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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 2018-01-07 to 2018-01-11, with the definitive exposure phase from 2018-01-08 to 2018-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Principles of method if other than guideline:
None
GLP compliance:
yes
Analytical monitoring:
no
Remarks:
TOC Analysis
Vehicle:
no
Details on test solutions:
Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is an UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018) Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the WAF
One water accommodated fraction (WAF) was prepared with a nominal loading rate of 100 mg test item/L.
For the loading rate, an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of dilution water. This dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fraction (WAF) was used in the test.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.
The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Not specified
Test temperature:
min.: 21.0 max.: 22.5 mean value: 21.8
pH:
Nominal test item loading pH-values
[mg/L] Start; 0 hours End; 72 hours
100 8.03 8.88
Control 8.08 9.07
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
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 10^3 cells/mL, Actual: mean 6442 cells/mL
- Control end cells density: Mean 1532094 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm)
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 6003 lux,measured: 5223 - 6709 with a mean of 6033 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-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 a background signal. No self-fluorescence was found in the preliminary range finding test at the loading rate of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate und yield inhibition
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield inhibition
Duration:
72 h
Dose descriptor:
other: EL10/20/50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
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: The test solutions were clear and colourless during the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2005, and the limits were set using the threefold standard deviation of these values.

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 0.799 0.753 ± 0.544
95% confidence interval 0.707 – 1.30
Yield inhibition
EyC50 0.607 0.407 ± 0.313
95% confidence interval 0.404 – 0.694
SD = Standard deviation
Reported statistics and error estimates:
EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design).

NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The Brown-Forsythe test was done for analysis of variance homogeneity. 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 are computer-generated and have been rounded for presentation from the full 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.

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the nominal test item loadings.

Cell Densities

 

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6442

21498

160029

1415350

2

6442

23343

182114

1476966

3

6442

22345

179442

1469618

4

6442

24567

170754

1604963

5

6442

24656

186135

1509536

6

6442

18989

172829

1422202

Mean

6442

22566

175217

1483106

Control

1

6442

24808

176682

1633562

2

6442

24520

198568

1512614

3

6442

23132

196373

1491613

4

6442

22665

194536

1547767

5

6442

20637

197028

1455219

6

6442

22514

210643

1551788

Mean

6442

23046

195638

1532094

 


Evaluation after 72 hours

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

Nominal test item loading

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.80

1

 

1408908

8

2

                                                                                        

1.81

1

 

1470524

4

3

 

1.81

1

 

1463176

4

4

 

1.84

-1

 

1598521

-5

5

 

1.82

0

 

1503094

1

6

 

1.80

1

 

1415760

7

Mean

(-)

1.81

1

(-)

1476664

3

Control

1

 

1.85

 

 

1627120

 

2

 

1.82

 

 

1506172

 

3

 

1.82

 

 

1485171

 

4

 

1.83

 

 

1541325

 

5

 

1.81

 

 

1448777

 

6

 

1.83

 

 

1545346

 

Mean

 

1.82

 

 

1525652

 

 

 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

1.35

1.96

2.22

1.85

0.450

24.4

26.7

2

1.34

2.09

2.03

1.82

0.419

23.0

3

1.28

2.14

2.03

1.82

0.468

25.8

4

1.26

2.15

2.07

1.83

0.495

27.1

5

1.16

2.26

2.00

1.81

0.571

31.6

6

1.25

2.24

2.00

1.83

0.514

28.1

 

 

 

Mean

1.82

 

 

 

 

 

SD ±

0.01

 

 

 

 

CV [%]

0.73

 

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, Licocare RBW 300 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.
Effect values were determined as follows (based on nominal test item loadings):
The EL50-values for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 100 mg/L.
Executive summary:

The toxicity of Licocare RBW 300 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-01-07 to 2018-01-11, with the definitive exposure phase from 2018-01-08 to 2018-01-11 at the test facility. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. As the test item is a UVCB substance, a water accommodated fraction (WAF) with the nominal loading rate of 100 mg test item/L was prepared.

Six replicates were tested for the limit loading and six for the control. The test media were clear and colorless throughout the test period. The environmental conditions were within the acceptable limits.

 

Due to the low solubility of the test item, no analytical monitoring was carried out.

 

All effect values given are based on the nominal test item loading of Licocare RBW 300.

NOEL, LOEL, ELx-values ofLicocareRBW 300(0 - 72 hours)

based on the nominal test item loadings

 

Growth Rate Inhibition
nominal test item loadings [mg/L]

NOEL

  100

LOEL

> 100

ErL10

> 100

ErL20

> 100

ErL50

> 100

 

Yield Inhibition

nominal test item loadings [mg/L]

NOEL

  100

LOEL

> 100

EyL10

> 100

EyL20

> 100

EyL50

> 100

Description of key information

The toxicity ofLicocare RBW 300 to the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3

In this study, Licocare RBW 300 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.

Effect values were determined as follows (based on nominal test item loadings):

The EL50-values for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information