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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

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, EL_x-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