Polar modified Rice Bran Wax

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:

2016-01-11 to 2016-01-15

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

GLP compliance:

yes

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.

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 (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

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

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.

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

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)

Description of key information

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.