5 mg/L.