A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 January 1997 - 17 January 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Details on test solutions:

The test article formulates of ANOX BF were prepared by transferring suitable volumes of a test article stock solution (1% acetone) into the study glass flasks. The containers were made up to 50 ml with algal growing medium in order to obtain the final nominal concentrations of 0.1, 0.1 8, 0.32, 0.56 and 1 mg/l.
Formulate analysis: samples were removed at 0 and 72 hours from all treatment formulates and were analyzed by the Sponsor.
The two lowest concentrations tested (0.1 and 0.18 mg/l) could not be checked because lower than the detection limits. For the two highest concentrations tested (0.56 and 1.00 mg/l) the measured values at 0 h time analysis were above the solubility limit in water, this is probably due to the sorption of the substance onto colloidal species present in solution.
Because of this analytical result it was decided to make statistical calculations using the nominal concentrations.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Selenastrum capricomutum ATCC 22662.

Justification for the selection of the test system: Selenastrum capricomutum was chosen as a species of unicellular fresh-water-green-alga since it is widely accepted by Health Authorities as an experimental model with documented susceptibility to a wide range of toxic substances.

Supplier: laboratory breeding algae cultured from the line purchased from American Type Culture Collection, 1230 1 Parklawn Drive - Rockville, Maryland 20852 USA.

Culturing apparatus: Algae were cultured in a chamber with a temperature range of 23 +/- 2˚ C and with continuous uniform illumination of approximately 6000 Lux in the spectral range 400-700 nm.

Culturing medium: the algae were cultured in the double- strength EPA algal assay medium (EPA/600/4-85/014, Section 14,method 1003.0).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

As per guideline

Hardness:

40 mg CaCO3/L

Test temperature:

Not specified

pH:

7.5 - 7.8

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: 0.10,0.18,0.32,0.56 and 1 mg/l.

Details on test conditions:

Experimental design: the test system was treated with the following nominal concentrations: 0.10,0.18,0.32,0.56 and 1 mg/l. One control with acetone as solvent control was tested. One control only with water was also tested.
Exposure period: 72 hours
Test containers : silylated glass flasks (100 ml capacity). The algae were kept in suspension by aeration.
Initial cell concentration: Approximately l0^4 cells/ml. The inoculum was withdrawn from a preculture incubated about 3 days under the test conditions.
Cell concentration counting: the cell concentration was determined at 24, 48 and 72 after the start of the study. A microscope with counting chamber was used to determine cell concentrations.
Parameter checking: at 0 and 72 hours pH of the test and control solutions was measured by means of the pH meter Radiometer PHM 80 standard.
EbC50, ErC50 and its statistical limits: were calculated by the modified probit method set up by the Flemish Institute for Technolocical Research (VITO).
NOEC: by ANOVA procedure.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Basis for effect:

biomass

EbC50 AND ErC50 AND ITS STATISTICAL LIMITS AND NOEC

Observation time (h)	24 h	48 h	72 h
EbC50 (mg/l)	0.469	>1	>1
95% conf.	0.462-0.476
NOEC (b) (mg/l)	0.32	0.1	0.1
ErC50 (mg/l)	0.96	>1	>1
95% conf.	0.93-0.99
NOEC (r) (mg/l)	0.32	0.1	0.18

 

 

Control 0 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	3.12	31.2	171.1
	0.68	4.69	32	170
	0.68	3.12	34.4	171.9
	0.68	3.9	32.8	171.1
	0.68	4.69	32	171.9
	0.68	4.69	35.9	171.9
	N(0)	N(1)	N(n-1)	N(n)
	0.68	4.035	33.05	171.317
pH	7.48	/	/	7.66
Mean area under the growth curve A	/	40.26	468.96	2905.04
Mean specific growth rate µ	/	0.074	0.081	0.077

 

Group 1 -Solvent control

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	4.69	31.2	171.9
	0.68	3.9	34.4	170
	0.68	3.9	31.2	171.9
	N(0)	N(1)	N(n-1)	N(n)
	0.68	4.6	33.05	171.317
pH	7.50	/	/	7.75
Mean area under the growth curve A	/	41.80	462.64	2888.72
% inhibition: IA	/	3.9	1.3	0.6
Mean specific growth rate µ	/	0.075	0.080	0.077
% mean specific growth rate inhibition: Iµ	/	-1.8	0.62	0.01

 

Group 2 - 0.1 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	3.12	30.5	168
	0.68	3.12	30.5	168
	0.68	3.9	31.2	171.9
	N(0)	N(1)	N(n-1)	N(n)
	0.68	3.38	30.7	169.3
pH	7.55	/	/	7.76
Mean area under the growth curve A	/	32.40	425.44	2809.52
% inhibition: IA	/	19.5	9.3	3.3
Mean specific growth rate µ	/	0.067	0.079	0.077
% mean specific growth rate inhibition: Iµ	/	9.9	1.9	0.2

 

Group 3 - 0.18 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	3.12	26.6	154.7
	0.68	3.9	28.9	168
	0.68	3.12	31.2	157
	N(0)	N(1)	N(n-1)	N(n)
	0.68	3.38	28.9	159.9
pH	7.60	/	/	7.77
Mean area under the growth curve A	/	32.48	403.60	2652.88
% inhibition: IA	/	19.3	13.9	8.7
Mean specific growth rate µ	/	0.069	0.078	0.076
% mean specific growth rate inhibition: Iµ	/	9.8	3.5	1.2

 

Group 4 - 0.32 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	3.13	28.9	162.5
	0.68	2.34	26.6	157
	0.68	3.13	28.9	154.7
	N(0)	N(1)	N(n-1)	N(n)
	0.68	2.87	28.13	158.07
pH	7.60	/	/	7.87
Mean area under the growth curve A	/	26.24	381.92	2600
% inhibition: IA	/	34.8	18.6	10.5
Mean specific growth rate µ	/	0.060	0.078	0.076
% mean specific growth rate inhibition: Iµ	/	19.2	4.1	1.5

 

Group 5 - 0.56 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	1.56	28.9	166.4
	0.68	1.56	31.2	161.7
	0.68	2.34	32.6	140.6
	N(0)	N(1)	N(n-1)	N(n)
	0.68	1.82	30.9	15.23
pH	7.60	/	/	7.83
Mean area under the growth curve A	/	13.68	390	2619.28
% inhibition: IA	/	66	16.8	9.8
Mean specific growth rate µ	/	0.041	0.08	0.076
% mean specific growth rate inhibition: Iµ	/	44.7	1.7	1.7

 

Group 6 - 1 mg/l

	Cell density 10^4 cells/ml
	T(0) 0	T(1)24	T(n-1)48	T(n)72
	0.68	1.56	29.7	151.5
	0.68	1.56	26.6	150
	0.68	2.3	29.7	152.3
	N(0)	N(1)	N(n-1)	N(n)
	0.68	1.807	28.67	151.27
pH	7.60	/	/	7.82
Mean area under the growth curve A	/	13.52	362.88	2505.76
% inhibition: IA	/	66.4	22.6	13.7
Mean specific growth rate µ	/	0.041	0.080	0.075
% mean specific growth rate inhibition: Iµ	/	45.1	3.7	2.3

 

Validity criteria fulfilled:

yes

Conclusions:

Experimental data from an algal inhibition growth study in which the unicellular green algae Selenastrum capricornutum were treated with the test article ANOX BF at the nominal concentrations of 0.10, 0.1 8, 0.32, 0.56 and 1 mg/l
The algae were treated and observed for 72 hours.
The EbC50 at 24 hours was 0.469 and at 48 and 72 hours was >1 mg/l .
The ErC50 at 24 hours was 0.96 and at 48 and 72 hours was >l mg/l .
The NOEC (b) at 24 hours was 0.32 and at 48 and 72 hours was 0.1 mg/l.
The NOEC (r) at 24 hours was 0.32, at 48 hours was 0.1 and at 72 hours was 0.18 mg/l .

Executive summary:

The purpose of the study was to evaluate the effects of the test article ANOX BF on

the growth of the unicellular fresh-water green alga Selenastmm capricornutum and

to determine, if any, the percentage of inhibition.

Experimental data from an algal inhibition growth study in which the unicellular green algae Selenastrum capricornutum were treated with the test article ANOX BF at the nominal concentrations of 0.10, 0.1 8, 0.32, 0.56 and 1 mg/l

The algae were treated and observed for 72 hours.

The EbC50 at 24 hours was 0.469 and at 48 and 72 hours was >1 mg/l .

The ErC50 at 24 hours was 0.96 and at 48 and 72 hours was >l mg/l .

The NOEC (b) at 24 hours was 0.32 and at 48 and 72 hours was 0.1 mg/l.

The NOEC (r) at 24 hours was 0.32, at 48 hours was 0.1 and at 72 hours was 0.18 mg/l .

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June 2014 - 10 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables"

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables"

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables"

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 504489.
On the day of analysis, the test samples were defrosted at room temperature. The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 50/50 (v/v) acetonintrile/M2-medium to obtain concentrations within the calibration range.

Stock and spiking solutions
Stock solutions of the test substance were prepared in acetonitrile at concentrations of 2000 and 3001 mg/l.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was acetonitrile.

Calibration solutions
Calibration solutions in the concentration range of 0.04 – 10 mg/l were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.

Procedural recovery samples
2 ml blank medium was spiked with the test substance at a target concentration of 0.1 or 100 mg/l.
The accuracy samples were treated similarly as the test samples (see paragraph 4.2 ‘Samples’).

Blank procedural recovery samples were prepared and treated similarly to the test samples.

Sample injections
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). No correction was made for the purity/composition of the test substance.
The batch of ANOX® 1315 tested was a clear yellow viscous liquid with a purity of 93.5% by GC and not completely soluble in test medium at the loading rates initially prepared.
Preparation of test solutions started with individual loading rates of 1.0, 10 and 100 mg/l. A two-day period of magnetic stirring was applied and was followed by a two- (combined limit/range-finding test) or one-hour (limit test) settlement period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off and used as test solutions. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/l

Test temperature:

22°C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Combined limit/range-finding test nominal concentration 1.0, 10 and 100 mg/l.
Limit test nominal concentration 1.0 mg/l.
Limit test measured concentration 0.17 mg/l.

Details on test conditions:

Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 94 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Combined limit/range-finding test
Mean cell densities, inhibition of growth rate and inhibition of yield
Based on these results samples taken from WAFs prepared at 10 and 100 mg/l were analysed. The initial concentration were 4.5 and 13 mg/l, respectively. Measured concentrations decreased to 45-75% of initial at the end of the test, respectively. Since significant effects were observed at concentrations strongly exceeding the water solubility of the test substance it was decided, in consultation with the sponsor, to perform the final test as a limit with WAF prepared at a loading rate of 1.0 mg/l.
Test conditions, except for temperature, were maintained within the limits prescribed by the protocol.

Limit test
Measured test substance concentrations
The actual concentration measured at the start of the test was 0.44 mg/l. The measured concentration decreased to 11% of initial at the end of the test. Based on these results, the average exposure concentration was calculated. It should be noted that the concentration measured at the end of the test was obtained by extrapolation and therefore, is considered an estimate. Therefore, the TWA concentration of 0.17 mg/l should be considered indicative. The concentration measured in the WAF prepared at 1.0 mg/l and incubated without algae showed similar results indicating that the observed decrease was not caused by algal biomass.

Inhibition of growth rate and inhibition of yield
No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Experimental conditions
The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 22°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 1.7 mg/l with a 95% confidence interval ranging from 1.6 to 1.8 mg/l. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/l. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.56 mg/l with a 95% confidence interval ranging from 0.50 to 0.64 mg/l. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Mean cell densities (x104 cells/ml) during the combined limit/range-finding test

Time (h)		ANOX® 1315, WAF prepared at (mg/l)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	4.5	4.2	4.3	3.2
48	14.1	15.7	13.6	7.6
72	51.8	55.6	49.1	18.0

 

Percentage inhibition of growth rate during the combined limit/range-finding test

ANOX® 1315, WAF prepared at (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.315	0.0281	6	0.0
1.00	1.339	0.0135	3	-1.8
10.00	1.297	0.0343	3	1.4
100.00	0.957	0.0657	6	27

 

Percentage inhibition of yield during the combined limit/range-finding test

ANOX® 1315, WAF prepared at (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	50.841	4.2840	6	0.0
1.00	54.633	2.2274	3	-7.5
10.00	48.101	5.0636	3	5.4
100.00	16.950	3.3799	6	67

 

Limit test - Measured concentrations versus nominal concentrations

ANOX® 1315, WAF prepared at (mg/l)	Measured concentration (mg/l)
	t=0h	t=24h	t=72 h	TWA (mg/l)
1.0	0.437	0.228	0.0460^1	0.17^2
1.0 WA	0.442	0.298	0.0960	0.23

WA – without algae

1. Value obtained by extrapolation of the calibration curve, it is considered an estimate.


2. Indicative value

 

Percentage inhibition of growth rate (total test period) during the limit test

ANOX® 1315 TWA concentration (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.764	0.0318	6	0.0
0.17	1.766	0.0227	6	-0.1

 

Percentage inhibition of growth rate at different time intervals during the limit test

ANOX® 1315 TWA concentration (mg/l)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.1	0.0	1.7	0.0	1.5	0.0
0.17	6	2.1	-0.4	1.8	-1.3	1.4	1.6

 

Percentage inhibition of yield during the limit test

ANOX® 1315 TWA concentration (mg/l)	Mean	Std. Dev.	n	%Decrease
Control	198.524	18.5500	6	0.0
0.17	199.481	13.2081	6	-1.5

 

Effect parameters

	Parameter (mg/l)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.17^1	0.17^1	0.17^1	0.17^1
Yield	Value	0.17^1	0.17^1	0.17^1	0.17^1

1. This concentration is considered the maximum soluble in test medium.

pH levels recorded during the final test

ANOX® 1315 TWA concentration (mg/l)	pH
	t=0h	t=72h
Control	8.2	8.3
0.17	8.1	8.2

 

REFERENCE TEST

Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7 (mg/l)	Mean	Std. Dev.	n	%Inhibition
control	1.817	0.0565	3	0.0
0.18	1.777	0.0562	3	2.2
0.32	1.737	0.0543	3	4.4
0.56	1.627	0.0273	3	10.4
1.0	1.221	0.0796	3	32.8
1.8	0.808	0.0246	3	55.5
3.2	0.566	0.0298	3	68.9

 

Overview of % inhibition of yield in the reference test:

Nominal conc. K2Cr2O7 (mg/l)	Mean	Std. Dev.	n	%Inhibition
control	234.296	39.6789	3	0.0
0.18	207.641	35.5620	3	11.4
0.32	183.677	31.3167	3	21.6
0.56	131.148	11.0573	3	44.0
1.0	38.692	9.5793	3	83.5
1.8	10.323	0.8208	3	95.6
3.2	4.476	0.5018	3	98.1

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at a TWA concentration being considered the maximum soluble in the medium.
The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) exceeded the maximum soluble concentration, i.e. was above 0.17 mg/l.
The 72h-NOEC for growth rate and yield inhibition was 0.17 mg/l i.e. maximum solubility of the test substance in test medium).

Description of key information

ANOX BF Algal growth inhibition study

The EbC50 at 24 hours was 0.469 and at 48 and 72 hours was >1 mg/l .

The ErC50 at 24 hours was 0.96 and at 48 and 72 hours was >l mg/l .

The NOEC (b) at 24 hours was 0.32 and at 48 and 72 hours was 0.1 mg/l.

The NOEC (r) at 24 hours was 0.32, at 48 hours was 0.1 and at 72 hours was 0.18 mg/l .

 

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with ANOX® 1315.

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at a TWA concentration being considered the maximum soluble in the medium.

The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) exceeded the maximum soluble concentration, i.e. was above 0.17 mg/l.

The 72h-NOEC for growth rate and yield inhibition was 0.17 mg/l i.e. maximum solubility of the test substance in test medium).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

ANOX BF Algal growth inhibition study

The purpose of the study was to evaluate the effects of the test article ANOX BF on the growth of the unicellular fresh-water green alga Selenastmm capricornutum and to determine, if any, the percentage of inhibition.

Experimental data from an algal inhibition growth study in which the unicellular green algae Selenastrum capricornutum were treated with the test article ANOX BF at the nominal concentrations of 0.10, 0.1 8, 0.32, 0.56 and 1 mg/l

The algae were treated and observed for 72 hours.

The EbC50 at 24 hours was 0.469 and at 48 and 72 hours was >1 mg/l .

The ErC50 at 24 hours was 0.96 and at 48 and 72 hours was >l mg/l .

The NOEC (b) at 24 hours was 0.32 and at 48 and 72 hours was 0.1 mg/l.

The NOEC (r) at 24 hours was 0.32, at 48 hours was 0.1 and at 72 hours was 0.18 mg/l .

 

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with ANOX® 1315.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 2012 and the OECD series on testing and assessment number 23, 2000.

The batch of ANOX® 1315 tested was a clear yellow viscous liquid with a purity of 93.5% by GC and not completely soluble in test medium at the loading rates initially prepared.

A limit test was performed based on the results of a combined limit/range-finding test. A Water Accommodated Fraction (WAF) was prepared at a loading rate of 1.0 mg/l. A two-day period of magnetic stirring was applied and was followed by a one-hour settlement period. Thereafter, the aqueous WAF was siphoned off and used as test solution. The final test solution was clear and colourless.

Six exponentially growing algal cultures per group were exposed to an untreated control and the limit concentration prepared at a loading rate of 1.0 mg ANOX® 1315 per litre. Initial cell density was 104 cells/ml. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

The actual concentration measured at the start of the test was 0.437 mg/l. The measured concentration decreased to 11% of initial at the end of the test. Based on these results, the average exposure concentration was calculated to correspond to 0.17 mg/l and was considered to be the maximum soluble concentration in test medium. It should be noted that the concentration measured at the end of the test was obtained by extrapolation and therefore, is considered an estimate. Therefore, the TWA concentration of 0.17 mg/l should be considered indicative.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

No inhibition of growth rate or inhibition of yield was recorded at a TWA concentration being considered the maximum soluble in the medium.

The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) exceeded the maximum soluble concentration, i.e. was above 0.17 mg/l.

The 72h-NOEC for growth rate and yield inhibition was 0.17 mg/l i.e. maximum solubility of the test substance in test medium.