Sodium anisate

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:

28 Aug - 29 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

Deviations:

yes

Remarks:

See section "Any other information on material and methods incl. tables"

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test item concentrations and the control were analysed
- Sampling method: At test start and test end, all samples were analysed in duplicates
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ -20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 320 mg test item/L was prepared by dissolving 319.9 mg test item into 999.69 mL test water by intense stirring for 15 min. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
- Controls: yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source: supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", Göttingen, Germany
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Algae were cultivated in the laboratories of test facility under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions: Same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21.6 - 25 °C

pH:

8.0 - 9.5 (Control)
8.0 - 9.6 (Test item concentration)

Nominal and measured concentrations:

control, 3.2, 10, 32, 100 and 320 mg test item/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with sterile stoppers)
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks; 50 mL of test volume; Continuously stirred with magnetic stirrers and incubated in a water bath.
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 107.88 x 10E+04
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water was used to perform the OECD meadia according to OECD 201
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes (all working steps were performed under the clean bench)
- Photoperiod: Continuous illumination
- Light intensity and quality: 5202 lux (range: 4840 to 5620 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell density was determined by spectrophotometric measurement, based on a calibration curve

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study: non-GLP range-finder was performed without any additional information

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Statistically significant but biological not relevant since the inhibition effect is below 10%.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

177 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

115 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 h of test duration did not show any difference between the highest test item concentration 320 mg test item/L and the algal cells in the control.
- Other: One replicate of the control was excluded from the calculation based on outlier-test after Dixon & Hartley

Results with reference substance (positive control):

Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate:
EC50: 1.12 mg test item/L (growth rate)
EC10: 0.541 mg test item/L (growth rate)
NOEC: 0.21 mg test item/L (growth rate)

Reported statistics and error estimates:

EC values were calculated by a 3-parametric concentration distribution function. The NOEC and LOEC were determined by Williams t-test (yield) and Step-down Jonckheere Terpstra test (growth rate). Moreover an outlier-test after Dixon & Hartley was performed leading to exclusion of one replicate in the control. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1: Growth rates µ and percentage Inhibition ofµ during the test period

Nominal concentration [mg test item/L]	Growth rates µ [1/day] and % inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control 1)	1.135	-	1.762	-	1.791	-
3.2	1.044	8.1	1.771	-0.5	1.789	0.1
10	1.091	3.8	1.758	0.2	1.779	0.7
32	1.175	-3.5	1.748	0.8	1.778	0.8
100	1.091	3.8	1.706	3.2*	1.761	1.7*2)
320	0.219	80.7	1.539	12.6*	1.613	9.9*2)

1) One replicate was excluded as an outlier (confirmed by Dixon & Hartley outlier test).

2) Statistically significant but biologically not relevant since the inhibition effect is below 10%.

Table 2: Summary of Analytical Results

Sample Description	% of nominal 1)	RSD
[mg test item/L]		[%]	n
Control	n.a.	n.a.	4
3.2	104	2	4
10	103	2	4
32	91	2	4
100	103	0	4
320	108	1	4

1 mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n: number of analysed samples

n.a.: not applicable

Table 3: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	216	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	35%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.9%	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please see section “Any other information on results incl. tables”.

Conclusions:

EC50 (72h) >320 mg/L (P. subcapitata, OECD 201, nominal)

Description of key information

EC50 (72h) >320 mg/L (P. subcapitata, OECD 201, nominal)

EC10 (72h) >320 mg/L (P. subcapitata, OECD 201, nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

320 mg/L

EC10 or NOEC for freshwater algae:

320 mg/L

Additional information

One experimental study is available investigating the toxicity of the test item sodium Anisate (CAS 536-45-8) to aquatic freshwater algae.

The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. The definitive test was carried out with the following concentration range: control, 3.2, 10, 32, 100 and 320 mg test item/L (nominal). The measured concentrations of the test item at test start (0h) and test end ranged between 90% and 109% of the nominal values. Thus, it is justified that the effect concentrations are based on the nominal concentrations. Inhibition of growth was recorded resulting in an ErC10 and ErC50 (72h) of >320 mg/L. Inhibition of yield was recorded resulting in an EyC10 (72 h) of 115 mg/L and EyC50 (72 h) of >320 mg/L.